Tcf21MerCreMer or in activated cardiac fibroblasts using periostin (Postn)MerCreMer. We show that β-catenin loss of function in cardiac fibroblasts after trans-aortic constriction significantly preserves cardiac function, and reduces interstitial fibrosis but does not alter the numbers of activated or differentiated cardiac fibroblasts in vivo. However, β-catenin is specifically required in resident cardiac fibroblasts for fibrotic excessive extracellular matrix gene expression and binds Col3a1 and Postn gene sequences in cultured cardiac fibroblasts after induction of Wnt signaling. Moreover, cardiomyocyte hypertrophy is blunted with cardiac fibroblast-specific loss of β-catenin after trans-aortic constriction in vivo. Thus, Wnt/β-catenin signaling in resident cardiac fibroblasts is required for excessive extracellular matrix gene expression and collagen deposition after trans-aortic constriction.Cardiac fibrosis is characterized by excessive extracellular matrix deposition that contributes to compromised cardiac function and potentially heart failure. Cardiac pressure overload resulting from trans-aortic constriction in mice leads to cardiac fibrosis and increased Wnt/β-catenin signaling in cardiac fibroblasts. Here, we conditionally induce β-catenin loss of function in resident cardiac fibroblasts using  Understanding the mechanisms causing cardiac fibrosis is key to prevention and therapy development of many heart diseases. Here, the authors show that Wnt/β-catenin signaling in resident cardiac fibroblasts is required for deposition of fibrotic extracellular matrix and the regulation of cardiomyocyte hypertrophy in a mouse model of heart fibrosis. The hallmark of cardiac fibrosis is increased fibrillar collagen, which contributes to reduced cardiac output and can ultimately lead to heart failure4. Cardiac fibroblasts (CFs) that arise from epicardial and endothelial progenitors in the developing heart are the predominant collagen-producing cell type in pathologic cardiac fibrosis7. Although these resident CFs maintain a quiescent phenotype under physiological conditions, they can be activated in response to various types of cardiac injury8. Importantly, the regulatory mechanisms that lead to increased collagen production from resident CFs under pathophysiologic conditions, ultimately leading to heart failure, have not been fully elucidated.Cardiac fibrosis, commonly seen with a variety of cardiac injuries, can significantly reduce tissue compliance and disrupt cardiac conduction, thus contributing to morbidity and mortality associated with heart disease11. However the role of Wnt/β-catenin signaling in myocardial interstitial fibrosis independent from scar formation has not been determined. In addition, the requirement for Wnt/β-catenin signaling specifically in resident CFs and direct downstream targets related to cardiac fibrosis have not been reported previously. Recently developed inducible Cre-expressing lines are effective for manipulation of gene expression in resident CF lineages. A tamoxifen (TAM)-inducible Tcf21MerCreMer (Tcf21MCM) Cre-expressing transgene is highly effective for manipulation of gene expression in adult cardiac resident fibroblasts, which are the primary source of activated fibroblasts in the adult mouse heart13. Kanisicak et al.7 have developed an inducible knock-in Cre line periostin (Postn)MCM that efficiently marks activated myofibroblasts in the fibrotic heart and other organs. These Cre drivers are not expressed in infiltrating immune cells, endothelial cells, or cardiomyocytes, and thus can be used for specific manipulation of resident and activated CFs7. Specific regulatory mechanisms that contribute to myofibroblast activation vs. fibroblast differentiation and collagen synthesis are not well-defined, particularly in hypertensive heart failure models14. Thus, the use of Tcf21MCM for genetic manipulation of resident CFs and PostnMCM to specifically target activated CFs is an effective approach for studies of CF-specific regulatory mechanisms in cardiac fibrosis.Wnt/β-catenin signaling is induced in areas of inflammation, scar formation, and epicardial activation in mouse models of autoimmune endocarditis and ischemic injuryTcf21MCM12- and PostnMCM7-mediated loss of β-catenin (Ctnnb1fl/fl). Here, we demonstrate that cardiac pressure overload leads to increased Wnt/β-catenin signaling in CFs, and loss of β-catenin in either Tcf21MCM or PostnMCM lineage results in improved cardiac function, blunted cardiac hypertrophy, reduced interstitial fibrosis and decreased expression of fibrotic extracellular matrix (ECM) protein genes 8 weeks post trans-aortic constriction (TAC). However, β-catenin loss of function (LOF) in CFs directly reduces Col1a1, Col3a1, and Postn gene expression and indirectly blunts cardiomyocyte hypertrophy. Together, these data support a regulatory role for Wnt/β-catenin signaling in fibrotic ECM gene expression and collagen deposition in CFs after TAC-induced pressure overload.The requirements for Wnt/β-catenin signaling specifically in resident and activated CFs after cardiac pressure overload were examined using 7. TAM-inducible Cre-mediated recombinase activity in Tcf21 and Postn lineages was examined in Tcf21MCM12 and PostnMCM7 mouse lines bred with the Rosa26mTmG (R26mTmG) green fluorescent protein (GFP)+ reporter line15. Mice were subjected to trans-aortic constriction (TAC) resulting in pressure overload compared to sham-operated controls 1/3 deposition after TAC ice Fig. , but litury Fig. . Thus, CTAC Fig. , as evid+ cells after TAC. β-catenin protein activation is increased in isolated CFs after pressure overload in Tcf21MCM;R26mTmG hearts, relative to sham-operated controls, as determined by western analysis   staining  in Tcf21MCM;Ctnnb1fl/fl and PostnMCM;Ctnnb1fl/fl mice compared to controls after TAC  CFs stimulated with angiotensin (Ang) II. Treatment with Ang II (100 nM) for 48 h significantly induced CF activation, as determined by αSMA staining  expression was observed. Collectively, these results demonstrate that loss of β-catenin in Tcf21 lineage CFs after TAC specifically leads to reduced collagen mRNA and protein expression in response to pressure-overload injury.Since no change in myofibroblast activation or overall numbers of CFs was observed, regulation of fibrillar collagen protein and gene expression was examined as a direct consequence of β-catenin LOF in CFs. By immunofluorescence, Col1/3 Fig.  protein ols Fig. . FibrotiTAC Fig. . mRNA exActa2 mRNA expression  CFs. Treatment with Wnt1 (100 ng/ml) for 48 h significantly induced CF activation as determined by αSMA staining Fig.  and Actaion Fig. . This inion Fig.  and incrion Fig.  indicatiion Fig. , providiCol1a1, Col3a1, and Postn, which was attenuated by XAV939  after injury21. In the adult, these induced EPDCs remain on the surface of the heart and express angiogenic factors after cardiac injury23. During cardiac fibrosis resulting from autoimmune myocarditis, Wnt1 and Wnt5a expression is induced in regions of immune cell infiltration, and inhibition of Wnt signaling via administration of soluble pathway inhibitors prevents cardiac fibrosis and improves cardiac function11. In contrast, β-catenin loss of function in collagen-expressing cells followed by ischemic injury inhibits scar formation and cardiac remodeling with adverse effects on cardiac function10, supporting a role for Wnt/β-catenin signaling in initial scar formation during the acute injury phase, as has also been demonstrated for periostin24. Here, we demonstrate that β-catenin is specifically required in resident CFs of Tcf21 and Postn lineages for fibrillar collagen deposition and gene expression after TAC-induced pressure overload. Thus, it is likely that Wnt/β-catenin signaling is activated in multiple cardiac resident and infiltrating cell types depending on the specific type of cardiac injury or stage of fibrotic remodeling25.Here, we demonstrate a requirement for Wnt/β-catenin signaling specifically in resident CFs in cardiac fibrosis after TAC. Previously, Wnt/β-catenin signaling has been reported to contribute to cardiac fibrosis in mice after ischemic injury in adults, cryoinjury in neonates, or in autoimmune endocarditis2. Here, we demonstrate that Wnt/β-catenin signaling is activated by Ang II or Tgfβ in CFs and is required for CF activation and fibrotic gene expression. Ang II induction of cardiac fibrosis can be mediated by Tgfβ signaling8, and our data show that the Wnt/β-catenin pathway is activated by both pathways. Wnt/β-catenin signaling also is required in TGFβ-mediated skin, lung, and liver fibrosis28. In mouse dermal fibroblasts and human lung fibroblasts, TGFβ signaling induces β-catenin nuclear localization via non-canonical intermediates, including TAK126. This is consistent with our observation that TGFβ induces β-catenin protein stabilization and activation in cultured CFs. Together, these studies support a role for Wnt/β-catenin downstream of Ang II and TGFβ signaling in regulation of CF activation and collagen gene expression in CFs.Multiple signaling pathways, notably Ang II and Tgfβ, have been implicated in aspects of CF activation, myofibroblast differentiation, and collagen remodeling during the cardiac fibrotic response29. Wnt1 treatment also indirectly induces Col1a2 promoter activity in dermal fibroblasts26. Stabilized β-catenin in dermal fibroblasts is sufficient to increase Col1a1 expression and promote skin fibrosis in mice29. Additionally, loss of β-catenin suppresses bleomycin-induced dermal fibrosis with reduced collagen deposition27. Our data show that β-catenin signaling specifically affects fibrillar collagen and periostin expression in CFs. Periostin has a critical role in collagen fibril formation and remodeling that likely contributes to the lack of fibrotic ECM deposition with β-catenin LOF30. The specific reduction of fibrotic ECM expression with reduced Wnt/β-catenin signaling in resident or activated CFs suggests that this pathway could be a therapeutic target in the reduction of fibrotic progression in heart failure.Regulation of ECM gene expression directly by Wnt/β-catenin has been reported in fibroblasts from different organs, notably skinPostnCre also results in decreased cardiomyocyte hypertrophy after pressure overload through regulation of Igf1 expression31. In addition, indicators of cardiac fibrotic remodeling are detectable prior to myocardial hypertrophy or dysfunction in mice or human patients with sarcomeric protein mutations that cause familial hypertrophic cardiomyopathy33. Taken together, these studies provide evidence for crosstalk between CFs and myocytes very early in the progression of cardiac dysfunction and failure. Multiple signaling pathways including Tgfβ, FGF (fibroblast growth factor), and CTGF (connective tissue growth factor) have been implicated in this crosstalk, but specific signaling and recipient cell types have not been clearly defined34. In the current study, cardiomyocyte hypertrophy is attenuated in hearts with reduced fibrotic ECM due to loss of β-catenin specifically in resident CFs. Thus, targeting fibrotic remodeling mediated by resident CFs is important for the development of effective therapeutic approaches for heart failure.In the current study, we demonstrate that inhibition of fibrotic ECM deposition with loss of β-catenin in resident CFs leads to reduced cardiac hypertrophy and improved cardiac function after TAC. Similarly, CF-specific loss of the transcription factors KLF5 using a transgenic All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee at Cincinnati Children’s Research Foundation.Tcf21MCM (Tcf21MCM) mice have been described previously by Acharya et al.12PostnMCM (PostnMCM) knock-in mice were generated in Dr Jeffery Molkentin’s laboratory at Cincinnati Children’s Hospital Medical Center7. Mice with loxp sites inserted into the β-catenin gene (Ctnnb1fl/fl) were used for β-catenin loss-of-function studies16. ROSA26mTmG (R26mTmG) reporter  mice were purchased from Jackson Laboratory. Genotyping was performed using