In-depth genetic molecular characterization of qWS8/ipa1-2D reveals that this newly identified QTL results from three distal naturally occurring tandem repeats upstream of IPA1, a key gene/locus previously shown to shape rice ideal plant architecture and greatly enhance grain yield. The qWS8/ipa1-2D locus is associated with reduced DNA methylation and a more open chromatin state at the IPA1 promoter, thus alleviating the epigenetic repression of IPA1 mediated by nearby heterochromatin. Our findings reveal that IPA traits can be fine-tuned by manipulating IPA1 expression and that an optimal IPA1 expression/dose may lead to an ideal yield, demonstrating a practical approach to efficiently design elite super rice varieties.Super hybrid rice varieties with ideal plant architecture (IPA) have been critical in enhancing food security worldwide. However, the molecular mechanisms underlying their improved yield remain unclear. Here, we report the identification of a QTL,  et al. show that, in the super rice variety YY12, this ideal plant architecture trait arose from three distal tandem repeats that are associated with an open chromatin state and increased expression of the IPA1 gene.Breeding of rice with ideal plant architecture has increased rice yield worldwide. Here Zhang  Global food demand is expected to double by the year 2050 ref. . Rice is24sd1, which led to the Green revolutionsd1 was identified as a mutation in GA20ox-2 that results in reduced biosynthesis of the hormone giberrellinGn1a, Ghd7, ipa1/WFP, SCM2, dep1 and SPIKE, all of which show great potential in improving rice yield91011121314IPA1 encodes the SBP-domain transcription factor OsSPL14. A point mutation in OsSPL14 relieves its repression by the miRNA OsmiR156 and affects all three IPA characteristics simultaneouslyThe advances in high-quality rice genetics, genomics and molecular markers have accelerated the dissection of key genes for yield improvement. The first leap of rice productivity was attributed to the finding and application of the semi-dwarf locus indica × japonica) exhibiting the IPA phenotypes, but we did not find the ipa1 allele in these varietiesThe utilization of hybrid vigour (heterosis) has greatly enhanced rice productivity, however, the underlying mechanisms have not been well-characterized. A global view of heterosis has been proposed by re-sequencing more than 1,000 hybrid varieties, which found that a number of superior alleles underlie the high yield potential of hybridsqWS8/ipa1-2D, from the original breeding stock of YY12 which explains the major IPA phenotypes. The cloned qWS8/ipa1-2D locus reveals a previously unidentified large tandem repeats which attenuate the epigenetic repression of IPA1 promoter and release IPA1 expression through chromatin remodelling. Our study demonstrates that IPA traits can be optimized by fine-tuning the expression of IPA1, and the novel qWS8/ipa1-2D locus will enable molecularly designing new super rice varieties with high yield.Here, we report the identification and characterization of a major QTL, 2 mapping population by crossing the IPA breeding stock YYP1 with the japonica variety Nipponbare (NIP). YYP1 bears extremely strong culms, large panicles and modest tiller numbers compared to NIP . To identify the gene underlying qWS8, we carried out fine mapping using backcross populations (BC2F2), with little interference of other loci  bearing heterozygous qWS8 alleles. A significant difference was identified among segregating genotypes and the locus explains as much as 50% of the phenotypic variance for panicle branch and 44% for stem diameter , we did not uncover sequence polymorphisms between YYP1 and NIP by PCR-based sequencing of the mapping region. However, Southern blotting revealed that YYP1 contains a large sequence insertion at the mapping region . Interesffective . We therIPA1 and qWS8 from both parents, and found only seven SNPs in addition to the repeats  carrying an ∼80-kb segment with ipa1-2D and IPA1 , and named them NILipa1-2D and NILIPA1 respectively.Given the proximity of the P allele . TherefoIPA1 and of two miRNAs thought to target IPA1 at different stages, miR156 and miR529 (ref. IPA1 was expressed mainly in the inflorescence meristem (IM), and was hardly detected in the seedling, opposite to the pattern of the two miRNAs  reporter and found that the fusion reporters did not show different GUS expression , which are usually silenced by DNA methylation and form heterochromatin. Histone H3K9m2, a modification associated with heterochromatin, was also enriched in this region  construct of IPA1 with its native promoter and coding region containing an artificial SNP to distinguish it from the native gene  site  and seveelements  that couelements . We founr region , in NILiive gene . Indepenive gene , confirmipa1-2D are caused by tissue-specific up-regulation of IPA1, we hypothesized that optimal IPA traits can be achieved by manipulating IPA1 expression dosage. Supporting the hypothesis, varying IPA traits were observed for stable lines with different transgenic copy numbers of two different IPA1 gDNA constructs  with strong culms could not be further reduced, suggesting a limit of IPA1 dosage effect in tillering control  and Hainan (southern China). Three genotypes with 0, 1 (heterozygous) and 2 (homozygous) copies of the ipa1-2D allele were analysed from the NIL population. The IPA phenotypes with larger panicle were observed in plants with ipa1-2D allele  inclines toward the level of homozygous plants (ipa1-2D/ipa1-2D) and produces mid-parent heterosis of the hybrid of NILs  bear ipa1-1D and another two (JYZK-3 and JYZK-4) bear ipa1-2D. Field tests showed that the new varieties, either with ipa1-1D or ipa1-2D, greatly improved yield performance in comparison with the control variety at two locations of different seasons  and seedlings  IPA1 allele. This epigenetic modification may represent a universal mechanism of de-repression for genes proximal to heterochromatin as suggested by recent genome-wide studies25Breeding rice with new plant type or IPA to break through the yield ceiling has been proposed since 1990s ref. . Howevercis or in trans, as indicated by the studies of the maize loci tb1 and b1 , but tb1 confers up-regulation whereas b1 confers down-regulation of the downstream ORF, respectively. It has been shown that tb1 covers a functional transposon insertion that up-regulates downstream ORF trascriptionb1 was identified as tandem repeats that exert function via trans-acting RNAs, and the phenotype can be regenerated by transforming the repeat sequence only30GL7 leads to up-regulation of GL7 and down-regulation of its nearby negative regulator, resulting in an increase in grain length and improvement of grain qualityipa1-2D exert neither trans-acting activity nor enhancer activity, presenting a novel case for non-coding sequence that mediates gene regulation. We provided evidence that the ipa1-2D repeats induce an open chromatin status to release the position-dependent repression of IPA1 in wild-type varieties, probably through an unrecognized chromatin complex. Nevertheless, the mechanisms need to be clarified in depth with advanced techniques in future work.Several QTLs underlying important agronomic traits have been mapped to non-coding regions that modify nearby gene transcription b1 refs , 31, 32.IPA1 functions as a pleiotropic regulator and determines different IPA traits in a dosage-dependent manner, which provides an alternative mechanistic explanation of heterosis. As IPA1 expression or copy number increases in transgenic lines, the panicle size increases to a plateau rapidly, whereas the tiller number continues to decrease, indicating that an optimal IPA1 dosage will confer the best yield potential. Thus, the F1 plants with big panicles and moderate productive tillers can be generated by crossing plants with contrasting IPA1 dosage, which even forms single-gene heterosis with specific allele combination . Tiller number was counted as all the fertile panicle in 13.34 m2 region and normalized to number per mu (667 m2).For yield test of JYZK hybrid varieties, total grain weight of plants in a plot of 13.34 m2 population derived from YYP1 and NIP were first genotyped by 145 genome-wide polymorphic markers and linkage analysis was performed by composite interval mapping module of the software WinQTLCart 2.5. Linkage analysis of two BC2F2 populations with 185 and 166 plants were then performed by single maker analysis module. Progenies of eight BC2F2 recombinants were planted to clarify the mapping region with valid phenotype. A larger BC3F2 population with 3,404 plants was genotyped using the markers RM23399 and RM23426 to find additional recombinants, and more markers were developed to discriminate the recombination sites  after agarose gel electrophoresis. Probes were synthesized by DIG PCR labelling system and hybridization was performed at 55 °C overnight in DIG Easy Hyb granules (Roche). The membranes were washed in 2 × SSC and 0.1% SDS at 65 °C for 15 min twice and then subjected to chemiluminescent detection following the instruction of Detection Starter Kit (Roche). High-quality images were captured by the Tanon-5500 Chemiluminescent Imaging System (Tanon Science & Technology).Genomic DNA was extracted by the CTAB method and digested by restriction enzymes in 400 μl volumes  for 18 h. The digested DNA was extracted using phenol:chloroform treatment followed by ethanol precipitation and transferred onto Hybond NIPA1 transcripts, 1 μg of total RNAs was used for cDNA synthesis with a reverse transcription kit (TaKaRa). qRT-PCR was performed in a 20 μl volume with 2 μl cDNA, 0.5 μM gene specific primers (Actin gene (LOC_Os03g50885) was used as the internal control. Northern blot analysis was carried out to confirm IPA1 transcript levels with the rice Actin probed as loading control. For detection of miR156 and miR529, the same RNA were reverse-transcribed by the miRNA RT-PCR Kit (TaKaRa) and qRT-PCR was performed following the same procedure described above, and universal adaptor primers (supplied by the Kit) and Uni-miRNA primers were used with 5 s rRNA as internal control  for RNA preparation. Total RNAs were prepared using a TRIzol kit according to the user's manual (Invitrogen). For detection of  primers  and 10 μ control .IPA1 promoter regions from the different genotypes by PCR using primers listed in Genomic DNA from IM tissues was extracted using CTAB method. Bisulfite treatment was performed using the EpiTect Bisulfite kit (Qiagen). Bisulfite-treated DNA was then used to amplify 0) harbouring the IPA1 construct with an artificial SNP (see below) were amplified separately using primers in IPA1). The number of colony with respective SNP type was subjected to the ratio calculation.For transgene ratio detection, the genomic DNAs and cDNAs from IM tissues of transgenic plants ) with gentle rotation for 15 min. The suspension was filtered through 30-nm CellTrics, and the elute was centrifuged for 10 min at 1,500 g at 4 °C. The pellet was resuspended as crude nuclei extract with digestion buffer ). Equal aliquot of crude nuclei extract was subject to digestion with increasing MNase amounts at 30 °C for 20 min and DNA was extracted using phenol:chloroform treatment followed by ethanol precipitation and used for PCR analysis.Open chromatin was detected using a published protocolqWS8/ipa1-2D was cloned into binary vector pCambia1301 and transformed into NIP for analysis of possible function of the repeats. The same repeats region was then fused with CaMV35S mini promoter driving the GUS reporter for reporter analysis. The single repeat from NIP was also cloned and fused with the reporter construct. These constructs were transformed in NIP callus to get independent transformants, which were grown in medium supplemented with X-Gluc to detect reporter activity. Three genomic fragments containing IPA1 with size of 8, 11.9 and 13.9 kb from the NIP BAC AP006049 were cloned into pCambina1301, respectively, and an artificial SNP was introduced into the 8-kb construct by site-directed mutagenesis. The IPA1 cDNA AK107191 was also cloned into pCambia1301 with the CaMV35S promoter to obtain ectopic IPA1 over-expression construct. All the gDNA and cDNA constructs of IPA1 were transformed into NIP to generate more than 15 independent lines. Stable transgenic lines (T2 generation) were used for gene expression and phenotype analysis.The region containing all the three repeats of 2 and coated with gold, followed by visualization with a scanning electron microscope . For histological sections, samples were embedded in paraffin or resin, and transverse sections were made at 10 μm for paraffin samples and 2 μm for resin samples. Sections were examined microscopically  and photographed. The IM size was measured from pictures of scanning electron microscopy using ImageJ software (NIH).IM tissues at different stages were sampled and fixed in the FAA solution . For scanning electron microscopy, the samples were critical-point dried in liquid COThe authors declare that all data supporting the findings of this study are available within the manuscript or its supplementary files or are available from the corresponding authors upon request.How to cite this article: Zhang, L. et al. A natural tandem array alleviates epigenetic repression of IPA1 and leads to superior yielding rice. Nat. Commun.8, 14789 doi: 10.1038/ncomms14789 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Tables


The adventitious roots (AR) of plants share the same function as primary and lateral roots (LR), although their development is mainly an adaptive reaction to stress conditions. Regeneration of grafted plants is often accompanied by AR formation thus making the grafting technique a good model for studying AR initiation and development and their means of emergence. Pectins and arabinogalactan proteins (AGP) are helpful markers of particular cellular events, such as programmed cell death (PCD), elongation, proliferation or other differentiation events that accompany AR development. However, little is known about the distribution of pectins and AGPs during AR ontogeny, either in the primordium or stem tissues from which AR arise or their correspondence with these events during LR formation.AR were developed from different stem tissues such as parenchyma, xylem rays and the cambium, depending on the stem age and treatment (grafting versus cutting) of the parental tissue. Immunochemical analysis of the presence of pectic  and AGP  epitopes in AR and AR-associated tissues showed differential, tissue-specific distributions of these epitopes. Two pectic epitopes  were developmentally regulated and the occurrence of the LM8 xylogalacturonan epitope in the root cap of the AR differed from other species described so far. AGP epitopes were abundantly present in the cytoplasmic compartments (mainly the tonoplast) and were correlated with the degree of cell vacuolisation. JIM8 and JIM13 epitopes were detected in the more advanced stages of primordium development, whereas the JIM16 epitope was present from the earliest division events of the initial AR cells. The comparison between AR and LR showed quantitative  and qualitative (pectins) differences.The chemical compositions of adventitious and lateral root cells show differences that correlate with the different origins of these cells. In AR, developmental changes in the distribution of pectins and AGP suggest the turnover of wall compounds. Our data extend the knowledge about the distribution of pectin and AGP during non-embryogenic root development in a species that is important from an agronomic point of view. Plant roots have two important functions – to anchor the plant in the soil and to absorb water and mineral nutrients. During normal development, a plant produces a primary root during embryogenesis , 2, follGrafting is widely used in various aspects of plant biological research . It is rHomogalacturonan (HG), a major pectin of the dicotyledonous primary cell walls , contribAGP belong to the hydroxyproline-rich glycoprotein superfamily with a high level of type II arabinogalactan glycosylation . AGP areDaucus carota, Zea mays, Pisum sativum, Brassica napus, Benicasa hispida, Alnus spp., Oryza sativa and Arabidopsis thaliana, the presence of different pectin and AGP epitopes has been analysed . Thtail see . Many oftail see , while otail see  and elontail see , the rootail see , 41.From the data summarised above, it appears that the chemical composition with particular emphasis on the pectic and AGP epitopes of primary roots is well described. In contrast, the different pectic and AGP epitopes in the AR tissues and in the tissues that are penetrated by AR during their development and emergence from parental tissues has not been described to date. Thus, the aims of the study were: 1/to identify any morpho-histological changes that accompany AR development, 2/to analyse the chemical composition of the cell walls of AR and surrounding tissues, 3/to check whether adventitious-derived LR have a distribution pattern that is similar to the pectic and AGP epitopes in AR.Solanum lycopersicon L. ‘Moneymaker’ were germinated in Petri dishes with wet blotting paper for 5–7 d at 23 ± 1 °C in darkness. Germinated seedlings were transferred to pots with soil and grown at a temperature of 23 ± 1 °C, relative humidity 35% and 16 h photoperiod. 30–40-d-old plants with undamaged cotyledons and an epicotyl length of about 1–1. 5 cm were autografted, as described in [Seeds of ribed in . The midFragments of grafted stems were collected for analysis during two time periods – from 5 to 10 d and between 20 to 30 d after grafting. Concurrently, control plants of similar ages were cut and put in beakers with tap water to check whether there were any differences between the developing and emerging AR from plants that had been wounded by cutting and the AR that were developing on grafted stems. The latter were designated for further investigations.Analysis of plants morphology was carried out using an Olympus  SZH10 stereomicroscope. Surface staining with aqueous solutions of 0. 05% (w/v) Toluidine Blue 0 (Sigma) and 0. 02% (w/v) Ruthenium Red (Sigma) were applied to the hand-cut sections .Fragments of the grafted epicotyls were fixed in a mixture of 3% (w/v) paraformaldehyde (Polysciences), 1. 25% (v/v) glutaraldehyde (Sigma-Aldrich) in phosphate-buffered saline (PBS), pH 7. 2. Samples were de-aerated in fixative for 2 h and incubated in fixative at 4 °C overnight. After rinsing with PBS , the material was dehydrated in an ethanol series  and embedded in Steedman’s wax . LongituSections were de-waxed, rehydrated in a successive ethanol series  and designated for the following histochemical schedules:Sections were oxidised in a 0. 5% (w/v) aqueous periodic acid (Sigma-Aldrich) solution for 1 h at room temperature, washed in running water for 10 min and rinsed once with distilled water. Next, the slides were placed in Schiff’s reagent (Sigma-Aldrich) for 15 min in darkness, rinsed with distilled water and transferred to a 0. 5% (w/v) sodium sulphite solution for 1–2 min. After washing with running tap water for 5 min, sections were placed in a 0. 5% (w/v) Toluidine Blue 0 aqueous solution for 5 s to visualise the meristematic cells of the developing AR. They were then dehydrated in an ethanol series  and 100% isopropanol for 3 min. Slides were shaken dry and mounted in Euparal (Roth).Sections were stained with a 0. 5% % (w/v) Sudan III (Sigma) solution  was then added and the solution was filtered again). Slides were stained for a minimum 3 h , rinsed with 50% (v/v) ethanol, rinsed with distilled water and mounted in 50% (v/v) glycerol.2HPO4 solution for 20 min, rinsed three times with distilled water and mounted in 50% (v/v) glycerol.Sections were stained with 0. 1% (w/v) Methyl blue (Sigma) in a 0. 15 M KFor the immunolabelling procedure, sections were de-waxed and rehydrated in an ethanol series . The area occupied by the sections on the microscope slides was marked using a hydrophobic PAP pen (Sigma-Aldrich). The detailed steps of procedure were performed exactly as described by Sala et al . The priAll observations and photography were carried out using a Nikon Eclipse Ni-U microscope equipped with a Nikon Digital DS-Fi1-U3 camera with corresponding software , and a maximum excitation wavelength of 490 nm (AlexaFluor 488) or 330 nm (Methyl blue). Photographs and diagrams were edited using the CorelDrawX7 graphics program.inset). The ‘envelope’ consisted of parenchymatous cells which originated from the proliferation of the collenchyma . The root and these parenchymatous cells remained in close contact during the early stage of root growth . The formation of the “envelope” occurred in older grafts, i.e. at approx. 