genomic DNA extracted from clipped tails with primers listed in Supplementary Table 2 inhalation followed by cervical dislocation at the specified time point for each experiment.35. Briefly, male mice  were anesthetized with 2% isoflurane inhalation. The animals were then placed in a supine position, and orally intubated with 20-gauge tubing and ventilated  at 120 breaths per minute . The chest cavity was exposed by cutting open the proximal portion of the sternum. After the aortic arch between the innominate and left common carotid arteries was isolated, the transverse aortic arch was ligated (7–0 Prolene) with an overlying 27-gauge needle, and then the needle was removed, leaving a discrete region of stenosis. Sham-operated mice underwent the identical surgical procedure, including isolation of the aorta, but without placement of the suture. Mice were fed with TAM diet  from day 0 post operation until the time of sacrifice to induce Cre activity.TAC was performed to create pressure overload in a mouse heart36. M-mode tracings of the LV were acquired using the short-axis view, with the ultrasound beam perpendicular to the LV at the midpapillary level to determine EF, FS, wall thickness, LV inner diameter, and LV volume. LV dimensions averaged over a minimum of five consecutive cardiac cycles per heart. Doppler echocardiography was performed on all mice subjected to TAC to ensure equal pressure gradients across the aortic constriction between groups38.Eight weeks after operation, mice were lightly anesthetized by 0.8–1% isoflurane inhalation and analyzed by B-mode and M-mode echocardiography using a Vevo2100 instrument with an 18–38 MHz transducer R26mTmG background, heart tissues were further fixed in 4% PFA overnight at 4° C and subsequently processed for paraffin embedding and sectioned at 5 μm. Heart sections were deparaffinized and hydrated through a graded ethanol series , then stained with Sirius Red to visualize fibrosis39 or hematoxylin and eosin40 to show the heart structure. For R26mTmG mice, fixed hearts were infused with 30% sucrose overnight at 4 °C and embedded in OCT  medium (SAKURA) prior to cryosectioning (5 µm). Heart sections from R26mTmG mice were incubated with 0.3% H2O2 in PBS at room temperature (RT) overnight in a ultraviolet chamber to quench the endogenous fluorescent signal before immunofluorescence staining40. The following antibodies were used for immunofluorescence: anti-collagen 1 , anti-collagen 3 , anti-αSMA , MF20 , anti-vimentin , anti-endomucin , anti-GFP , anti-phospho-histone H3 (pHH3) , anti-α-actinin , and biotinylated anti-lectin I . Corresponding anti-rabbit/mouse/chicken fluorescent-conjugated secondary antibodies  were used. Alexa Fluor 488-conjugated WGA  was used to visualize cell membranes. Nuclei were counterstained with DAPI.At the time of collecting, mouse hearts were perfusion-washed with ice-cold 1× phosphate buffered saline (PBS), followed by 0.1 ml of 10% KCl to induce diastolic arrest. The hearts were then perfusion-fixed with 4% paraformaldehyde  for 15 min. For mice not in the + cells exclusive of smooth muscle cells surrounding defined blood vessels was determined by investigators blinded to genotype in microscopic fields at ×400 in the LVFW. A minimum of 10–15 fields were analyzed and averaged to obtain the number of αSMA+ cells per field for each sample. Similar analyses were performed for the number of vimentin+ and vimentin+;endomucin− cells per field in LVFW.Low-magnification color images were obtained using a Nikon SMZ1500 microscope, DXM1200F digital camera, and NIS-Elements BR 3.2 software. High-magnification color images were obtained using an Olympus BX51 microscope, Nikon DS-Ri1 digital camera, and NIS-Elements BR 3.2 software. Fluorescent images were captured with Zeiss LSM 510 confocal microscope and LSM version BR 3.2 software. For confocal microscopy, four to six controls and mutants were stained and visualized using Nikon confocal microscope (Nikon) with consistent settings for each group. The number of individual αSMA39. Stained sections were rinsed twice in 1% acetic acid, dehydrated through ethanol washes, cleared in xylene, and mounted with Cytoseal (Electron Microscopy Sciences). Samples were analyzed using Nikon light microscope and NIS Elements Basic Research software (Nikon) with the same light setting and pixel classifier. Interstitial fibrosis was determined as the percent of collagen-stained area/total myocardial area.To detect collagen content, rehydrated heart sections were stained in Sirius Red solution for 1 h at RT+ cells from heart tissue. One was through GFP antibody -coated magnet beads  pull-down41. Magnet beads of 2 × 108 were washed three times in 0.1% BSA  in PBS and then incubated with 5 μl anti-GFP antibody  at 4 °C overnight. After three washes, GFP antibody-coated magnet beads were re-suspended in 500 μl 0.1% BSA in PBS (4 × 108/ml) and stored at 4 °C. Cardiac cell suspensions were prepared from collected mouse hearts perfused with cold PBS31. The LV was isolated, cut into small pieces, and digested in collagenase type 2 /HBSS  (Hyclone) two times at 37 °C for 20 min. The cell suspension was filtered with 100 μm mesh into 5% fetal bovine serum /HBSS. Cardiomyocytes were depleted by centrifuging at 50×g for 5 min. The supernatant was then transferred to another tube and centrifuged at 500×g for 10 min. The cell pellet was re-suspended in 0.1% BSA in PBS (4 ml for one adult heart). Magnetic beads that are 107 GFP antibody-coated were incubated with 1-ml cell suspension at 4 °C for 30–60 min and then magnetically pulled down by a magnetic rack (Invitrogen). After three washes, isolated GFP+ cells were used for protein isolation and western blot analysis.Two methods were used to isolated GFP+ cell sorting was performed using a FACSAriaII flow cytometer (BD)42. Cardiac cell suspensions of individual hearts were prepared as described above. Anti-red fluorescent protein antibody -coated magnet beads were used to deplete the non-recombined GFP-negative cells. The cell suspension was then sent for sorting using a FACSAriaII flow cytometer in the flow cytometry core at Cincinnati Children’s Medical Center. Sorted GFP+ cells were collected in Trizol reagent (Invitrogen) for mRNA analysis.Alternatively, GFP+ cells or cultured CFs. Standard western blots were performed. Briefly, 15–30 μg of protein was separated by 10–15% SDS-PAGE gel and transferred to PVDF membranes. After blocking in Odyssey blocking buffer , blots were probed with primary antibodies: activated β-catenin , total β-catenin , and GAPDH . After incubation with corresponding anti-mouse/rabbit secondary antibodies , immunoblots were developed using ODYSSEY Infrared Imaging System (LI-COR). Signal intensities were quantified with ImageStudio software (LI-COR). GAPDH was used as a loading control. Uncropped images of western blots are included in Supplementary Fig. Protein lysates were obtained from isolated GFP40. RNA of 500 ng was used to synthesize complementary DNA using Moloney murine leukemia virus reverse transcriptase  and random primers . Quantitative real-time PCR was conducted using SYBR Green PCR Master Mix  or Taqman probes . The oligonucleotide primer sequences used with SYBR green are listed in Supplementary Table Gapdh (rat) or β-2-microglobulin (B2M) were determined using the comparative ΔΔCt method40.For mRNA analysis, total RNA was extracted with Trizol reagent (Invitrogen)Tcf21MCM;R26mTmG;Ctnnb1fl/fl β-catenin LOF and Tcf21MCM;R26mTmG;Ctnnb1fl/+ control hearts isolated 4 weeks post TAC were stained using a TUNEL kit  according to the manufacturer’s instructions. Briefly, sections were permeabilized with 0.1% Triton X-100 at RT for 10 min followed by incubation of TUNEL reaction mixture at 37 °C for 1 h. Sections were then co-stained with GFP antibody.Heart sections from 31. Cardiac cell suspensions were prepared for six to eight P0 hearts or two P60 hearts pooled for each preparation as described above for cell-sorting experiments. Cardiac cell suspensions were then plated into a 10 mm cell culture dish at 37 °C. Attached CF cells (1 h for P0 CF attachment and 4–6 h for P60) were cultured in DMEM  supplemented with 15% FBS, 1% penicillin/streptomycin (Life Technologies), and MEM non-essential amino acid solution . Passage 2 CFs were used for all the in vitro experiments. Passage 2 P60 CFs were starved with serum-free DMEM overnight 24 h after seeding. The following treatments were used to manipulate CF activation and Wnt/β-catenin signaling: recombinant human Wnt1 , porcine TGFβ1 , and XAV-939 . DMSO (Sigma) was added at 0.1% as the vehicle control in DMEM.CFs were cultured from post-natal day 0 (P0) and day 60 (P60) mouse hearts40. DNA/protein complexes were cross-linked for 10 min in formaldehyde  at a final concentration of 1%. The fixed cells were lysed and sonicated twice for 15 s each at output 10  and a 2 min refractory period. For immunoprecipitation, cell lysates were incubated with an antibody against β-catenin  and incubated overnight at 4 °C with gentle rocking. Immunoprecipitation with normal rabbit IgG  was used as control. ChIP assays were performed according to the manufacturer’s instructions . The immunoprecipitations were subjected to quantitative real-time PCR with primer sets that amplify Col1a1, Col3a1, and Postn gene regions43 (murine genome GRCm38/mm10) containing potential LEF/TCF-binding sites20. The primer set flanking an Axin2 intron1 region that contains previously reported LEF/TCF-binding sites was included as a positive control20. Primers are listed in Supplementary Table 40.β-catenin protein complex binding to chromosomal DNA was evaluated in mouse P0 CFs treated with 6-bromoindirubin-3′-oxime  by ChIP assayU tests. For data with multiple groups, Kruskal–Wallis tests were performed, followed by Mann–Whitney U tests for pairwise comparisons using Bonferonni adjustments to control for multiple testing. Fold enrichment of DNA binding in ChIP assays was analyzed using one sample Wilcoxon signed-rank test compared to one. A two-tailed p value < 0.05 was considered to be statistically significant.Data were summarized using median and interquartile ranges. All data sets were analyzed using Graphpad Prism . Statistical analyses between two groups were analyzed with Mann–Whitney Data that support the reported findings are available from the corresponding author upon reasonable request.Supplementary information