20 d or more after grafting, when the stems were exhibiting secondary growth. The AR that were surrounded by this parenchymatous ‘envelope’ had a bulbous outline , depending on the stage of stem development and the type of procedure that is used to induce their growth Fig. . In the inset 2). Within a given scion, multiple AR at different stages of development were found , independently of the point of emergence of the latter roots. There was no significant occurrence of callose during AR or LR emergence. Callose was present, however, in some areas within the thick layer between the AR and the scion tissues . In the AR, callose was detected in the primary pit fields and cell plates of dividing cells . When the AR or LR were still enclosed within the maternal tissues, no lipid substances were detected in their cell walls (data not shown). After root emergence, deposition of polyphenolics in the rhizodermal walls commenced and cells beneath the rhizodermis divided periclinally . Identification of this tissue was based on its phenotypic features such as the presence of lipid substances within the cell wall, the presence of polyphenols and cell shape.At the earliest stage of AR development, initial cells, which were rich in starch, showed cell divisions, thereby forming multicellular complexes Fig. . The celund Fig. . Moreoveund Fig. , inset 1ues Fig. , or at tues Fig. , inset 2lls Fig. , inset 1lly Fig.  giving rlly Fig. , inset. inset). Cells of a root cap could already be distinguished within the dome-shaped primordium  was detected in the walls of all of the initial cells Fig . Howeverinset). In the walls of the cortical cells, which were adjacent to a developing root, the LM19 epitope occurred abundantly and was distributed in a punctate manner throughout the primary walls and the middle lamellae; it was also detected in the intercellular spaces  was less abundant than the LM20 epitope and was distributed in a punctate manner in the walls of the initial cells Fig. . In the ces Fig. . In oldeces Fig. . The LM1ces Fig. . In betwces Fig. ; howeverces Fig. .In the AR that developed within the scion tissues, the pectic epitope that was recognised by the LM8 antibody  occurred only in the walls of root cap cells, which were adjacent to the primordium Fig.  and i’. inset). The JIM8 epitope did appear, however, at the root primordium stage, though here it occurred less abundantly than it did in the scion collenchyma or cortical cells  as a punctate signal and this type of distribution was displayed throughout the entire root development. At the primordium stage, the JIM16 epitope occurred in all of the cells of the developing root with the exception of the future vasculature. This was in contrast to the scion tissues, collenchyma and cortex, where it was hardly present . The occurrence of the JIM16 epitope was associated with the tonoplast, cytoplasm and plasma membrane .At the earlier stage of AR development, the AGP epitope that was recognised by the JIM16 antibody was detected in the walls/plasma membrane of the dividing initial cells Fig. inset asent Fig. . In content Fig. inset 2.Upon the emergence of the LR, the LM19 epitope was found in the walls of the cells enclosing the future vascular tissue and in the outer periclinal walls of the rhizodermis  Fig. . In the The AGP epitope that was recognised by the JIM8 antibody occurred abundantly in the AR tissue Fig. . Within An arabinogalactan protein epitope that was recognised by the JIM13 antibody was observed in similar regions of the lateral root primordia Fig. , and alsThe arabinogalactan protein epitope that was recognised the by JIM16 antibody was detected in all of the tissues of the developing lateral root, either as a continuous or punctuate signal, although in the central region of the primordium, the fluorescence signal was punctate and mostly weak Fig. . In contCeratopteris richardii [The distribution of the LM19 and LM20 epitopes during AR primordium development indicates that these epitopes have distinctive localisations at the corresponding developmental stages in both the developing AR and the scion. At the earliest phase of AR development, the LM20 epitope occurred abundantly, while at a more advanced stage, its presence decreased and its distribution became less uniform. Concurrently, the LM19 epitope increased and, at the primordium stage, it was more widespread than the LM20 epitope. Similarities in the distribution also concerned the AR/cortex border where both epitopes were abundantly detected in a thick layer, whereas neither occurred, or were only faintly detected, in the new cell walls of the divided cells. This is consistent with observations that meristematic cells have methyl-esterified HG domains whereas differentiating cells exhibit more non-methyl-esterified domains . A distiichardii , 49 who ichardii , which wichardii . DifferePisum sativum, Daucus carota, Zea mays and Arabidopsis [Xylogalacturonan, which is represented by the LM8 epitope, was present in the root cap cells of AR during the primordium stage but not at a more advanced stage of development. This epitope is thought to be specifically associated with the root cap cells in a range of angiosperm species , thus inbidopsis . The LM8bidopsis . The occbidopsis  and alsobidopsis . Moreovebidopsis . It is obidopsis , thus foIn the case of the AGP epitopes, it appeared that their presence and distribution depended on the stage of AR development and the cell type. JIM8 and JIM13 antibodies were detected in the more advanced primordium stage of AR development, whereas the JIM16 epitope was present from the earliest division events.Arabidopsis thaliana, Pisum sativum and Brassica napus [Arabidopsis thaliana [Daucus carota [versus adventitious roots), they still point to the important role of AGP in root tissue development.Results from various studies concerning the presence of AGP epitopes in the roots of a range of species are listed in , 35. JIMca napus , whereasthaliana  and in ts carota . In our In Brasica napus flowers, a temporal and spatial regulation of the plasma membrane AGP epitope that is recognised by the JIM8 antibody was postulated [stulated . During stulated . This instulated  or JIM13stulated , 36, 41 stulated . TherefoThe distribution of all of the AGP epitopes that were applied within primordium in the presented study is schematically presented in Fig. The comparison of LR and AR at the corresponding stages of development showed some differences as well as similarities in the occurrence of the pectic and AGP epitopes. No xylogalacturonan was detected in the LR; moreover, the LM20 epitope  was hardly present in LR in comparison to AR. The LM19 epitope  occurred in the outer periclinal cell walls of the primordium and was especially abundantly in the regions where LR had already emerged. AGP epitopes had a similar distribution pattern in both types of roots. However, they were more expressed in the LR than in the AR. AR share the same function as LR but develop from aerial tissues . The parWe performed histo- and immunohistochemical analyses of the cell wall to study the development of tomato AR. The AR that formed on the grafted stems differed from the AR that were induced by stem cutting in terms of the shape of the primordium and the way they emerged. Two HG epitopes, LM19 and LM20, had different localisations at the corresponding AR developmental stages with an increase in LM19 occurrence along with the progress of AR growth. Of the three AGP epitopes that were applied, JIM8 and JIM13 showed an association with the tonoplast in particular cells of the primordium, whereas the JIM16 epitope occurred from the earliest stage, and therefore it can be considered to be a marker of initial cells. Finally, the differences observed between the AR and LR cell wall composition might be origin- or environment-dependent.


We welcome Jesus et al.’s paper, which makes an important contribution to the under-researched area of the physical rehabilitation workforce. The authors present recommendations to “advance a policy and research agenda for ensuring that an adequate rehabilitation workforce can meet the current and future rehabilitation health needs” (p. 1). We argue that their perspective could however be strengthened by adopting a stronger global perspective, including consideration of the needs of low-resource settings. In particular, we highlight the integral role of more effective sector and inter-sectoral governance, the opportunity to support the development of community-based rehabilitation (CBR), the lessons that can be learnt from human resources for health (HRH) research and practice more generally, and the recent developments in the global provision of assistive technologies. Each of these issues has important implications and contributions to make to advance the policy and research agenda for the global rehabilitation workforce. We welcome Jesus et al.’s paper , which mJesus et al. propose a policy agenda, highlighting the need to “adapt policy options to different contexts , even within a country” (p. 1), and provide recommendations for both local and global policymakers. However, the six rehabilitation workforce challenges outlined by the paper do not sufficiently recognise the integral role of governance and leadership in addressing these, including strategies to increase the priority of rehabilitation on the health agenda. Dieleman and Hilhorst  specify With the rapidly growing ageing population, and the majority of persons with disabilities living in low- and middle-income countries (LMICs), there is a need to consider the global context when addressing rehabilitation workforce challenges , 10. JesWhile Jesus et al. focus on setting an agenda for physical rehabilitation, it is important to consider that often professionals working with people with physical disabilities may need to have a multi-faceted skillset . This caWhile the role of task-shifting in providing services was alluded to by Jesus et al., this approach deserves much more attention. The evidence for task-shifting in rehabilitation is slowly growing . HoweverOne of the most significant global initiatives in the field of physical rehabilitation is WHO’s GATE  programme, which seeks to produce a step-change in the provision and use of affordable quality assistive technology (AT) . FollowiJesus et al. draw attention to the important and under-researched area of physical rehabilitation health workforce challenges. It is important that while setting a global agenda, such an effort should encompass the challenges and potential solutions for high-, medium- and low-resource settings, as well as urban and rural settings. We have sought to complement Jesus et al.’s important article by highlighting some of the ways in which this can be done.


Hepatocellular carcinoma (HCC) is a male-dominant disease with poor prognosis. Sorafenib is the only approved systemic chemotherapeutic drug for patients with advanced HCC. Previous studies have shown that androgen and androgen receptor (AR) are involved in human hepatocarcinogenesis and the development of HCC. Here, we discuss the recent data on AR and HCC, and the combination of sorafenib and inhibitors of AR for advanced-HCC patients. Androgen-dependent and androgen-independent AR activation exist in human hepatocarcinogenesis. AR could directly control hepatocarcinogenesis and regulate the innate immune system to influence HCC progression. Combination of sorafenib with AR inhibitors might represent a potential treatment for patients with advanced HCC. Hepatocellular carcinoma (HCC) is one of the poor-prognosis cancers ,2. In JaTo surpass the treatment with sorafenib alone for advanced HCC, new treatments have been developed in recent years ,5,6. HisHCC is one of the male-dominant cancers . We and 2-terminal transactivation domain, DNA-binding domain (DBD), hinge region and ligand-binding domain (LBD).Androgens act through AR, a 110-kDa ligand-inducible nuclear receptor A 14]. T. T14]. TAR regulates the expression of target genes that have androgen response elements (AREs) A 14,15],1514,15]AR co-regulators also influence a number of functional properties of AR, including ligand selectivity and DNA binding capacity . OncogenThe activation of Src kinase is involved in the ligand-independent activation of AR . Two UDPHuman HCC and normal liver express AR ,10,25. HIncreased expression of variant transcripts from the AR gene (ARVs) has been shown to be involved in the development of castration-resistant prostate cancer . The expAt present, sorafenib is the only approved drug for systemic chemotherapy of HCC. We observed that sorafenib-induced apoptosis was enhanced by the inhibition of AR and GRP78 in human hepatoma cell lines . SorafenWe have already reviewed clinical trials targeting androgen in HCC . However


Stroke is one of leading diseases causing adult death and disability worldwide. Home-based telerehabilitation has become a novel approach for stroke patients as effective as conventional rehabilitation, and more convenient and economical than conventional rehabilitation. However, there is no study assessing the mechanism of home-based telerehabilitation in promoting motor recovery among stroke patients with hemiplegic.This study is designed to determine the efficacy and explore the mechanism of motor recovery after home-based telerehabilitation in stroke patients with motor deficits.In a single-blinded randomized controlled pilot study, patients with acute subcortical stroke (n = 40) are assigned to receive home-based telerehabilitation or conventional rehabilitation. Task-based or resting-state functional magnetic resonance imaging (rs-fMRI), diffusion tensor imaging (DTI), and Fugl–Meyer assessment (FMA) score will acquired before and after rehabilitation. Activation volume of bilateral primary motor (M1), supplementary motor area (SMA), premotor cortex (PMC); lateralization index (LI) of interhemispheric M1, SMA, and PMC; functional connectivity of bilateral M1, SMA, PMC; fractional anisotropy (FA) will be measured; correlation analyses will be performed between neuroimaging biomarkers and FMA score pre- and postrehabilitation.We present a study design and rationale to explore the effectiveness and neural mechanism of home-based rehabilitation for stroke patients with motor deficits. The study limitations related to the small-amount sample. Moreover, home-based rehabilitation may provide an alternative means of recovery for stroke patients. Ultimately, results of this trial will help to understand the neural mechanism of home-based telerehabilitation among stroke patients with hand movement disorder. Furthermore, the therapeutic effects of rehabilitation treatment in stroke patients are poor because of relative high costs of traditional rehabilitation and the difficulty in adhering to rehabilitation treatment on account of the fatigue of commuting rehabilitation training institution and family. Telerehabilitation could facilitate the access to rehabilitation treatment, no matter where and when, improving patients’ compliance and chances of recovery.,10Stroke is characterized by its high morbidity, disability, and mortality rate, which brings serious damage on human health. Longitudinal task-based fMRI measurements could provide qualitative, quantitative, and dynamic information about conditions of remaining neurons during the process of recovery, including monitoring activation state of each related cerebral cortex qualitatively and dynamically, as well as measuring activation volume of motor area quantitatively and dynamically. Resting-state functional magnetic resonance imaging (rs-fMRI) is widely applied to investigate integration network of brain function. It could dynamically reflect the changes of brain motor function network connection. Diffusion tensor imaging (DTI) is a noninvasive method used to study microstructure, providing dynamic information about the recovery state of lesioned brain tissue qualitatively and quantitatively.Task-based functional magnetic resonance imaging provides an effective approach to studying brain active region.–18 To further explore the mechanisms of home-based telerehabilitation in improving neurological functions of stroke patients, our study investigated dynamic changes of motor activated region, changes of motor functional connectivity, and structural changes of white matter fiber in patients with motor deficits by means of task-based fMRI, rs-fMRI, and DTI.Our previous research and other numbers of studies have shown that acute stroke patients undertaking home-based rehabilitation could achieve equal effects compared with traditional rehabilitation, in terms of effectiveness of neurologic function improvement, degree of stress relief of caregivers and safety of rehabilitation implementation.We designed this study to provide more robost evidence of the application of home-based rehabilitation on patients of stroke with motor deficits.1.11.1.1The trial is an assessor blinded, paralleled randomized controlled trial (RCT). The overall flowchart is shown in Figure 1.2This trial follows the principal ethical guidelines for conducting clinical trials of Helsinki Declaration and has received approval from the Independence Ethics Committee of the Fifth Peoples’ Hospital of Shanghai, Fudan University  and has been registered in the Chinese Clinical Trial Registry (ChiCTR-IPR-17011757). Any issues related to clinical study safety will be reported to the Ethics Committee in time, such as the changes of clinical protocols or patient information. Participants will be acquainted with informed consent and asked to sign for it before being recruited.1.31.3.1In this pilot study, we aim to collect 40 patients diagnosed as stroke from the Department of Neurology, Shanghai the Fifth People's Hospital, Fudan University. Patients included in the study will be randomly allocated into 2 groups in a ratio of 1:1 after baseline assessment: home-based rehabilitation group and conventional rehabilitation group. An independent statistician will conduct randomized allocation by using a computer. The former will receive exercise training rehabilitation and electromyography-triggered neuromuscular stimulation with the assistance of caregivers at home. Specialist physicians will evaluate the recovery condition of patients via telerehabilitation system every week and give guidance on rehabilitation training and electro-stimulation therapy. The latter will receive the same rehabilitation training under the guide of specialist physicians face to face. Specialist physicians will evaluate the recovery condition of patients at outpatient clinic every week and adjust the rehabilitation strategies. Allocation concealment will be guaranteed, since allocation information will be protected in opaque sealed envelopes by a specially assigned person who is not involved in the study.1.3.2In this study, a specific device during home-based telerehabilitation will be adopted; the patients and home-based rehabilitation therapists cannot be blinded. To ensure the research quality, the assessors and data analysts are totally blind to allocation and rehabilitation intervention until they finish all the data collection and analyses.1.41.4.11.First-ever stroke and in stable condition after acute management of ischemic stroke.2.Aged between 35 and 85, male or female.3.Responsible focuses of the unilateral white matter area indicated by cranial CT or MRI.4.No more than 14 days from stroke onset, with unilateral limb movement dysfunction and be proved to be right handed after standardized testing.5.National Institute of Health Stroke Scale (NIHSS) scores from 2 to 20, Grading Muscle Strength of paralyzed upper limbs between grade 1 and grade 4, and have not received any standard rehabilitation intervention before inclusion.6.Sign the informed consent by the patient himself or voluntarily.1.4.21.Glasgow Coma Scale (GSC) scores less than 15, have dementia indicated by Minimum Mental State Examination (MMSE) assessment, have mental disorders and be unable to cooperate with examination or treatment.2.Motorial and sensory disturbance not induced by stroke or induced by previous ischemic stroke.3.Serious comorbidity like malignant tumor, primary cardiac, hepatic, renal or hematopoietic system diseases.4.History of cognitive disorder, psychiatric disorders, drug abuse and alcoholism.5.Infections