Tropomyosin-receptor kinase Fused Gene (TFG), histopathologically characterized by progressive spinal motor neuron loss with TFG cytosolic aggregates. Although the TFG protein, found as a type of fusion oncoprotein, is known to facilitate vesicle transport from endoplasmic reticulum (ER) to Golgi apparatus at ER exit site, it is unclear how mutant TFG causes motor neuron degeneration. Here we generated induced pluripotent stem cells (iPSCs) from HMSN-P patients, and differentiated the iPSCs into neural cells with spinal motor neurons (iPS-MNs). We found that HMSN-P patient iPS-MNs exhibited ubiquitin proteasome system (UPS) impairment, and HMSN-P patient iPS-MNs were vulnerable to UPS inhibitory stress. Gene correction of the mutation in TFG using the CRISPR-Cas9 system reverted the cellular phenotypes of HMSN-P patient iPS-MNs. Collectively, these results suggest that our cellular model with defects in cellular integrity including UPS impairments may lead to identification of pathomechanisms and a therapeutic target for HMSN-P.Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is caused by a heterozygous mutation (P285L) in The online version of this article (doi:10.1186/s13041-017-0286-y) contains supplementary material, which is available to authorized users. Tropomyosin-receptor kinase Fused Gene (TFG) was identified as the gene responsible for HMSN-P, and the pathological hallmark of HMSN-P is the formation of cytosolic inclusions of TFG protein in spinal MNs  with protease inhibitor cocktail  and phosphatase inhibitor (Roche) on ice for 30 min. After sonication with Bioruptor , samples were centrifuged at 20,000 g for 15 min at 4 °C. The supernatant was used as sample. Each 10- or 15-μg sample was subjected to SDS-PAGE , and separated proteins were transferred to PVDF. The membranes were incubated with primary antibodies, followed by appropriate secondary antibodies, and then visualized using ECL Prime . The images were acquired on LAS 4000 . The following primary antibodies were used in this assay: TFG , Ubiquitin , cleaved caspase-3 , and β-actin .For detection of cleaved caspase-3, iPS-MNs of each clone were exposed to vehicle or 1 μM MG132 on Day 35 for 24 h. Cells were lysed in RIPA buffer and used for immunoblot analysis with anti-cleaved caspase-3 antibody.Production, concentration and infection of HB9::GFP lentivirus vector were previously reported . All sorHB9::GFP-positive MNs or SMI-32-positive neurons were stained with anti-TFG antibody. The area of TFG puncta in cytoplasm was quantified by IN Cell Analyzer 6000  and IN CELL Developer toolbox software 1.92  in randomized 9- or 16-field images in each well.4cells per well on Matrigel-coated 96-well plates for 48 h after treatment with DMSO or 10 μM MG132, or 10 nM bortezomib . The nucleus was stained with DAPI. Each clone was analyzed using IN Cell Analyzer 6000  and IN CELL Developer toolbox software 1.92  in randomized 9-field images in each well. Cell counts treated with MG132 or bortezomib were normalized to cell counts treated with DMSO in each well.iPS-MNs of each clone on Day 35 were seeded at 3.0 x 105cells per well on Matrigel-coated 24-well plates. Whole cell lysates of iPS-MNs were lysed in 0.5% NP-40. After sonication with Bioruptor , cell lysates were centrifuged at 20,000 g for 15 min at 4 °C. The supernatant was used as sample. Proteasome activity was measured with a Proteasome Activity Assay Kit  following the manufacturer’s instructions.iPS-MNs of each clone on Day 35 were seeded at 5.0 x 10TFG P285L mutation by CRISPR-Cas9, we designed a guide RNA to target the 5′-CCTAACAGTAAGACTAATT-3′ site using CRISPR Design (http://crispr.mit.edu/). The guide RNA oligonucleotide was inserted into the BamHI-EcoRI site in pHL-H1-ccdB plasmids in order to express from human H1 polymerase III promoter [TFG gene sequence, and puromycin-resistant cassette flanked by piggyBac terminal repeats into pBluescript SK (+) for constructing the donor plasmid. Their nuclease activity was validated in 293 T cells by T7E1 method [6 cells using a NEPA 21 electroporator . Four days after transfection, puromycin selection was applied for 10 days. Surviving colonies were expanded and PCR was used to confirm proper cassette insertion by PCR using primers A, B, C, and D ; *p < 0.05, **p < 0.01.All data are shown as mean ± s.e.m. Comparison of two groups was analyzed using unpaired two-tailed Student’s


Benign airway stenosis is a clinical challenge because of recurrent granulation tissues. Our previous study proved that a Chinese drug, β-elemene, could effectively inhibit the growth of fibroblasts cultured from hyperplastic human airway granulation tissues, which could slow down the progression of this disease. The purpose of the present study is to find out the mechanism for this effect. We cultured fibroblasts from normal human airway tissues and human airway granulation tissues. These cells were cultured with 160 μg/ml normal saline (NS), different doses of β-elemene, or 10 ng/ml canonical Wnt/β-catenin pathway inhibitor . The proliferation rate of cells and the expression of six molecules involved in canonical Wnt/β-catenin pathway, Wnt3a, glycogen synthase kinase-3β (GSK-3β), β-catenin, α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), and Collagen I (Col-I), were measured. At last, we used canonical Wnt/β-catenin pathway activator (LiCl) to further ascertain the mechanism of β-elemene. Canonical Wnt/β-catenin pathway is activated in human airway granulation fibroblasts. β-Elemene didn’t affect normal human airway fibroblasts; however, it had a dose–responsive inhibitive effect on the proliferation and expression of Wnt3a, non-active GSK-3β, β-catenin, α-SMA, TGF-β, and Col-I of human airway granulation fibroblasts. More importantly, it had the same effect on the expression and nuclear translocation of active β-catenin. All these effects were similar to 10 ng/ml DKK-1 and could be attenuated by 10 mM LiCl. Thus, β-elemene inhibits the proliferation of primary human airway granulation fibroblasts by down-regulating canonical Wnt/β-catenin pathway. This pathway is possibly a promising target to treat benign tracheobronchial stenosis. Benign tracheobronchial stenosis is the narrow airway caused by nonmalignant factors, including tracheotomy, intubation, tuberculosis, systemic diseases, tracheobronchomalacia, and lung transplantation . In ChinElemene is a Chinese traditional medicine isolated from the essential oils of Rhizoma Curcumae. β-Elemene is the main constituent of elemene and has been proved to be able to slow down the progression of a wide spectrum of malignant tumors . HoweverCanonical Wnt/β-catenin pathway is an evolutionarily conserved signal pathway which plays an important role in promoting fibrosis in many organs, such as the lung, kidney, liver, heart, and skin . We hypo® premix Ex Taq™ (RR420A) reagent kits were purchased from TaKaRa (Japan). TGF-β and Col-I ELISA kits were purchased from Neobioscience (China) and Xlpcc (China) respectively. Primers of human GAPDH, Wnt3a, GSK-3β, β-catenin, α-SMA, TGF-β, and Col-I were purchased from Sangon Biotech . Primary monoclonal antibodies of human GAPDH (ab181602), Wnt3a (ab81614), and α-SMA (ab32575) were purchased from Abcam (U.S.A.) and GSK-3β (12456), phospho-GSK-3β (Ser9) (5558), β-catenin (8480), and non-phospho (active) β-catenin (Ser45) (19807) were purchased from Cell Signaling Technology (U.S.A.). Goat anti-rabbit and mouse horseradish peroxidase-conjugated secondary antibodies and goat anti-rabbit fluorescent secondary antibody were purchased from Beyotime (China). DAPI (1 mg/ml) was purchased from Sigma (U.S.A.). β-Elemene (98% in purity) purchased from Jingang Pharmaceutical Company  was dissolved in high glucose DMEM to make a stock solution (5 mg/ml). Canonical Wnt/β-catenin pathway inhibitor (DKK-1) and activator (LiCl) were purchased from RD Systems (U.S.A.) and Sigma (U.S.A.) respectively. They were both dissolved in the same media to make a stock solution with the concentration of 100 ng/ml and 100 mg/ml respectively.High glucose Dulbecco’s modified Eagle’s medium (DMEM), 0.25% trypsin-EDTA, and FBS were purchased from HyClone (U.S.A.). MTT cell proliferation and cytotoxicity detection reagent kit were purchased from Keygen Biotech . PrimeScript™ RT (RR037A) and SYBR2 and 37°C. They were passaged every 3 days and the cells for passage 5–7 were incubated in six-well plates, 96-well plates, or coverslips on six-well plates. After 48 h, these cells were cultured with 2% FBS supplemented with 160 μg/ml normal saline (NS), various doses of β-elemene , or 10 ng/ml DKK-1. The fibroblasts cultured from granulation tissues were also cultured in the same media with various doses of LiCl , or 160 μg/ml β-elemene plus 10 mM LiCl for 48 h.The research plan followed guidelines of Helsinki Declaration and was endorsed by the Ethics Committee of the Second Affiliated Hospital of Fujian Medical University . After normal people and patients diagnosed with benign tracheobronchial stenosis signed informed consent forms, normal airway tissues and airway granulation tissues were obtained by small biopsy forceps under bronchoscopy examination. Fibroblasts were cultured from these two tissues using a reformative method called a combination of “tissue adherent” and “trypsinization” [According to MTT cell proliferation and cytotoxicity detection reagent kit’s instructions, the cells cultured in 96-well plates were used for this experiment. Fifty microliters of 1× MTT was added into each well and the 96-well plate was maintained at 37°C for 4 h. Then the cell culture supernatant was removed and 150 μl of DMSO was added into each well. The optical density (OD) value for each well was measured by Microplate Reader  at a wavelength of 490 nm. The experiment was repeated for at least three times.® premix Ex Taq™ reagent kit’s instructions. The genes’ expression was detected by ABI PRISM 7500 sequence detection system . GAPDH was used as the internal control and fold changes of the genes’ expression were calculated by the method of 2C−ΔΔT. The experiment was repeated for at least three times. Primers for QRTPCR were shown in Total RNA was extracted from fibroblasts using trizol under the manufacturer’s instructions. cDNA was synthesized from 500 ng of total RNA under PrimeScript™ RT reagent kit’s instructions. Two hundred nanograms of cDNA from each sample was used for quantitative real-time polymerase chain reaction (QRTPCR) under SYBRThe cells were digested by 0.25% trypsin-EDTA and total proteins were extracted by cell lysis buffer supplemented with protease inhibitors. Equal amount of total proteins from each group was separated by SDS/PAGE and the proteins needed were transferred onto PVDF membranes. The membranes were incubated with 5% skimmed milk for 2 h, followed by primary antibodies of Wnt3a (1:2000), GSK-3β (1:1000), phospho-GSK-3β (Ser9) (1:1000), β-catenin (1:1000), and α-SMA (1:5000) overnight at 4°C. Subsequently, the membranes were washed and incubated with goat anti-rabbit (1:100000) or mouse (1:5000) horseradish peroxidase-conjugated secondary antibody for 1 h. Finally, the membranes were washed again and ECL detection reagent was added to make protein bands to be seen under ImageQuant LAS4000 chemiluminescence detection system . The protein levels were quantified by ImageJ software and the gray values were normalized to GAPDH. The data were obtained from at least three independent experiments.The cells on coverslips after treatment were fixed by 4% paraformaldehyde for 20 min. Then, 0.1% Triton X-100 was added on the cells for 20 min to make cell membrane perforated. The cells were incubated with 5% BSA for 30 min, followed by primary antibodies of non-phospho (active) β-catenin (1:1600) overnight at 4°C. Subsequently, the cells were washed for three times and incubated with goat anti-rabbit fluorescent secondary antibody (1:400) in dark for 1 h. The cells were washed three times again and incubated with DAPI (0.1 μg/ml) in dark for 10 min to make nuclear dyed. Finally, the cells were washed for four times and mounting medium containing anti-fluorescence quenching agent was added after the coverslips were dried. The cells’ fluorescence was observed under fluorescence microscope .The cell culture supernatant was used for ELISA assay. The concentrations of TGF-β and Col-I were measured according to ELISA kits’ protocols. The experiment was repeated for at least three times.post hoc test for three or more groups’ comparison or Student’s t test for two groups’ comparison using Statistical Product and Service Solutions (SPSS) software 21.0  and were expressed as the mean and standard deviation (SD). P<0.05 indicated that the difference between two groups was statistically significant and P<0.01 indicated that the difference was highly significant.The data were analyzed by one-way variance analysis followed by Tukey’s Fibroblasts cultured from normal human airway tissues and human airway granulation tissues were almost the same. However, after a careful observation, we could see a bit different. Primary human airway granulation fibroblasts are bigger and longer and have more branches compared with primary human airway fibroblasts A. We firAfter treatment with different concentrations of β-elemene for 48 h, we observed distinct morphological changes of primary human airway granulation fibroblasts under the microscope. The cells not treated with β-elemene showed a typical spindle-shaped morphology and arranged orderly and closely A and B. To determine whether this pathway is underlying the inhibitive effect of β-elemene on primary human airway granulation fibroblasts, mRNA and proteins were extracted from primary human airway granulation fibroblasts treated with 160 μg/ml NS, different concentrations of β-elemene, or 10 ng/ml DKK-1. The above six molecules were assessed by QRTPCR, Western blot, immunofluorescence staining, and ELISA.β-Elemene had a significant dose–responsive inhibitive effect on the mRNA and protein levels of Wnt3a A and B. The mRNA and protein levels of GSK-3β of primary human airway granulation fibroblasts were not affected by β-elemene. However, β-elemene had a significant dose–responsive inhibitive effect on the phosphorylated protein level of GSK-3β A and B. β-Elemene had a significant dose–responsive inhibitive effect on the mRNA and protein levels of β-catenin A and B. β-Elemene had a significant dose–responsive inhibitive effect on the mRNA and protein levels of α-SMA A and B. β-Elemene had a significant dose–responsive inhibitive effect on the mRNA and protein levels of TGF-β A and C. β-Elemene had a significant dose–responsive inhibitive effect on the mRNA and protein levels of Col-I A and C. It was found that β-elemene up-regulated the activity of GSK-3β and down-regulated the activity of Wnt3a, β-catenin, α-SMA, TGF-β, and Col-I, all in a dose-dependent manner. All these effects were similar to the pathway inhibitor, 10 ng/ml DKK-1  and 6. TAt last, we used the pathway activator, LiCl, to further ascertain whether inhibiting canonical Wnt/β-catenin pathway is the key mechanism by which β-elemene inhibits the proliferation of primary human airway granulation fibroblasts. We first cultured primary human airway granulation fibroblasts with different concentrations of LiCl and found out 10 mM LiCl has the largest promotive effect on this pathway . Then, wBenign airway stenosis is easily to recur because of exuberant granulation tissues. One way to solve this problem is to inhibit the growth of airway granulation fibroblasts. β-Elemene is a widely used anticancer Chinese drug. Recently, many studies proved that it can also inhibit fibrosis. In the present study, we cultured normal human airway fibroblasts and human airway granulation fibroblasts with different concentrations of β-elemene and found out at the concentration equal or below 160 μg/ml, β-elemene had a dose–responsive inhibitive effect on the proliferation of human airway granulation fibroblasts. When the concentration reached to 160 μg/ml, about half of the cells were dead. And in this concentration range, β-elemene almost had no inhibitive effect on the proliferation of normal human airway fibroblasts. This indicated that β-elemene is a safe drug, and has specific inhibitive effect on human airway granulation fibroblasts. This drug can be a promising drug to treat benign airway stenosis.Canonical Wnt/β-catenin pathway plays a vital role in regulating tissues’ regeneration . This paβ-Catenin is an important molecule related to fibrosis. Sustained activation of β-catenin would promote the proliferation of fibroblasts  and leadα-SMA is another molecule closely related to fibrosis. It is the main molecular marker of myofibroblasts . MyofibrTGF-β and Col-I are two important molecules secreted by fibroblasts. TGF-β is one of the most potent cytokines to promote fibrosis and can promote fibroblasts to turn into myofibroblasts . Col-I iWhen canonical Wnt/β-catenin pathway is inactive, β-catenin will be phosphorylated by a destruction complex which includes adenomatous polyposis coli protein (APC), casein kinase 1 (CK1), AXIN, and GSK-3β. Then, β-catenin will be phosphorylated at serine residue 45 and then be ubiquitylated and degraded by proteasomes . When caOur study proved that β-elemene inhibited canonical Wnt/β-catenin pathway of primary human airway granulation fibroblasts, and this effect could be attenuated by the pathway activator, LiCl. This indicated that down-regulating the activity of canonical Wnt/β-catenin pathway is possibly the key mechanism by which β-elemene inhibits the proliferation of human airway granulation fibroblasts. This pathway may become a promising target to treat benign tracheobronchial stenosis.Canonical Wnt/β-catenin pathway is activated in primary human airway granulation fibroblasts. β-Elemene had a dose–responsive inhibitive effect on the proliferation of human airway granulation fibroblasts and didn’t affect normal human airway fibroblasts. It also had a dose–responsive inhibitive effect on the activity of canonical Wnt/β-catenin pathway of human airway granulation fibroblasts. All these effects were similar to 10 ng/ml DKK-1 and could be attenuated by 10 mM LiCl. These results indicated that down-regulating canonical Wnt/β-catenin pathway is possibly the key mechanism by which β-elemene inhibits the proliferation of primary human airway granulation fibroblasts.


Mechanistically, IKKα plus IKKβ enhanced and IKKγ inhibited the interplay among HP1α, HP1β and HP1γ that competes for the interaction among HP1α, SUZ12, HEZ2. Therefore, IKKα plus IKKβ inhibited and IKKγ enhanced the activity of H3K27 methyltransferase SUZ12 and EZH2, which methylates H3K27 immediately sites on HOTAIR promoter region. Therefore, IKKα plus IKKβ increased and IKKγ decreased the HOTAIR expression. Strikingly, IKKα plus IKKβ decreases and IKKγ increases the HP1α interplays with DNA methyltransferase DNMT3b, which increases or decreases TERRA promoter DNA methylation. Thus IKKα plus IKKβ reduces and IKKγ increases to recruit TRF1 and RNA polymerase II deposition and elongation on the TERRA promoter locus, which increases or decreases TERRA expression. Furthermore, IKKα plus IKKβ decreases/increases and IKKγ increases/decreases the interplay between TERT and TRRRA/between TERT and TREC. Ultimately, IKKα plus IKKβ increases and IKKγ decreases the telomerase activity. On the other hand, at the telomere locus, IKKα plus IKKβ increases/drcreases and IKKγ decreases/increases TRF2, POT1, pPOT1, Exo1, pExo1, SNM1B, pSNM1B/CST-AAF binding, which keep active telomere regulatory genes and poised for telomere length. Strikingly, HOTAIR is required for IKKα plus IKKβ and IKKγ to control telomerase activity and telomere length. These observations suggest that HOTAIR operates the action of IKKα, IKKβ, IKKγ in liver cancer stem cells. This study provides a novel basis to elucidate the oncogenic action of IKKα, IKKβ, IKKγ and prompts that IKKα, IKKβ, IKKγ cooperate to HOTAR to be used as a novel therapeutic targets for liver cancer.Cancer stem cells are associated with tumor recurrence. IKK is a protein kinase that is composed of IKKα, IKKβ, IKKγ. Herein, we demonstrate that IKKα plus IKKβ promoted and IKKγ inhibited liver cancer stem cell growth in  IκB kinase (IKK) is composed of three subunits, IKKα, IKKβ, IKKγ, where IKKα and IKKβ are catalytic subunits, and IKKγ is the regulatory subunit. Many diseases are related to IKK . IKKα haHeterochromatin protein1 is involved in chromatin packing and epigenetic gene regulation . The α, Accumulating evidence indicates that HOTAIR plays a critical role in cancer progression. HOTAIR could promote migration and invasion of hepatocellular carcinoma (HCC) cells by inhibiting RBM38 . The PolTRF2 activity alteration is absorbing and of great concern. Our previous findings suggest that SET1A and CUDR increased TRF2 expression on the transcriptional and translational level through H3K4me3 . The shevitro and in vivo. Mechanistically, IKKα plus IKKβ enhanced and IKKγ inhibited the interplay among HP1α, HP1β and HP1γ. Therefore, IKKα plus IKKβ increased and IKKγ decreased the TOTAIR expression. Ultimately, IKKα plus IKKβ increases and IKKγ decreases the telomere length and telomerase activity. Of significance, HOTAIR is required for IKKα plus IKKβ and IKKγ to control telomere. This study provides a novel basis to elucidate the oncogenic action of IKKα, IKKβ, IKKγ.In this report, our findings suggest that IKKα plus IKKβ promoted and IKKγ inhibited liver cancer stem cell growth in in vitro.To address whether synergy of IKKα, IKKβ, IKKγ altered the liver cancer stem cells growth in vitro, we first constructed stable cell lines transfected with plasmid with IKKα, IKKβ, IKKγ. As shown in Figure in vivo.To determine whether IKKα, IKKβ, IKKγ, IKKα+IKKβ, IKKβ+IKKγ, IKKα+IKKγ, IKKα+IKKβ+IKKγ could affect the tumorigenesis in vivo, the aforementioned eight stable cell lines were injected subcutaneously at armpit into Balb/C mice. As Expected, significant differences in tumor weights were observed among some groups. As shown in Figure To prove whether IKKα, IKKβ, IKKγ influence on the methylation of HistoneH3 on lysine 27, we performed western blotting and IP assay in these groups. As shown in Figure To investigate whether IKKα, IKKβ, and IKKγ influence on HOTAIR expression, we first performed IP assay in liver cancer stem cells. As shown in Figure To investigate whether IKKα, IKKβ, IKKγ influence on telomere, we first consider to analyse the alteration of telomerase activity. As shown in Figure To explore whether IKKα, IKKβ, and IKKγ influence on telomere, we also detected the alteration of telomere length in these liver cancer stem cell. As shown in Figure To determine whether HOTAOR regulated the IKKα, IKKβ, and IKKγ's function on telomeres, we performed rescued-test in liver cancer stem cells. As showed in the Figure To address whether HOTAIR controls the synergy action of IKKα, IKKβ, and IKKγ, we constructed stable cell lines transfected with plasmid with IKKα, IKKβ, IKKγ together with HOTAIR knocked down. As shown in Figure vitro and in vivo. Mechanistically, IKKα plus IKKβ enhanced and IKKγ inhibited the interplay among HP1α, HP1β and HP1γ that competes for the interaction among HP1α, SUZ12, HEZ2. Therefore, IKKα plus IKKβ inhibited and IKKγ enhanced the activity of H3K27 methyltransferase SUZ12 and EZH2, which methylates H3K27 immediately sites on HOTAIR promoter region. Therefore, IKKα plus IKKβ increased and IKKγ decreased the TOTAIR expression. Strikingly, IKKα plus IKKβ decreases and IKKγ increases the HP1α interplays with DNA methyltransferase DNMT3b, which increases or decreases TERRA promoter DNA methylation. Thus IKKα plus IKKβ reduces and IKKγ increases to recruit TRF1 and RNA polymerase II deposition and elongation on the TERRA promoter locus, which increases or decreases TREEA expression. Furthermore, IKKα plus IKKβ decreases/increases and IKKγ increases/decrease the interplay between TERT and TRRRA/between TERT and TREC. Ultimately, IKKα plus IKKβ increases and IKKγ decreases the telomerase activity. On the other hand, at the telomere locus, IKKα plus IKKβ increases/drcreases and IKKγ decreases/increases TRF2, POT1, pPOT1, Exo1, pExo1, SNM1B, pSNM1B/CST-AAF binding, which keep active telomere regulatory genes and poised for telomere length. Strikingly, HOTAIR is required for IKKα plus IKKβ and IKKγ to control telomerase activity and telomere length. HOTAIR depletion blocks the function of IKKα plus IKKβ, IKKγ on telomere. These observations suggest that HOTAIR operates the action of IKKα, IKKβ, IKKγ in liver cancer stem cells. This study provides a novel basis to elucidate the oncogenic action of IKKα, IKKβ, IKKγ and prompts that IKKα, IKKβ, IKKγ cooperate to HOTAR to be used as a new therapeutic targets for liver cancer.Cancer stem cells (CSCs) are involved in tumorigenesis. The IKK is critical in cellular proliferation and in tumor-initiating . HoweverIt is worth mentioning that IKKα, IKKβ, IKKγ may play an important role in hepatocarcinogenesis. In this report, we focused mainly on the view how IKKα, IKKβ, IKKγ function during liver cancer stem cells malignant growth. To this date, accumulating evidence indicates that IKKα, IKKβ, IKKγ influence on cell proliferation. For examples, inhibition of IKK/NF-κB pathway controls stem cell proliferation . IKKβ plvitro; (2) IKKα plus IKKβ promoted and IKKγ inhibited liver cancer stem cell growth in vivo.It is worth noting that our findings in this study provide novel evidence for an active role of IKKα plus IKKβ promotion or IKKγ inhibition of liver cancer stem cell growth. Herein, the involvement of promotion or inhibition of liver cancer stem cells growth based on IKKα, IKKβ, IKKγ is