or ruptures on the skin of forearms or legs.6.Have a heart pacemaker, metal stent, plate or an implant that is susceptible to the current pulse in the body.7.Be pregnant or lactant or have birth plans recently.8.Be long-term musical conductors or keyboard operators before stroke onset.9.Have claustrophobia.10.Be unable to finish basic course, or be hard to maintain compliance and follow-up.1.5 Rehabilitation therapies include exercise rehabilitation training and electromyography-triggered neuromuscular stimulation (ETNS).Intervention measures were described in detail in our previous studies.1.5.1All patients will receive Brunnstrom motor function grading evaluation by the same rehabilitation therapist after discharged from hospital. Individualized rehabilitation training plans will be made based on specific conditions of each patient, including physical therapy (PT) and occupational therapy (OT). Patients will be given corresponding treatment based on the Brunnstrom stages. Patients receive the designated exercise rehabilitation for 45 minutes, once a day for 5 days a week for 3 months.1.5.2ETNS is a novel method developed from the combination of myoelectric biofeedback technology and neuromuscular electrical stimulation (NMES), which has outstanding features compared to NMES. It can increase stroke patients’ initiatives in taking rehabilitation training and effectively improve motor function of the affected limb and ability of daily living, so it has becomes a new therapeutic option in treating paralyzed patients. Patients in both groups will receive ETNS 20 minutes each time, twice a day for 5 days a week for 3 months. The MyoNet-AOW biofeedback equipment used in our study has been approved by China Food and Drug Administration (Shanghai food drug supervision character 2010 no. 221114).1.61.6.1M1+SMA+PMC); lateralization index (LI) of interhemispheric M1, SMA, and PMC (LIM1+SMA+PMC)(LI = (ΣVC − ΣVI)/(ΣVC+ ΣVI), subscripts C and I refer to contralateral and ipsilateral hemisphere responses, respectively. The ΣV measures the regional activation level associated with the activated voxel); functional connectivity of bilateral M1, SMA, PMC, and the value of fractional anisotropy (FA) in corticospinal tract (CST) at ipsilesional posterior limb of internal capsule. Data will be collected before (at basement) and after (3 months) rehabilitation.Primary outcomes will be conducted before and after rehabilitation therapies, including sum of bilateral activated volume of primary motor (M1), supplementary motor area (SMA), premotor cortex (PMC) (V1.6.2Fugl–Meyer assessment (FMA) score will be assessed as secondary outcome. The FMA score consists of 17 items, ranging from 0 to 34, with lower scores demonstrating poorer movement function.1.73, 21 axial slices. During rs-fMRI scan, patients will be asked to keep away with the eyes closed and think of nothing. The motor task scans used a block design paradigm consisting of alternating 30-seconds periods of performance of an isometric precision grip task and rest for 4 minutes. DTI images will be acquired with the following parameters: matrix size = 94 mm × 94 mm, field of view = 240 mm × 240 mm, 2.6 mm slice thickness, 56 slices. We acquired 1 set of 30 diffusion weighted images with a b-value = 1000 s/mm2, and 5 no diffusion weighted images.Data will be acquired by a Siemens 3.0T Magnetom Trio scanner . Subjects will receive 2 MRI sessions (1 before and 1 after rehabilitation therapy). Each session included 1 rs-fMRI scan, 1 task functional scan and 1 DTI scan. For fMRI scan, parameters are TR/TE/FA = 1500 ms/30 ms/90°, 200 time points, resolution = 3.4 × 3.4 × 5 mmAll data will be analyzed using Functional MRI of the Brain (FMRIB's) Software Library (FSL) tools. Functional data sets will undergo slice timing correction, motion correction, scaling to percent signal change, smoothed with a Gaussian kernel of 8 mm full-width-at-half maximum, bandpass temporal filtering (0.01–0.08 Hz), and grand mean intensity normalized. A whole brain analysis of white matter integrity between groups will be performed to investigating FA. FA quantifies the degree to which water diffusion will be restricted and calculated based on the diffusion tensor. The data will be corrected for head motion and eddy current distortion, and brain extracted using Brian Extraction Tool. FA images will be then created by fitting the data to a tensor model at each voxel using DTI fit within the FMRIB's Diffusion Toolbox. Lesion will be masked out in each subject's data so that all analyses are performed on nonlesioned data.1.8,21 The early involvement of the SMA in the process of stroke recovery and correlation of initial activation of the SMA with motor recovery were demonstrated.,23 PMC might influence other cortical areas in the surviving motor network to support residual motor output and higher order processes required for motor function. A 6-mm radius sphere will be placed at the above 3 pairs of seeds to generate the regions of interest (ROI).We choose three pairs of seeds in sensorimotor network, the bilateral primary motor (M1), supplementary motor area (SMA), and premotor cortex (PMC). Hand movement is most closely related to M1.Lesions will be drawn in each patients’ structural space on the T1-weighted images using the co-registered T2-weighted diffusion images as a reference for lesion extension. The lesion mask is nonlinearly registered into standard space using FMRIB's Non-linear Registration Tool (FNIRT) with an affine transformation (12 degrees of freedom). The lesion mask will be also used during the registration steps of the structural, resting state fMRI, and DTI data so that voxels in the lesioned area would be excluded from the normalization procedure.1.9t-test or independent sample rank test will be used for grouped comparison of quantitative data. Pearson correlation coefficients (r) will be used to determine whether patients’ functional connectivity correlated with their FMA score and be computed after application of Fisher's r-to-z transform to yield variates that are approximately normally distributed (z = 0.5 ln[(1+r)/(1−r)]). All the statistical analysis will be carried out by the SPSS software . For DTI data, statistical analyses will be performed using nonparametric permutation testing (Randomize in FSL) with 5000 Monte Carlo simulations. We looked for differences in FA between the groups accounting for age, time of stroke onset, and NIHSS as regressors of no interest in the general linear model.Data analysis will be handled by independent statisticians who are blind to the groups. The statistical tests will be 2-side and at the 5% significance level. Means and standard deviation will be used for statistical description of continuous variables. For fMRI data, independent sample 1.10The researchers will receive specific training about protocols before the implementation of the study. They will try their best to make the subjects have a thorough understanding of the trial requirements, so that the subjects could cooperate better during the trial. The related rehabilitation equipment and examinations should be free to the subjects. The medication compliance of the subjects will be supervised by researchers. Superiors appointed by the sponsor will inspect and visit the process of the study at regular intervals to check the original data to ensure its accordance with the content on the case report form (CRF).1.11Adverse events are unavoidable in the course of rehabilitation treatment. Conditions may appear in few patients during the treatment period, such as unintentional injury, rupture and bleeding of skin, muscle soreness, joint pain, and skin rash. Any adverse events (AE) during the course of rehabilitation treatment should be recorded about time of occurrence, severity, relationship of duration with intervention, and prognosis, which should be recorded in the specific CRF. Researchers should take necessary measures to deal with all AE to ensure the safety of subjects and follow-up the subjects until their physical conditions come back to normal level.1.12Results of the research will be published in peer-reviewed journals and the publications will be on “open access” terms, with datasets will be accessible to the research investigators and statistical assessors. The research results will also be disseminated to the participants, stakeholders, and policymakers . The investigators will be responsible for the publication of the research to share results with broader scientific community, regardless of the magnitude or direction of effect.2We present a study design and rationale to explore the effectiveness and neural mechanism of home-based rehabilitation for stroke patients with motor deficits based on fMRI and DTI imaging. A total of 40 stroke patients will be eligibly enrolled in this clinical trial. The primary outcomes involve neural biomarkers; the secondary outcome is FMA to assess motor function; correlation between functional connectivity and motor function will also be calculated. Home-based telerehabilitation may provide an alternative means of recovery for stroke patients. Results of this trial will help to understand the neural mechanism of home-based telerehabilitation among stroke patients with hand movement disorders.3CCR conceptualized the study, and drafted and revised the manuscript. JC, MLL, and DLS drafted and revised the manuscript, and YJ contributed to the rehabilitation therapies, TRW contributed to the neuroimage methods and statistical analysis. All authors revised the manuscript and contributed to the writing.


The most striking feature of molecular apocrine breast cancer (MABC) is the expression of androgen receptor (AR). We report here the mechanism of the AR in regulating the behavior of MABC.The MABC cell line, MDA-MB-453, and the nonMABC cell line, MCF7, were used in this study. The effect of dihydrotestosterone (DHT) and heat shock protein 27 (HSP27) on cell proliferation was quantified using the cell counter kit-8 (CCK8) and clonogenic assays in vitro and by a xenograft tumor model in vivo. The expression of the AR and HSP27 was analyzed using western blot, qPCR, and immunofluorescence assays. Complexes of the AR and HSP27 were detected by co-immunoprecipitation (Co-IP).HSP27 knock-down decreased the proliferative ability of MDA-MB-453 cells, which could be rescued by DHT, while HSP27 and DHT had synergistic effects on MCF7 cells. HSP27 phosphorylation was a prerequisite for AR translocation into the nucleus, especially phosphorylation on serine 82. In addition, DHT stimulated the tumorigenic and metastatic capacities of MDA-MB-453 cells, while HSP27 knock-down decreased the rate of tumor formation and induced apoptosis in cells.In MDA-MB-453 cells, DHT promoted cell proliferation and stimulated AR and HSP27 translocation from the cytoplasm to the nucleus, whereas, it inhibited MCF7 cell growth, and only the AR translocated into the nucleus. The results suggest that HSP27 assists the AR in regulating the malignant behavior of MABC, and these findings might be helpful in the treatment of MABC.The online version of this article (10.1186/s13046-018-0762-y) contains supplementary material, which is available to authorized users. As a member of the nuclear receptor family, the androgen receptor (AR) plays an important role in breast cancer, and has been identified as a biomarker for a specific molecular subtype of breast cancer. The AR gene expression profile can be used for further classifying receptor-negative tumors as molecular apocrine breast cancer (MABC) . MDA-MB-In the absence of ligand, the AR remains in a non-active state, which forms a protein complex with heat shock proteins (HSPs) and other co-chaperones in prostate cancer, while the AR becomes active when ligand binds, and it translocates to the nucleus as a transcriptional regulator . Based oHSP27 were used to analyze the location of the AR and HSP27 in vitro, and their effects on the tumorigenic capacity of MABC cells in vivo. The results of this study could determine the mechanism of the AR in regulating the malignant potential of MABC. Additionally, it aims to explore potential therapeutic targets for patients with MABC.To detect the specific mechanism of AR cytoplasmic and nuclear translocation in MABC cells, androgen and siRNAs specific for 2.Because the MDA-MB-453 cell line is classified as a model of MABC , and MCFHSP27 siRNAs and control plasmids were constructed by Genechem (China). Three target sequences for the HSP27 siRNAs were studied, which included siRNA#4892-1: 5′-CTGTGAGGACTGTGGATAA-3′, siRNA#4893-1: 5′-CCCAGCAAATCCCTCTCTA-3′ and siRNA#4894-2: 5′-GGCAAGTTCCAGGCATTT-3′.The E. coli, and then extracted by using the Endotoxin-free Plasmid Size Kit .The deletion of HSP27 phosphorylation sites  were carried out by GeneCopoeia , respectively, and incubated for 5 min at 37 °C. Further, the FuGENE Transfection Reagent  was added and mixed. After incubating for 15 min, the complex solution was added to the cells, and replaced with complete medium 8 h later. The reactions were incubated for 48 h.Cell transfections were performed as follows: firstly, cells were seeded in 6-well plates at a density of 1.0 × 10GAPDH was adopted as an internal control, and the primers used were as follows: GAPDH: 5′-GGAAGGTGAAGGTCGGAGTC-3′ and 5′-GTCTTCTGGGTGGCAGTGAT-3′; AR: 5′-GGAATTCCTGTGCATGAAA-3′ and 5′-CGAAGTTCATCAAAGAATT-3′; HSP27: 5′- GCGTGTCCCTGGATGTCAAC-3′ and 5′-TGTATTTCCGCGTGAAGCAC-3′. Each sample was assayed in triplicate.The RNAs used in this study were extracted using Trizol reagent . Reverse transcription was carried out using the SuperScript RT kit . qPCRs were performed according to the manufacturer’s protocol using the SYBR Green PCR kit . The transcript level of Total proteins were extracted with RIPA buffer  and 1 mM PMSF. Cytoplasmic and nuclear subcellular fractionation was performed according to the manufacturer’s instructions using the Nuclear and Cytoplasmic Isolation Kit . All proteins were separated on 10% SDS-PAGE  gels, transferred onto PVDF membranes , and then blocked using 5% skim milk. The proteins were detected by incubating the following primary antibodies: anti-AR , anti-estrogen receptor , anti-progesterone receptor , anti-HSP27 , anti-HSP27 (phospho S15) , anti-HSP27 (phospho S78) , anti-HSP27 (phospho S82) , anti-β-actin , and anti-Histone H3  overnight; and incubated with horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG, followed by detection using the ECL detection kit . Each sample was analyzed in triplicate.Co-IP of the AR and HSP27 was carried out according to the manufacturer’s protocol using the Pierce Co-IP kit . Briefly, AR or HSP27 antibody (10 μL) was first incubated with the AminoLink Plus coupling resin. The antibody-coupled resin was incubated with protein lysates overnight. Subsequently, the resin was washed, followed by elution of the protein complexes, which bound to the AR or HSP27 antibody. Subsequently, the samples were detected by western blot using the HSP27 or AR antibody as described previously. Each sample was assayed in triplicate.HSP27 knock-down) were suspended and seeded in 96-well plates at 5000 cells per well and incubated for 24 h. Further, 10 μL of CCK8  solution was added into each well on day 1, 2, 3, 4 and 5. After 1–4 h, the absorbance of each well was measured at 450 nm using a microplate reader. Clonogenic assays were carried out using 6-well plates with 1000 cells per well. After 15 days, cells were collected and stained with crystal violet, and then the number of cell colonies was counted. Each sample was assayed in triplicate.Cells  was used to stain the nucleus. Images were visualized and analyzed using a fluorescence microscope. Each sample was analyzed in triplicate.HSP27 knock-down, and the control group. Cells (2 × 106) were inoculated subcutaneously in the groin. Care and procedures of the mice were provided by the Institution of Animal Use and Care Committee of Tianjin Medical University Cancer Institute and Hospital. All mice were sacrificed until 55 days. Tumor volumes were calculated as previously reported [Eighteen female BALB/c-nude mice  were purchased and randomly divided into three groups: MDA-MB-453 cells with DHT treatment, reported .All the tumors, livers, and lungs were paraffin-embedded and stained with hematoxylin-eosin (HE). HSP27 and Ki67 expression of mouse tumors were analyzed by immunohistochemistry as previously reported . In ordeP < 0.05 was considered as statistically significant.Statistical analyses were performed using SPSS 19.0 software. The data were recorded as means ± standard deviation from at least three independent experiments, and analyzed by one-way ANOVAs and T-tests. Two breast cancer cell lines were used in this study, MDA-MB-453 and MCF7, which are representative of MABC and nonMABC, respectively. Cells were tested by western blot to determine the expression levels of ER, PR, and AR. MDA-MB-453 cells demonstrated no detectable levels of ER or PR, but high levels of AR, while all of these proteins could be detected in MCF7 cells. However, the level of AR protein was 1.73-fold higher in MDA-MB-453 than in MCF7 cells Fig. .Fig. 1DH− 6, 10− 7, and 10− 8 M) for 24, 48, or 72 h. CCK8 assays showed that the effects of DHT were dose and time dependent, as the growth inhibitory effects could work best at a concentration of 10− 8 M and simultaneously for 48 h  could affect the proliferation of breast cancer cells, MCF7 cells were cultured in 96-well plates in medium containing 10% dextran-coated charcoal-treated FBS (DCC-FBS) incubated with or without different concentrations of DHT , the phosphorylated forms of HSP27 after DHT treatment were analyzed. Interestingly, the expression of Ser82 significantly increased, while the others remained unchanged Fig. . The chaate Fig. .Fig. 2DHFurthermore, the protein levels of the AR and HSP27 in the cytoplasm and nucleus after subcellular fractionation were analyzed. In MDA-MB-453 cells, the levels of both the AR and HSP27 were significantly increased in the nucleus and decreased in the cytoplasm by DHT treatment, while only the level of the AR increased in the nucleus and decreased in the cytoplasm in MCF7 cells Fig. . In addiTo further confirm the mechanisms of DHT-induced AR and HSP27 translocation from the cytoplasm to the nucleus, the interaction between HSP27 and AR was analyzed by Co-IP methods. Firstly, total proteins were extracted from MDA-MB-453 and MCF7 cells treated with DHT. The results indicated that the AR could be detected in HSP27 immunoprecipitated complexes, and vice versa Fig. . SubsequHSP27 were examined to explore the effect of HSP27 on AR expression. qPCR and western blot assays determined that HSP27 siRNA#4893–1 could clearly decrease the expression of HSP27 compared with the others . When cells knocked-down for HSP27 were treated with DHT again, the number of colonies increased (75%). For MCF7 cells, HSP27 knock-down decreased the number of colonies, almost to the same extent as with DHT , and the number was much lower when cells were treated with both HSP27 siRNAs and DHT  were divided into three groups and injected into the mammary fat pad of mice. DHT treatment accelerated tumor growth significantly, while HSP27 knock-down decreased the tumor formation rate compared to the control group (83.3% vs. 100%) and inhibited tumor volume significantly  staining, no obvious difference was found in the tissue structure and cell morphology of xenograft tumors generated from these three groups; however, necrosis could be found in the oup Fig. . In addioup Fig. .. The results showed that HSP27 phosphorylation induced by androgen played a vital role in the process of AR translocation from the cytoplasm to the nucleus, which could affect the proliferation of tumor cells and tumorigenic capacity.Breast cancer is a heterogeneous tumor that can be reflected in different aspects, such as the classical histopathological features and the more modern molecular classification. MABC is defined as