supported by results from two parallel sets of experiments: (1) IKKα plus IKKβ promoted and IKKγ inhibited liver cancer stem cell growth in Strikingly, our observations suggest that IKKα plus IKKβ increased and IKKγ inhibited HOTAIR expression dependent on tri-methylation of Histone H3 on the twenty-seven lysine. This assertion is based on several observations in IKKα plus IKKβ or IKKγ overexpressed liver cancer stem cells: (1) IKKα plus IKKβ enhanced and IKKγ inhibited the interplay among HP1α, HP1β and HP1γ that competes for the interaction among HP1α, SUZ12, HEZ2. (2) IKKα plus IKKβ inhibited and IKKγ increased methylation of histoneH3 on lysine 27 dependent on the tri-complex of HP1. (3) IKKα plus IKKβ increased and IKKγ decreased the H3K27Ac and NF-κB through H3K27me3. (4) IKKα plus IKKβ increased and IKKγ decreased HOTAIR expression dependent on H3K27me3. Researches indicated heterochromatin causes epigenetic repression that control gene expression and function . HP1α isOn the other hand, we find that IKKα, IKKβ, and IKKγ control telomerase activity and telomere length. The stability of telomeres depends upon TRF2, which prevents inappropriate repair . Upon teSignificantly, HOTAIR is required for IKKα plus IKKβ and IKKγ to control telomerase activity, telomere length and tumorigenesis. HOTAIR is associated with a variety of human cancers, such as breast, liver and endometrial carcinoma . HOTAIR Especially, our results showed IKKα plus IKKβ enhanced the interplay among HP1α, HP1β and HP1γ by an unknown mechanism, and then inhibited the activity of H3K27 methyltransferase SUZ12 and EZH2 and increased the HOTAIR expression. However, single IKKα or IKKβ overexpression could not alter HOTAIR expression. Because of excessive HOTAIR, IKKα plus IKKβ increases the interplay between TERT and TREC, the telomerase activity and telomere length. However, single IKKα or IKKβ overexpression could not alter the telomerase activity, increases telomere length. Moreover, because of the increase of telomerase activity and telomere length, IKKα plus IKKβ simultaneous overexpression significantly increased liver cancer stem cells's growth, while single IKKα or IKKβ overexpression could not promote liver cancer stem cells's growth.In addition, our results showed that HOTAIR depletion blocks IKKα plus IKKβ, IKKγ function on telomere. It suggest HOTAIR is required for IKKα plus IKKβ and IKKγ to control telomerase activity and telomere length. In this study, we found IKKα plus IKKβ enhanced and HP1γ decreased the interplay among HP1α, HP1β and HP1γ by an unknown mechanism, and then IKKα plus IKKβ inhibited and HP1γ enhanced the activity of H3K27 methyltransferase SUZ12 and EZH2. Ultimately, IKKα plus IKKβ increased and HP1γ decreased the HOTAIR expression. Because of HOTAIR, IKKα plus IKKβ increases and HP1γ decreased the interplay between TERT and TREC, the telomerase activity and telomere length. IKK is also a crucial protein kinase that activates NF-κB translocating from cytoplasm to nucleus for DNA binding. Especially, IKK phosphorylates NF-κB to modulate its promoter specificity and promote genes expression. NF-κB could regulates and controls telomere related genes. Although IKK could regulate NF-κB and HOTAIR, both abnormal NF-κB and HOTAIR could alter telomeres through different pathways.We should explore the function of IKKα plus IKKβ, IKKγ in liver cancer stem cells. For example, what causes strong oncogenic action of IKKα plus IKKβ and triggers IKKγ's actions? How does IKKα plus IKKβ, IKKγ cooperates with HOTAIR? Does IKKα plus IKKβ, IKKγ regulates a series of molecular events liver stem cells malignant growth? Answering these questions will help understand the mechanism about liver stem cell malignant differentiation. In summary, our present data indicated that IKKα plus IKKβ, and IKKγ promotes/inhibits liver cancer stem cells malignant progression through altering telomere length and telomerase activity dependent on HOTAIR, with clinic implications. These observations provide insight into a novel link between IKKα, IKKβ, IKKγ and liver cancer stem malignant transformation.8 total cells after aspirating supernatant completely. Add 100 μL of FcR Blocking Reagent per 108 total cells and 100 μL of CD133/CD44/CD24/EpCAM MicroBeads per 108 total cells. Mix well and incubate for 30 minutes in the refrigerator (2−8 °C). Wash cells by adding 1−2 mL of buffer per 108 cells and centrifuge at 300×g for 10 minutes. Resuspend up to 108 cells in 500 μL of buffer. Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of CD133+/CD44+/CD24+/EpCAM+ cells.CD133/CD44/CD24/EpCAM MicroBead Kits were purchased from Miltenyi technic  and MACS® Technology operation according to and the operation according to the manufacturer. In brief, centrifuge cell suspension at 300×g for 10 minutes and. Resuspend cell pellet in 300 μL of buffer per 102 incubator at 37°C. pCMV6-A-GFP, pGFP-V-RS were purchased from Origene . pcDNA3.1-IKKα, pcDNA3.1-IKKβ, pcDNA3.1-IKKγ were purchase from Addgene . pCMV6-A-GFP-HOTAIR, pGFP-V-RS-HOTAIR, pGFP-V-RS-HP1α, pGFP-V-RS–HP1β, pGFP-V-RS–HP1γ, pGL4-HOTAIR/promoter were prepared by ourselves.Liver cancer stem cell lines were maintained in Dulbecco's modified Eagle medium (Gibco BRL Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL Life Technologies) in a humidified atmosphere of 5% COR 2000 (Invitrogen) according to manufacturer's instructions.Cells were transfected with DNA plasmids using transfast transfection reagent lipofectamine17-18, random primers, and a SuperScript First-Strand Synthesis System (Invitrogen). PCR analysis was performed under the specical conditions. β-actin was used as an internal control.Total RNA was purified using Trizol (Invitrogen) according to manufacturer's instructions. cDNA was prepared by using oligonucleotide (dT)The logarithmically growing cells were washed twice with ice-cold phosphate-buffered saline  and lysed in a RIPA lysis buffer. Cells lysates were centrifuged at 12,000g for 20 minutes at 4°C after sonication on ice, and the supernatant were separated. After being boiled for 5-10 minutes in the presence of 2-mercaptoethanol, samples containing cells proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membranes . Then blocked in 10% dry milk-TBST  for 1 h at 37°C. Following three washes in Tris-HCl pH 7.5 with 0.1% Tween 20, the blots were incubated with 0.2 μg/ml of antibody (appropriate dilution) overnight at 4°C. Following three washes, membranes were then incubated with secondary antibody for 60 min at 37°C or 4°C overnight in TBST. Signals were visualized by ODYSSEY infrared imaging system . IRDye 680LT secondary antibodies were purchased from LI-COR scientific company.Cells were lysed in 1 ml of the whole-cell extract buffer A . Five-hundred-microliter cell lysates was used in immunoprecipitation with antibody. In brief, protein was pre-cleared with 30μl protein G/A-plus agarose beads  for 1 hour at 4°C and the supernatant was obtained after centrifugation  at 4°C. Precleared homogenates (supernatant) were incubated with 2 μg of antibody and/or normal mouse/rabbit IgG by rotation for 4 hours at 4°C, and then the immunoprecipitates were incubated with 30μl protein G/A-plus agarose beads by rotation overnight at 4°C, and then centrifuged at 5000rpm for 5 min at 4°C. The precipitates were washed five times×10min with beads wash solution  and then resuspended in 60μl 2×SDS-PAGE sample loading buffer to incubate for 10 min at 100°C. Then Western blot was performed with a another related antibody indicated in Western blotting.2, 10 mM HEPES [pH 7.0], 0.5% NP40, 1 mM DTT, 100 units/ml RNase OUT (Invitrogen), 400 μM vanadyl-ribonucleoside complex and protease inhibitors (Roche). The lysates were incubated with specific antibody or normal mouse/rabbit IgG overnight at 4°C, followed that the lysates were incubated with protein A/G-plus agarose beads  4 hours at 4°C. Then the beads were subsequently washed four times with 50 mM Tris-HCl (pH 7.0), 150 mM NaCl, 1 mM MgCl2, and 0.05% NP-40, and twice after addition of 1M Urea. RNA is isolated from the Immunoprecipitates (IPs) and RT-PCR is performed.Cells were lysed  in 100 mM KCl, 5 mM MgCl2, 100% glycerol, 1 mM DTT, and 0.5% NP40) containing protease and phosphatase inhibitors for the preparation of nuclear exact. Equal amount of cell nuclear extracts were precleared with Streptavidin-agarose Resin (Thermo) for 1 hours, and then were incubated with 1μg biotinylated double-stranded-oligonucleotides and together with 10μg poly(dI-dC) at 4°C for 24 hours. DNA-bound proteins were collected with the incubation with streptavidin-agarose Resin for 1 hour with gently shaking to prevent precipitation in solution. Following 5 washings of the resin bound complex with 0.5-1.0 ml of binding buffer, the samples were boiled and subjected to SDS-PAGE and Western blot analysis.Cells were lysed by sonication in HKMG buffer  formaldehyde (Sigma) for 10 min at room temperature and stopped with 125 mm glycine for 5 min. Crossed-linked cells were washed with phosphate-buffered saline, resuspended in lysis buffer, and sonicated for 8-10 min in a SONICS VibraCell to generate DNA fragments with an average size of 500 bp or so. Chromatin extracts were diluted 5-fold with dilution buffer, pre-cleared with Protein-A/G-Sepharose beads, and immunoprecipitated with specific antibody on Protein-A/G-Sepharose beads. After washing, elution and de-cross-linking, the ChIP DNA was detected by PCR.Cells were washed and scraped in ice-cold PBS to prepare nuclei for electrophoretic gel mobility shift assay with the use of the gel shift assay system modified according to the manufacturer's instructions (Promega).The telomerase activity was measured by using Quantitative Telomerase Detection Kit (MT3010) according to manufacturer's instructions .Telomere length assay using Telo TAGGG PCR ELISApuls kit according to manufacturer's instructions (Roche). A standard curve is established by dilution of known quantities of a synthesised 84 mer oligonucleotide containing only TTAGGG repeats.mthylated DNA Immunoprecipitation (MeDIP)-Dot blot-western blotting with anti-5-Methylcytosine (5-mC) and ethylation analysis by MspI plus BamHI digestion.Cells were synchronized in G0 phase by serum deprivation and then released from growth arrest by reexposure to serum, and then cells were grown in complete medium for assay. The cell proliferation reagent CCK8 is purchased from Roch and the operation according to the manufacturer instruction.2 cells were plated on a 6 well plate containing 0.5% (lower) and 0.35% (upper) double layer soft-agar. The 6 well plates were incubated at 37°C in humidified incubator for 14 days. The cells were fed 1-2 times per week with cell culture media (DMEM). Soft-agar colonies on the 6 well plates were stained with 0.5 ml of 0.05% Crystal Violet for more than 1 hour and the colonies were counted.2 × 1070-80% confluent cells were cultured for 24 hour before treatment with 10μl BrdU (Roche) for 4 hours. Immunofluorescent staining with an anti-BrdU antibody was performed according to the manufacturer's instructions (Becton Dickinson). BrdU positive cells from ten random chosen fields of at least three independent samples were counted.The Four-weeks athymic Balb/C mouse was injected the liver cancer cells at the armpit area subcutaneously. The mice were observed over 4 weeks, and then sacrificed to recover the tumors. The wet weight of each tumor was determined for each mouse. A portion of each tumor was fixed in 4% paraformaldehyde and embedded in paraffin for histological hematoxylin-eosin (HE) staining. The use of mice for this work was reviewed and approved by the institutional animal care and use committee in accordance with China national institutes of health guidelines.The significant differences between mean values obtained from at least three independent experiments. Each value was presented as mean±standard error of the mean (SEM) unless otherwise noted, with a minimum of three replicates. Student's t-test was used for comparisons, with P<0.05 considered significant.