ER-negative, PR-negative, and AR-positive ; furtherHSP27 could reduce the androgen promotion effect on MDA-MB-453 cells. Additionally, HSP27 can decrease the toxic effects of oxidized proteins and reduce reactive oxygen species, which results in inhibiting cancer cell apoptosis [As a model of MABC, the proliferation of MDA-MB-453 cells is regulated by androgen in an AR-dependent manner –15. In Mpoptosis –20. ThesHSP27 knock-down significantly decreased the level of AR protein, but no difference could be found in the level of AR mRNA, which suggests that HSP27 might only regulate the protein translation of AR in MABC cells. However, Stope et al. [HSP27 is a member of the HSPs family and has been stated to regulate the functions of the AR, such as AR cytoplasmic/nuclear translocation and AR transactivation , 21. Stue et al.  stated tIn malignant tumor cells, the expression of HSP27 is obviously high , 25. TheSeveral studies have confirmed that the positive expression of AR is significantly associated with poor survival, and increased mortality in AR-positive triple negative breast cancer (TNBC) , 29–31. In conclusion, this study confirmed that activation of AR and HSP27 by androgen could increase the proliferative ability of MABC cells and the growth of xenograft tumors. HSP27 phosphorylation on residue serine 82, induced by androgen, played a critical role in the process of AR translocation from the cytoplasm to the nucleus. The AR and HSP27 could form a protein complex, which was the main factor in AR regulating the malignancy behavior of tumor cells, and could present a new therapeutic regimen in clinical therapies of MABC.Additional file 1:The sequence of the deleted HSP27 phosphorylation sites. (DOCX 13 kb)


Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) is a validated treatment target for the treatment of metastatic castration-resistant prostate cancer (CRPC). Abiraterone acetate (AA) inhibits both 17α-hydroxylase (hydroxylase) and 17,20-lyase (lyase) reactions catalyzed by CYP17A1 and thus depletes androgen biosynthesis. However, coadministration of prednisone is required to suppress the mineralocorticoid excess and cortisol depletion that result from hydroxylase inhibition. VT-464, a nonsteroidal small molecule, selectively inhibits CYP17A1 lyase and therefore does not require prednisone supplementation. Administration of VT-464 in a metastatic CRPC patient presenting with high tumoral expression of both androgen receptor (AR) and CYP17A1, showed significant reduction in the level of both dehydroepiandrosterone (DHEA) and serum PSA. Treatment of a CRPC patient-derived xenograft, MDA-PCa-133 expressing H874Y AR mutant with VT-464, reduced the increase in tumor volume in castrate male mice more than twice as much as the vehicle (P < 0.05). Mass spectrometry analysis of post-treatment xenograft tumor tissues showed that VT-464 significantly decreased intratumoral androgens but not cortisol. VT-464 also reduced AR signaling more effectively than abiraterone in cultured PCa cells expressing T877A AR mutant. Collectively, this study suggests that VT-464 therapy can effectively treat CRPC and be used in precision medicine based on androgen receptor mutation status. Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1), a key enzyme for sex steroid biosynthesis expressed primarily in the testes and adrenal gland, is a validated target for treatment of metastatic castration-resistant prostate cancer (CRPC). Recent experimental evidence in preclinical and clinical settings has demonstrated that expression of both androgen receptor (AR) and CYP17A1 predicts the active intracrine androgen signaling that drives CRPC1Abiraterone acetate  in combination with prednisone is approved for treatment of CRPC. AA specifically and irreversibly inhibits both CYP17A1 hydroxylase and lyase126in vitro CYP17A1 enzyme assay showed that VT-464 lyase inhibition was 10-fold as potent as hydroxylase inhibition; in contrast, abiraterone inhibited hydroxylase 6-fold more potently than lyaseA selective inhibitor of CYP17A1 lyase has the potential to improve the side effect profile of the AA-prednisone therapy. In this study, we investigated VT-464, a small-molecule, CYP17A1 inhibitor that, as recently reported, was optimized for lyase selectivity and oral activityThe objective of the current study was to assess the effects of VT-464 on a CRPC patient as well as two different experimental models, in a CRPC patient-derived xenograft (PDX) tumor in mice and in a prostate cancer cell line in culture. In the model experiments, the effects of VT-464 were compared to those of abiraterone or its orally available acetate form.Prostate biopsies identified adenocarcinoma with a Gleason score 8 (4 + 4) in the left peripheral prostate lobe of a 68 year old man in 2002. The patient was treated with LHRH antagonist, Leuprolide, and androgen receptor (AR) antagonist Bicalutamide to January 2012 followed by 3 months of somatostatin analog, Lanreotide, due to rising PSA. Computed tomography scans confirmed metastasis to bone and VT-464 therapy (300 mg twice a day) without prednisone was initiated. Immunohistochemistry analysis of the prostate biopsies showed expression of high level of both AR and CYP17A1 . MeasureThe impact of oral VT-464 and AA on growth of MDA-PCa-133 prostate cancer PDX tumors was determined in castrate mice. The MDA-PCa-133 PDX tumor, which expresses an AR with a H874Y mutation within the ligand-binding domain and secretes PSA, was initially established and maintained as subcutaneous tumors in noncastrated SCID male mice. At passage 5, the xenograft tumor tissue was implanted subcutaneously in castrate male SCID mice, in which the tumor was established and maintained. The rates of tumor growth and serum PSA increases were very similar in noncastrate and castrate mice . ExpressNKX3.1. This indicates the induction of adaptive androgen signaling due to depletion of intratumoral androgen, as previously described9Castrate mice bearing MDA-PCa-133 tumors were treated with oral VT-464, AA, or vehicle for 28 days. Oral VT-464 or AA at 100 mg/kg twice-daily significantly reduced tumor growth (P < 0.05) ,B. AnalyTo determine the impact of VT-464 on AR signaling, we measured the effects of VT-464 on a AR reporter construct, FKBP51-ARE-luciferase, which is activated in the presence of the synthetic androgen 1nM R1881 in C4-2B prostate cancer cells . For comPSA and NKX3.1, stimulated expression of AR, and did not affect expression of UBE2C. Abiraterone treatment at the same concentrations resulted in very similar effects, except that VT-464 treatment displayed a stronger inhibition of PSA and NKX3.1 gene expression. These experiments showed that VT-464 treatment specifically inhibited AR-stimulated PSA and NKX3.1 gene expression in C4-2B cells. The inhibition of NKX3.1 gene expression by 5 μΜ VT-464 or 5  μΜ abiraterone was abrogated by the addition of 10 nM DHT, demonstrating that the direct or indirect (resulting from the inhibition of androgen biosynthesis) AR-inhibitory effects of VT-464 that lead to NKX3.1 suppression are negated by exogenous DHT  activity with overexpression of wild-type AR, two mutant ARs,  and AR-V11It was previously shown that the oral, nonsteroidal, small-molecule CYP17 lyase inhibitor VT-464, reduced circulating androgens in animal models thereby not promoting the steroid changes that can increase AR signaling and that contribute to abiraterone resistance, without disrupting the mineralocorticoid or glucocorticoid steroidogenic pathwaysAnalysis of blood plasma of VT-464 treated patient demonstrated VT-464 effectively inhibited adrenal androgen production but not cortisol during entire 40 months of treatment. The treatment also decreased PSA for 30 months but the last 10 months of treatment a slight increase in PSA levels was observed, indicating development of treatment resistant tumors. Because of very limited amount biopsy samples, we were unable to analyze status of AR or its variants. Thus we analyzed effectiveness of VT-464 in two different CRPC models.in vivo.In agreement with other studies of patient-derived xenografts, MDA-PCa-133 displayed similar complex tumor adaptation characteristics of those that occur in the absence of circulating testosterone, including increased expression of CYP17A1, wild-type full length AR and its ligand-independent variant AR-V7, and a gain-of-function mutation in the full-length AR ligand-binding domain sequence (H874Y)1014VT-464 strongly inhibited androgen-dependent reporter activity in C4-2B cells, which express a full-length AR that harbors the gain-of-function mutation T877A within the ligand-binding domain. The increased inhibition of the luciferase reporter by VT-464 compared to abiraterone was observed in the presence of the synthetic androgen R1881. Both VT-464 and abiraterone also inhibited the expression of the AR-mediated transcripts PSA and NKX3.1 as well as NKX3.1 protein and stimulated transcription of the AR gene. Neither VT-464 nor abiraterone altered the expression of UBE2C, which is activated by AR in a ligand-independent mannerThe stimulation of AR gene expression by VT-464 or abiraterone is likely due to derepression of the AR gene at a lower level of androgen in C4-2B cells. Similarly, addition of DHT repressed AR gene expression in C4-2B cells. A previous report identified an androgen-dependent enhancer located in the second intron of the AR gene, which mediates repression of AR gene expression at a higher level of androgen through recruitment of the lysine-specific demethylase 1 repressor complexThe intracellular suppression of exogenous DHT stimulation in C4-2B cells strongly suggests that VT-464 had AR antagonist activity. A recent published study compared the AR antagonist and agonist effects of VT-464 to those of abiraterone and the nonsteroidal inhibitor TAK-70019The same medicinal chemistry rationale that was applied to the design of VT-464 as a selective CYP17A1 lyase inhibitor can be used to explain the wild-type AR and mutant AR binding activity of VT-464The AR-dependent reporter activity in PC3 cells, which does not express AR was stimulated by transient overexpression of wild-type AR and two mutant full length AR with respect to the vector plasmid control. This analysis at best can compare the effects of Abi and VT-464 with each other and the three AR receptors. The activation of reporter activity by wild-type AR or H874Y mutant AR was not inhibited by