To report a case of impending extrusion of Ex-PRESS shunt treated by shunt-position adjustment.A 56-year-old Asian woman presented with impending extrusion and malposition of Ex-PRESS shunt in her left eye. The bleb of the left eye was shallow and diffuse. In the past, the patient was treated by Ex-PRESS shunt implantation under the scleral flap in both eyes. There had been no Ex-PRESS shunt-related complication in her right eye, and she reported no history of left-eye trauma. Based on these findings, we hypothesized that the source of the left-eye problem was a loosely fixed Ex-PRESS shunt spur. It was thought, furthermore, that this inadequate scleral resistance during the Ex-PRESS shunt implantation was due to the low scleral rigidity resulting from high myopia and insufficient maintenance of the anterior chamber. We proceeded to make an incision in the area adjacent to the Ex-PRESS shunt using a super sharp blade. The shunt was then pushed into the anterior chamber with forceps, and the spur was fixed firmly. Pushing the shunt to the anterior chamber was found to have been sufficient to fix it firmly. In fact, when the sclera was palpated with a sponge, aqueous outflow was observed with no shunt displacement. Postoperative intraocular pressure (IOP) was managed well, and the bleb had formed with diffuse, prominent shapes. The Ex-PRESS shunt was well sustained with good positioning.When an Ex-PRESS shunt operation is performed on a patient who shows a tendency for low scleral rigidity, shunt implantation should be accomplished carefully and with force adequate for firm spur fixation. The Ex-PRESS shunt  is a stainless steel non-valve tube. It has a spur designed for fixation in the trabecular meshwork without suturing. Originally implanted under the conjunctiva to connect the anterior chamber to the subconjunctival space , it is nIn order to regulate intraocular pressure (IOP) adequately and prevent complications including shunt extrusion or exposure, the Ex-PRESS shunt must be properly positioned and securely fixed. There have been reports of Ex-PRESS shunt removal necessitated by shunt extrusion or exposure resulting from improper fixation or trauma, or simply by spontaneous extrusion or exposure –7. We heA 56-year-old Asian woman visited our clinic. Her best-corrected visual acuity (BCVA) was 20/20, and the IOP in the problem eye (the left) was 13 mmHg. Slit-lamp examination showed impending Ex-PRESS shunt extrusion or exposure. The internal opening was tilted to the corneal endothelium obliquely in the anterior chamber, and the external plate was prominent in the subconjunctival space  months previously, IOP in both eyes was uncontrolled, ranging from 25 to 28 mmHg. The spherical error was −9.5 diopters(D) and axial length (AXL) was 26.98 mm in the right eye. The spherical error was −5.0D and AXL was 27.06 mm in the left eye. BCVA was 20/200 in the right eye and 20/25 in the left eye, due to nuclear cataracts. This had necessitated combined surgery (cataract + Ex-PRESS shunt implantation) in our hospital. A 3 × 3 mm scleral flap of half thickness was made at superotemporal area. Then, phacoemulsification and lens implantation was performed through a 2.2 mm corneal incision site located in the temporal area. The tract was made with a 25-gauge needle in one-third of the medial area of the blue-gray transition zone, under a preformed scleral flap. The Ex-PRESS shunt  was then pushed into the anterior chamber. After surgery, right-eye visual acuity, IOP, bleb shape and Ex-PRESS shunt positioning were all satisfactory. In the left eye, however, the spur of the Ex-PRESS shunt was not firmly fixed in the trabecular meshwork, and the shunt had been implanted obliquely. These left-eye problems were thought to have been caused by low scleral rigidity resulting from high myopia and insufficient maintenance of the anterior chamber, which presumably had rendered scleral resistance inadequate during Ex-PRESS shunt implantation. We decided to observe carefully without surgical intervention, because the external plate of the shunt was covered well by scleral flap and there was no extrusion sign.Seven (7) months postoperatively, we observed an impending extrusion sign that the external plate was prominent in the subconjunctival space, though it was covered by the scleral flap. Because Ex-PRESS shunt exposure can necessitate shunt removal or additional operation for IOP control, surgery was performed prior to any exposure. A limbus-based conjunctival incision was made followed by careful dissection of the adhesions between the subconjunctival and Tenon tissue. A subsequent incision was made along the previous scleral-flap wound with a super sharp blade, and then the scleral flap was opened carefully until the Ex-PRESS shunt was visible. After dissection of adhesions between the external plate of the EX-PRESS shunt and the surrounding tissue was performed, the shunt was grasped with forceps. However, as the shunt was fixed stronger than expected and, thus, was not easily removable by forceps, we decided against removal and opted for adjustment of its position instead. To make sufficient space for easy adjustment, an incision with the super sharp blade was made from the external-plate-inserted site, in both lateral directions, into the anterior chamber parallel to the iris plane. Then, the shunt was inserted perpendicularly to the limbus, parallel to the iris plane, and well pushed in anterior chamber by forceps for firm fixation of the spur. The internal opening was parallel to the iris, and the external plate was tight on the sclera Fig. . When scGlaucoma is a leading cause of irreversible blindness worldwide . If IOP In the early days of its application, the Ex-PRESS shunt was implanted under the conjunctiva; however, complications such as hypotony, conjunctival erosion, shunt extrusion or exposure were encountered. To prevent such complications, Dahan and Carmichael performed implantation under the scleral flap . In the Ex-PRESS shunt scleral-flap implantation has, in most cases, provided resistance adequate for prevent shunt extrusion. The potential for spontaneous post-operative displacement, accordingly, is low. This means that Ex-PRESS shunt extrusion occurs more readily as a consequence of intraoperative improper fixation. In our case, a patient with senile cataract and open-angle glaucoma underwent combined surgery (cataract + Ex-PRESS shunt implantation) in both eyes. Considering that the right-eye shunt sustained good function without displacement, and keeping in mind all of the above-noted findings, we supposed that the left-eye shunt extrusion had occurred by intraoperative improper spur fixation. The determined causes of the improper spur fixation are as follows: first, the patient had low scleral rigidity originating from high myopia; second, in the left-eye operation, unlike the right-eye case, the viscoelastic material utilized in the anterior chamber was insufficient. These factors combined to compromise scleral resistance to the implanted Ex-PRESS shunt.According to the standard procedure, when malposition or exposure on conjunctiva of the Ex-PRESS shunt occurs, the shunt is removed and secondary glaucoma surgery is performed. However, we treated a patient with impending extrusion of Ex-PRESS shunt by shunt-position adjustment. To our best knowledge, this is first case of the treatment of malpositioned EX-PRESS shunt by shunt-position adjustment. This treatment is considered to be a particularly effective method, because it renders shunt removal and secondary glaucoma surgery unnecessary.Ex-PRESS shunt removal is always quite traumatic and in cases of impending erosion it impossible to try to reposition it instead of straight forward removal. So to prevent this situation, Ex-PRESS shunt should be inserted perpendicularly to the limbus, parallel to the iris plane and well pushed in anterior chamber for firm fixation of the spur.In conclusion, when Ex-PRESS shunt implantation is performed on a patient who shows a tendency to low scleral rigidity, care should be taken to ensure that the shunt spur is firmly fixed.


RNAs (lncRNAs) are non‐protein‐coding transcripts in the human genome which perform crucial functions in diverse biological processes. The abnormal expression of some lncRNAs has been found in tumorigenesis, development and therapy resistance of cancers. They may act as oncogenes or tumour suppressors and can be used as diagnostic or prognostic markers, prompting their therapeutic potentials in cancer treatments. Studies have indicated that many lncRNAs are involved in the regulation of several signal pathways, including Wnt/β‐catenin signalling pathway, which has been reported to play a significant role in regulating embryogenesis, cell proliferation and controlling tumour biology. Emerging evidences have suggested that lncRNAs can interact with several components of the Wnt/β‐catenin signalling pathway to regulate the expression of Wnt target genes in cancer. Moreover, the expression of lncRNAs can also be influenced by the pathway. Nevertheless, Wnt/β‐catenin signalling pathway‐related lncRNAs and their interactions in cancer are not systematically analysed before. Considering these, this review emphasized the associations between lncRNAs and Wnt/β‐catenin signalling pathway in cancer initiation, progression and their therapeutic influence. We also provided an overview on characteristics of lncRNAs and Wnt/β‐catenin signalling pathway and discussed their functions in tumour biology. Finally, targeting lncRNAs or/and molecules associated with the Wnt/β‐catenin signalling pathway may be a feasible therapeutic method in the future.Long non‐coding  It has become a great threat to human health. Though having been extensively studied, multidrug resistance (MDR) of tumour cells remains to be an obvious barrier of cancer treatments. Therefore, a better understanding of mechanisms in tumorigenesis, progression and drug resistance in cancer will be imperative for the development of efficient therapeutic strategies. Recently, more and more reports revealed that mutations within the non‐coding genome have a close relationship with tumour biology.Results from the latest GENCODE have indicated that there are only approximately 2% of the human genome are protein‐coding genes, whereas the vast majority of the human genome is made up of non‐coding RNAs (ncRNAs).The comprehensive interrelationship of lncRNAs and Wnt/β‐catenin signalling pathway has appeared as a significant tumorigenic signalling network in various cancers. In this review, we summarized some lncRNAs which are associated with Wnt/β‐catenin signalling pathway to describe their interactions and underlying mechanisms in cancer formation, development and response to treatments.2LncRNAs represent a novel heterogeneous group of ncRNAs that usually exhibit cell‐type–specific expression manner.Functionally, lncRNAs are not the “transcriptional junk” any more. They play an important role in normal cellular proliferation and differentiation.32+ pathway and Wnt/planar cell polarity (PCP) pathway. Among them, Wnt/β‐catenin signalling pathway (Wnt pathway for short) has obtained particular attentions due to its versatile functions in cells. In this section, we will provide an overview of the molecular mechanism of Wnt pathway.Wnt signalling pathway is an evolutionarily highly conserved pathway which controls critical biological processes including embryonic development, normal homoeostasis of adult tissues and cell fate.The primary components of Wnt pathway are as follows: Wnt ligands, Frizzled receptors, LRP5/6 co‐receptors (low density lipoprotein receptor‐related protein 5/6), Dsh (Dishevelled), β‐catenin and TCF/LEF (T‐cell factor/lymphoid enhancer factor) as well as one “degradation complex” composed of APC (adenomatous polyposis coli), axin (the scaffolding protein) and two kinases: CK1α (casein kinase 1α) and GSK‐3β (glycogen synthase kinase 3β). Wnt ligand and β‐catenin play a vital role in this pathway. In the absence of Wnt ligand, the signalling cascade is repressed. Cytoplasmic β‐catenin is phosphorylated and kept in low levels via proteasome‐mediated destruction, which is controlled by the “degradation complex”. As a result, the translocation into nucleus is inhibited. However, when Wnt ligand binding to receptors at the surface of target cells, a chain of events is initiated that disrupt the degradation complex via Dsh (Dishevelled) phosphorylation. Then β‐catenin is separated from the destruction complex and results in the accumulation and stabilization of β‐catenin in cytoplasm.Multiply dysfunctions and mutation of the pathway have suggested a strong connection with different kinds of human diseases including cancer.4The Wnt pathway has been recognized as a pivotal pathway participated in the regulation of cell proliferation, tumorigenesis and progression as well as chemoresistance in various cancers.4.1As is known to all, the ability to propagate continuously is one of the primary characters of cancer, and the process contains various changes in gene expression. Previously, studies into mechanisms of tumorigenesis mostly focused on protein‐coding genes. Recently, lncRNAs, with no protein‐coding ability, have been found to play widespread functions in regulating gene expression and other biological processes.As the functions of lncRNAs influence many signalling pathways involved in multiple cellular processes, they could also interact with Wnt pathway through several mechanisms in tumorigenesis and progression. Recently, Shao et al reported that lncRNA CRNDE is obviously over expressed in RCC cells and the heightened CRNDE could accelerate proliferation of RCC cells via activating Wnt pathway. They found that CRNDE could somehow modulate PI3K/Akt/GSK3β pathway, then the level of β‐catenin in nucleus is elevated, which ultimately controls target gene expressions. However, the detailed mechanism remains to be expounded in further studies.In summary, lncRNAs activate or suppress the Wnt pathway by targeting diverse components associated with the Wnt signalling cascade to modulate downstream gene expression, revealing a strong interaction between them in tumour formation and progression.4.2Chemotherapy is one of the major treatment methods of tumours. Nevertheless, chemoresistance remains an obvious obstacle against satisfactory treatment outcomes.Considering the functions of lncRNAs and Wnt pathway in drug resistance, we then explored the underlying mechanisms and crosstalk between them in chemoresistance of cancer. As is shown, XIA et alRadiotherapy is also considered as a vital component of treatment in malignant tumours. But some of them are seriously resistant to it, and the mechanism remains largely unknown, which further contributes to the poor prognosis of patients. Nowadays, several studies have indicated that some lncRNAs can affect the sensitivity of radiotherapy for human cancer.Collectively, these studies have revealed a potent interrelationship between lncRNAs and Wnt pathway in drug resistance and in radiotherapy of various cancers. Based on these findings, it is expected that further researches are needed to clarify molecular mechanisms between lncRNAs and Wnt pathway, which may act as candidates to develop novel strategies to reverse the chemoresistance and increase radiosensitivity of cancer.4.3Detection of the specific biomarkers of tumour is one of effective methods for the early diagnosis of tumours. LncRNAs apparently exhibit a tissue‐specific expression and can be measured non‐invasively, raising the possibility that they may serve as promising biomarkers and therapeutic targets in cancer.Considering the functions of lncRNAs in cancer, recently, emerging studies have indicated that biological therapies targeting these lncRNAs by the use of small interfering RNAs, ribozymes, antisense oligonucleotides, and by the small molecule inhibitors may be novel approaches to fight against cancer.On the other hand, several crucial components of the Wnt pathway could act as potential biomarkers for cancer diagnosis or prognosis and have been recognized as innovative targets for cancer therapy.5LncRNAs are a class of non‐protein–coding RNAs, which play important roles in diverse biological processes.To summarize, the close interaction between lncRNAs and Wnt/β‐catenin signalling pathway seems to play a significant role in tumorigenesis, progression and influencing the response of cancer cells to treatments. Potential therapies targeting lncRNAs or Wnt pathway have been mentioned in some documents. However, further studies about the underlying mechanisms are needed to promote an efficient therapeutic strategy into a reality by targeting lncRNAs and/or Wnt pathway in cancer.The authors declare that we have no conflict of interest.


The hepatic lobule is divided into three zones along the portal-central vein axis. Hepatocytes within each zone exhibit a distinctive gene expression profile that coordinates their metabolic compartmentalization. The zone-dependent heterogeneity of hepatocytes has been hypothesized to result from the differential degree of exposure to oxygen, nutrition and gut-derived toxins. In addition, the gradient of Wnt signaling that increases towards the central vein seen in rodent models is believed to play a critical role in shaping zonation. Furthermore, hepatic zonation is coupled to the site of the homeostatic renewal of hepatocytes. Despite its critical role, the regulatory mechanisms that determine the distinctive features of zonation and its relevance to humans are not well understood. The present study first conducted a comprehensive zone-dependent transcriptome analysis of normal human liver using laser capture microdissection. Upstream pathway analysis revealed the signatures of host responses to gut-derived toxins in the periportal zone, while both the canonical Wnt pathway and the xenobiotic response pathway govern the perivenular zone. Furthermore, we found that the hypoxic environment of the perivenular zone promotes Wnt11 expression in hepatocytes, which then regulates unique gene expression via activation of the non-canonical Wnt pathway. In summary, our study reports the comprehensive zonation-dependent transcriptome of the normal human liver. Our analysis revealed that the LPS response pathway shapes the characteristics of periportal hepatocytes. By contrast, the perivenular zone is regulated by a combination of three distinct pathways: the xenobiotic response pathway, canonical Wnt signaling, and hypoxia-induced noncanonical Wnt signaling. The environmental differences among each zone are hypothesized to modulate the gene expression profile, resulting in zone-dependent functional heterogeneity, namely, hepatic zonation.4 For example, zone-1 specializes in gluconeogenesis and β-oxidation, while zone-3 is dedicated to glycolysis and lipogenesis.5 Moreover, zone-3 hepatocytes abundantly express cytochrome P450s, which facilitate xenobiotic metabolism. Furthermore, recent studies in mice revealed that zone-3 might be designated for homeostatic renewal of hepatocytes through upregulation of canonical Wnt signaling.8Hepatic zonality divides the hepatic lobule into three zones along the line connecting the portal triads and the central vein.9 Therefore, it is presumed that tissue areas enriched in Wnt signaling activity are sites of self-renewal in terminally differentiated organs. The activation of canonical Wnt signaling results in the release of a signal transducer, β-catenin, from a ‘destruction complex.’ The complex, comprised of APC, Axin, CK1, and GSK3β, promotes constitutive proteasomal degradation of β-catenin via phosphorylation-dependent ubiquitination. Upon Wnt binding to the cell-surface receptor, unphosphorylated β-catenin is released from the destruction complex and translocates into the nucleus for gene transcription.10Wnt is a secreted protein that governs cell fate determination, particularly the maintenance of stem or progenitor cells in adults and embryonic development.8 Alternatively, it has also been reported that Apc expression is reduced in zone-3, resulting in constitutive activation of β-catenin.11 According to these studies, focal activation of the canonical Wnt pathway has two major implications,11 (1) sets zone-3 as the site of homeostatic renewal, and (2) patterns the metabolic compartmentalization of hepatocytes. Thus, the regulation of Wnt signaling activity is indispensable for homeostatic maintenance of organ mass and diversifying liver function. By contrast, deregulated Wnt signaling increases the risk of oncogenic transformation. Accordingly, Wnt signaling upregulation is noted in up to 40% of hepatocellular carcinomas.12 Therefore, furthering our understanding of the mechanisms governing focal Wnt signaling activity has great potential to be extrapolated to the development of stem cell and anti-cancer strategies.Rodent-based studies have demonstrated increasing amounts of unphosphorylated β-catenin in the hepatocytes of zone-3. This event appears to be regulated by Wnt ligands produced in the adjacent endothelial cells of the central vein.in vivo and in vitro studies were performed to further define the regulatory mechanisms that govern zonation.Despite the critical roles of the regulatory mechanisms that shape zonation, their relevance to humans has been poorly understood. Historically, a digitonin–collagenase perfusion approach has been the standard method used to study zonation. However, this method is associated with inevitable cell damage and is incapable of collecting hepatocytes under direct visualization. By contrast, laser capture microdissection (LCM) allows for the collection of all zones under the precise guidance of histological orientation. Consequently, the present study investigated the zone-dependent transcriptome of normal human liver tissue using an LCM approach. Subsequent Subjects who underwent curative hepatectomy for the removal of an isolated metastatic liver tumor were enrolled under an approved IRB protocol (HS-028017 and HS-12-00168). The uninvolved tissue within the resected liver was stored in FFPE at room temperature or OCT compound without fixation at −80 °C. The histology of the research material was reviewed and confirmed as ‘normal’ liver tissue by two independent anatomical pathologists. Samples from patients with significant abnormalities in biochemical liver function tests or with risk factors for primary liver diseases were excluded. The clinical characteristics of the enrolled subjects are summarized in M thickness and then mounted onto polyethylene naphthalate (PEN) membrane glass slides  and subsequently fixed in 100% ethanol at 4C for 15 min. After air-drying, the sections were stained with Hematoxylin QS  for 30 s. Thereafter, the slide was dipped in 95% ethanol. The air-dried slides were subjected to LCM at the parameters of power: 30, aperture: 20, speed: 10, specimen balance: 25, head current: 100, pulse frequency: 800. Multiple lobules from each subject were microdissected, and the samples from each zone were pooled in RLT buffer  and stored at −80C.A LMD7000  system was used for LCM. Liver tissue embedded in OCT was cut with a cryostat at 12–14 μRNA extraction of the pooled LCM samples was performed with an Quick-RNA MicroPrep . One ng of RNA per sample was subjected to first-stranded cDNA synthesis followed by PCR amplification (11 cycles) using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing . The cDNA was then used for deep sequencing with NextSeq500 (Illumina) at 75 single-end sequencing. The quality and quantity of all RNA and the amplified cDNA were assessed using a 2100 Bioanalyzer . RNA sequencing data are deposited at NCBI under accession code GSE83990.13 Gencode v23 annotation was used to quantify the aligned reads to genes/transcripts using the Partek E/M method. Finally, read counts per gene/transcript in all samples were normalized using Upper Quartile normalization13 and analyzed for differential expression using the Partek Gene Specific Analysis method (genes/transcripts with less than 10 reads in any sample among a data set were excluded). Analysis of variance (ANOVA) False Discovery Rate (FDR)<0.1, FC>2.5 were employed for differential expression analysis. IPA upstream analysis used its default Z score (>2 or <−2) as significant.RNA-seq data were analyzed with Partek Flow version 4 (Partek). Raw sequencing reads were first trimmed from both ends with a Quality Score method . Trimmed reads were then mapped to the human genome hg38 using Star version 2.4.1d with default parameter settings and Gencode v23 annotation as guidance.t-test. The differences were considered to be significant if P⩽0.05 (*).All data are presented as the mean±s.d. and were analyzed by two-tailed Student’s A hexagonal hepatic lobule with a portal triad at each corner and a central vein in the center has been defined as the functional unit of the liver. It is divided into 3 zones along the line of the portal-central vein, which spans cords of 15–25 hepatocytes . Normal 14Given the three-dimensional variation of the hepatic lobule to the plane of tissue sectioning, only those lobules with a PV-CV axis spanning 15–25 hepatocytes were utilized for sample collection with LCM. First, 5–6 hepatocytes from the edge of the portal triad with the exclusion of connective tissue near Glisson's capsule were collected as zone-1 tissue . Next, 3The cDNA library of each zone and whole-liver tissue was applied to deep sequencing followed by transcriptome analysis. ANOVA analysis, which integrates both subject and zonation factors as the fixed attribute, was used to illustrate zone-dependent differential gene expression. The volcano plot revealed 139 transcripts that are differentially expressed between zone-1 and zone-3 . Upon th15The gene expression profile between zones suggests that zone-1 and zone-3 tissues are indeed exposed to substantially different environments. To better understand the regulatory factors of zonation, we conducted an upstream analysis of the genes differentially expressed in zones 1 and 3 using IPA software. The analysis predicts that genes uniquely upregulated in zone-1 are largely governed by lipopolysaccharide (LPS), IL-1β, IL-6 signaling and their downstream transcription factors such as NFκB, STAT3, and CEBPB . It is iUpstream analysis of the genes upregulated in zone-3 tissue predicted the contribution of the canonical Wnt/β-Catenin pathway  and the 11 Accordingly, our IHC analysis of APC in normal human liver showed a gradual loss of expression towards zone-3 (Studies in rodent models showed that there is a decreasing gradient of expression of adenomatous polyposis coli (Apc) in zone-3 hepatocytes. This phenomenon may account for constitutive β-Catenin activation, as the loss of Apc releases it from the destruction complex.s zone-3 . As our s zone-3 . These f9 In agreement with this notion, our data suggest that Wnt11 is produced in zone-3 and may influence gene expression in surrounding hepatocytes.To delineate the mechanism of β-Catenin activation in zone-3, we analyzed the mRNA expression of Wnt genes in normal human liver. Our results suggest that Wnt2B, 3, 5B, and 11 were expressed in whole-liver tissue across all three subjects . ConversTo assess whether Wnt11 manifests the unique gene expression seen in perivenular hepatocytes, a custom qPCR array comprised of genes upregulated in zone-3 was employed . The qPC16 These results suggest that genes regulation by Wnt11 is most likely mediated by non-canonical responses, such as the planar cell polarity (PCP) pathway and/or Wnt/Ca2+ signaling. We then first tested whether Wnt11 promotes the phosphorylation of Jun amino-terminal kinases (JNK), which serves as an activation marker of the PCP pathway  reporter assay, and showed a negligible effect on the Ca2+ pathway (data not shown). Of note, our RNA sequencing result demonstrated upregulation of R-spondin 3 (RSPO3) in zone-3. RSPO3 is a well-known activator of the Wnt signaling pathway.17 Therefore, we extended our investigation to test whether Wnt11 and RSPO3 exhibit any synergistic or additive effects on either canonical or noncanonical Wnt signaling. We found that RSPO3 exhibited activity only when added to the ligand for canonical Wnt pathway activation but had negligible effects on the activation of Wnt11-mediated non-canonical signaling  in a hepatocyte-specific manner.19 VHL is the E3 ligase that promotes ubiquitin-mediated degradation of HIFs, and thereby a lack of VHL results in the constitutive activation of HIFs. These observations suggest that Wnt11 production may occur in hepatocytes under hypoxic conditions. Consequently, we tested whether MPH cultured in a condition mimicking hypoxia induces Wnt11 expression -1α and −1β.pression . The resle genes . These r21 By contrast, zone-3 is exposed to plasma of low oxygen tension and, as a result, HIF expression is upregulated in perivenular areas.23 Moreover, the upregulation of xenobiotic metabolizing genes suggests that zone-3 is exposed to relatively higher concentrations of endogenous ligands for CAR, PXR and AHR.18 Cellular adaptation to these environmental factors has been used to explain zone-dependent gene expression, which facilitates the divergence of liver function.25 Furthermore, the concept of hepatic zonation has been coupled to growing controversy over the site for the homeostatic renewal of hepatocytes.28 Thus, hepatic zonation is a critical regulatory mechanism for both the multifaceted liver function and the maintenance of homeostatic organ volume. However, our current understanding of the mechanism that regulates liver zonation is largely based on aforementioned assumptions or studies with the rodent model system. Consequently, its relevance in human liver biology is not well understood.Zone-1 is fed by plasma enriched with oxygen, nutrients and gut-derived toxins transported via the hepatic artery and portal vein.29 For example, high expression of SDS and GLS2 were seen in zone-1. In addition, the expression of GLUL and RHBG are enriched in zone-3, indicating that zone-1 specializes in urea production, while zone-3 utilizes ammonia for glutamine synthesis in both humans and mice. Moreover, genes that are known to be regulated via the canonical Wnt pathway, such as Gpr49 (LGR5), are also enriched in zone-3. Furthermore, the enzymes involved in xenobiotic metabolism appear to be abundant in zone-3 in both humans and mice. By contrast, we noted some interspecies disparity; for example, the expression of aldehyde dehydrogenases (ALDH), an enzyme involved in alcohol and retinoid metabolism,30 is enriched in zone 1 in the murine liver, while a higher abundance is seen in zone 3 of the human liver. Taken together, our study results generally reassert the usefulness of rodents for the study of human liver biology, with the caveat that there are potential disadvantages arising from interspecies differences.Our LCM approach has provided, for the first time, a comprehensive transcriptome analysis of human liver zonation. Our results combined with results from previous studies by others collectively found grossly similar patterns between humans and mice regarding the zone-dependent gene expression profile.Our pathway analysis of the human liver zone-based transcriptome found that most genes enriched in zone-1 tissue fit well within the innate inflammatory response network . This re32 Moreover, an in vivo study with hepatocyte-specific genetic deletion of Apc and β-Catenin suggested that the expression of nuclear receptors of xenobiotics, such as AhR and CAR, is directly regulated by the Wnt/β-Catenin pathway.33 These observations collectively suggest that there is reciprocal positive regulation between the xenobiotic response pathway and Wnt/β-Catenin in zone-3 hepatocytes.Pathway analysis of zone-3 genes predicted a substantial contribution of the Wnt/β-Catenin pathway and, to a lesser extent, the xenobiotic response pathway . Our RT-17 Consistent with observations in mice, we found reduced expression of APC in zone-3 in normal human liver. However, the gradient of APC expression does not appear to correlate well with the pattern of activated β-Catenin distribution. Our data showed that the expression of activated β-Catenin is restricted only in zone-3 hepatocytes adjacent to the central vein. This observation is also congruent with the pattern of expression of LGR5 or Axin2, which are activated β-Catenin-inducible genes, as seen in mouse models.31 Recent seminal studies demonstrated that the endothelial cells of the central vein express Wnt 2, 9, and Rspo3, thus suggesting that these ligands might activate β-Catenin in the adjacent proximal layer of zone-3 hepatocytes.17 This possibility is consistent with the fact that Wnt proteins do not offer long-distance bioactivity due to their hydrophobic nature.35 The focal activation of Wnt/β-Catenin in proximal zone-3 hepatocytes facilitates the homeostatic maintenance of organ mass in a fine-tuned balance between naturally occurring turnover and compensatory homeostatic renewal of hepatocytes.8 Thus, this explanation now replaces our longstanding belief in the ‘streaming theory’ in which newly proliferating hepatocytes in zone-1 move to zone-3.36 However, further investigations are required to determine whether this notion holds true in the human liver. Thus, our next investigation will require LCM-based collection of endothelial cells of the central vein to validate this phenomenon in the human liver.The exact mechanisms that facilitate focal upregulation of the canonical Wnt pathway in zone-3 remain elusive. Recent rodent-based studies proposed two potential explanations for the focal upregulation of Wnt/β-Catenin in proximal zone-3 hepatocytes: 1) autonomous activation of β-Catenin due to reduced APC expression and 2) exposure to Wnt ligands and Rspo3 supplied by endothelial cells of the central vein.22 The cellular response to hypoxia is largely facilitated by the transcription factors HIF-1α and -2α. Thus, we conducted a bioinformatics analysis with BioBase TRANSFAC to compare the occurrence of HIF-1α and -2α binding consensus sequences within the promoter region (−5000 to +500) of genes upregulated in zone-3 and compared to the results of zone-1 genes. We noted that the consensus binding sequences of HIF-1α and HIF-1β but not HIF-2α were more frequently found in the promoters of upregulated in zone-3 (37 Thus, further investigation is required to determine how hypoxic conditions influence gene expression of perivenular hepatocytes.An additional important environmental factor unique to zone-3 tissue is the relative hypoxia wherein perivenular oxygen tension is at least 30 mm Hg lower than that in the periportal region.n zone-3 . However19 leading us to hypothesize that the relative hypoxia contributes to this occurrence. Our study found that the hypoxic condition in hepatocytes robustly induce Wnt11 expression. Moreover, VHL−/− in the liver results in the upregulation of genes regulated via Wnt11. Wnt11 has been generally recognized as ‘non-canonical Wnt’ that exclusively activates only non-canonical signaling pathways.39 Accordingly, we found that Wnt11 has a negligible effect on β-Catenin activation regardless of RSPO3 treatment. Taken together, our results indicate that hypoxia-mediated induction of Wnt11, and subsequent non-canonical pathway activation controls a subset of gene expression in zone-3. Accordingly, our finding led us to speculate that Wnt11-mediated activation of the PCP pathway plays a partial role in shaping the characteristics of zone-3 hepatocytes. However, further biochemical and genetic investigations are required to determine the exact mechanism of how Wnt11 regulates zonation.We found that Wnt11 was upregulated in zone-3. A previous study demonstrated that hypoxia in the liver results in Wnt11 induction,40 The interaction between HIFs and β-Catenin is also implicated in the potential pathophysiology of liver cancer.41 Taken all together, furthering our understanding of hepatic zonation can be highly exploitable for the development of therapeutic strategies for a broad spectrum of liver diseases.On the basis of our study results, we propose that there are three separate regulatory mechanisms that govern the gene expression profile of the perivenular zone . These rIn summary, our study illustrates the distinctive pattern of gene expression of normal human liver in a zone-dependent manner. Furthermore, our study provides the first evidence that hypoxia-induced Wnt11 serves as a novel regulatory mechanism of the unique gene expression profile of zone-3, along with canonical Wnt and xenobiotic response pathways.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.