VT-464. The activity of H874Y is modestly inhibited by abiraterone. In contrast, the activity of the T877A mutant was inhibited more potently by VT-464 than by abiraterone, suggesting VT-464 antagonist selectivity for the T877A AR mutant. The relative activity in DMSO control (without VT-464 or Abi treatment) in the three assays differs possibly due to differential activation of the ARE reporter by AR or AR mutants.Activation of the reporter by the H874Y and T877A AR could occur possibly due to activation by intracellular promiscuous ligands, as noted previously11191522Altogether, our clinical and preclinical study provided insight into the effect of VT-464 on CRPC tumor growth, the steroid metabolome, and androgen signaling.Abiraterone and AA were prepared at Viamet Pharmaceuticals as described previously24PC3 prostate cancer cells were obtained from the American Type Culture Collection, and C4-2B prostate cancer cells were obtained from The University of Texas MD Anderson Cancer Center. Both PC3 and C4-2B cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin. Cell authentication and mycoplasma screening were performed according to institutional guidelines. Cells were transfected using FuGene 6 reagent (Roche) according to the manufacturer’s instructions.FKBP51-ARE-luciferase [FKBP51-(−3)], a reporter construct under control of androgen-responsive enhancer of the FKBP51 gene was kindly provided by Dr. Jorma J. Palvimo, University of Eastern Finland262) were implanted subcutaneously in the flank 10 days postcastration, and the mice were monitored weekly for tumor growth. The xenograft tumors were then maintained by serial passaging in the castrate mice.MDA-PCa-133-4 (MDA-PCa-133) prostate cancer xenograft tumors were derived from bone lesions of a patient with CRPC as previously described2829303. The mice were then randomly divided into three treatment groups (n = 4 or n = 5): control vehicle (0.5% carboxymethylcellulose), VT-464 (100 mg in vehicle/kg body weight/twice daily), and AA (100 mg in vehicle/kg body weight/twice daily). Both drugs were administered by oral gavage daily for 25 days. Tumor dimensions were measured with calipers twice weekly to calculate tumor volume using formula width2 × length/2. At day 25, the mice were euthanized, and the tumors were harvested, cut into small pieces, and snap-frozen in liquid nitrogen. The tumor tissues were used to prepare protein lysates for immunoblot analysis and also for steroid profile analysis by mass spectrometry. Tumor volume ratios were calculated by dividing the tumor volume measured on days 4–25 of treatment by the predose tumor volume (day 0). Tumor volume ratios were compared between treatment groups at each time point using a two-tailed, two-sample equal variance Student t-test. P ≤ 0.05 was considered significant. All animal experiments were conducted in accordance with accepted standards of animal care and were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center.To determine the impact of VT-464 and AA treatment, CRPC tumor pieces were implanted subcutaneously in the flank in castrate male SCID mice. The tumors were allowed to grow until they reached about 200 mmProstate specific antigen (PSA) levels were measured using Beckman Coulter Access Immunoassay System at HS Q Squared Solutions . Steroid levels, dehydroepiandrosterone (DHEA), DHEA sulphate, testosterone, androstenedione, progesterone, pregnenolone, cortisol, corticosterone, and ACTH were quantified using triple quadrupole mass spectrometry at Quest Diagnostics-Nichols Institute . The institutional review board approved this study, which was conducted according to the Declaration of Helsinki, the International Conference on Harmonization, and the Guidelines for Good Clinical Practice. All experimental protocols were approved by the institutional review board of The University of Texas MD Anderson cancer Center and The University of Athens. Written informed consent had been obtained from the patient for treatment and sample acquisition according to the approved protocol.Further experimental methods are described in detail in How to cite this article: Maity, S. N. et al. Targeting of CYP17 Lyase by VT-464 Inhibits Adrenal and Intratumoral Androgen Biosynthesis and Tumor Growth of Castration Resistant Prostate Cancer. Sci. Rep.6, 35354; doi: 10.1038/srep35354 (2016).


The role of long non-coding RNAs (lncRNAs) in colorectal cancer (CRC) progression has not fully been elucidated. This study was designed to report the identification of a novel lncRNA, lnc-GNAT1-1, and its functional role in CRC progression.lncRNA expression profile microarray was performed in three paired primary and liver metastatic tissues of CRC, and a novel lncRNA, lnc-GNAT1-1, was identified to be a potential functional lncRNA. Quantitative real-time PCR was used to detect its expression in CRC tissues, cell lines, and patients’ plasma, cell fractionation was used to evaluate its subcellular location. lnc-GNAT1-1 was knockdown by siRNA or overexpressed by a lentivirus vector, then in vitro an vivo experiments were performed to evaluate its biological role and the underlying mechanisms in CRC.Expression of lnc-GNAT1-1 was decreased in liver metastasis than the primary tumor, while the later one is lower than the paired normal mucosa. Decreased lnc-GNAT1-1 expression was associated unfavorable clinicopathological features and a poor prognosis of CRC patients. In multivariate analysis, lnc-GNAT1-1 was proved to be an independent prognostic factor. In plasma, lnc-GNAT1-1 was significant decreased in CRC patients than healthy donors, and with the TNM stages advanced, the plasma lnc-GNAT1-1 level decreased; Receiver operating characteristic curve (ROC curve) showed that plasma lnc-GNAT1-1 had a moderate to well diagnostic efficiency for CRC. In vitro experiments showed that knockdown of lnc-GNAT1-1 could inhibit the aggressive phenotypes of CRC cell lines. In vivo study showed that overexpression of lnc-GNAT1-1 could suppress the liver metastasis of CRC cells. Finally, we explored the underlying mechanism of the role lnc-GNAT1-1 plays in CRC, and found a positive correlation between lnc-GNAT1-1 and Raf kinase inhibitor protein (RKIP) expression both in cells and in patients’ tissues. We further found that lnc-GNAT1-1 could regulate the RKIP-NF-κB-Snail circuit in CRC.We have demonstrated in this study that a novel lncRNA, lnc-GNAT1-1, is low expressed in colorectal cancer tissues and plasma, and acts as a tumor suppressor through regulating RKIP-NF-κB-Snail circuit.The online version of this article (doi:10.1186/s13046-016-0467-z) contains supplementary material, which is available to authorized users. Colorectal cancer is one of the leading cause of cancer-related death around the world, and causes over a half million deaths every year . DespiteLong non-coding RNAs (lncRNAs) are greater than 200 nucleotides without protein-coding potential. Recent studies have shown that lncRNAs play a crucial role in diverse biological processes, including alternative splicing, nuclear import, imprinting, cell differentiation and RNA decay . The dysTo explore the role lncRNAs played in CRC liver metastasis process, we performed lncRNA microarray using the “LncRNA + mRNA Human Gene Expression Microarray V4.0”  according to standard protocol in our unpublished study. Three pairs of colorectal cancer and liver metastasis tissues were tested. Among the differentially expressed lncRNAs, lnc-GNAT1-1 was significantly low expressed in the liver metastasis tissues than the primary tumor site. It is a sense-overlapping long non-coding RNA located on Chromosome 3 with a total of 11 transcripts, of which four transcripts of lnc-GNAT1-1 were significantly decreased in the metastatic tumor tissue, which indicated that lnc-GNAT1-1 might have participated in the liver metastasis processes of CRC cancer.However, to the best of our knowledge, the biological functions and the roles lnc-GNAT1-1 plays in cancer has not been reported previously. Hence, in this study, we sought to determine the expression and the biological function of lnc-GNAT1-1 in CRC, especially its role in liver metastasis. Expression levels of lnc-GNAT1-1 were determined not only in the CRC primary tumor and liver metastasis tissues, but also in the plasma of CRC patients, and its correlations with clinicopathological parameters were analyzed. We further studied the influence of lnc-GNAT1-1 on the aggressive phenotypes of CRC cell lines in vitro and in vivo. The regulatory role of lnc-GNAT1-1 on RKIP were also explored to elucidate the potential mechanisms. We have demonstrated that a novel lncRNA, lnc-GNAT1-1, played a vital role in the progression of CRC.Sixty-eight CRC patients who were diagnosed and underwent surgery in Peking University People’s Hospital between 2007 and 2015 were included in this study. Fresh colorectal tumor tissues and matched normal colorectal mucosa tissues were obtained from all the 68 patients. Liver metastatic tumor tissues were obtained from 18 of the 68 patients. The specimens were obtained and immediately frozen in liquid nitrogen and stored at −80 °C until RNA or protein extraction. Plasma samples from another 62 CRC patients were collected just before surgery between 2014 and 2016. None of them received any antineoplastic treatment. Plasma from 37 healthy donors with matched age were collected as controls.The human CRC cell lines SW480, SW620, HT29, LoVo, HCT116, and RKO cells were purchased from the American Type Culture Collection  and subcultured and preserved by our lab. Cells were maintained at 37 °C with 5% CO2 in Dulbecco’s Modified Essential Medium (DMEM) medium supplemented with 10% fetal bovine serum , 100 U/mL penicillin , and 100 μg/mL streptomycin (Sigma-Aldrich).Total RNA from cell lines and tissue samples was extracted using Trizol (Invitrogen) according to the manufacturer’s instructions. For the plasma, the total RNAs were extracted using mirVanaTM PARISTM microRNA extraction kit (ABI) according to the manufacturer’s instructions. For lncRNA quantification, GAPDH was used as internal control, and PrimeScriptTM RT Master Mix (TAKARA) were used for reverse transcription and real-time PCR. The primer sequences were as follows: lnc-GNAT1-1 forward: 5′-ATGTGTCCCCAGGTTCCTGTT-3′, lnc-GNAT1-1 reverse: 5′-CCCCTGAGGACTTGAGTAGC-3′; RKIP forward: 5′-GGAACGGGGAGTGTACCAAG-3′, RKIP reverse: 5′-CCATCTGGTCGTAATCTTGAAGG-3′. GAPDH forward: 5′- GCAAGAGCACAAGAGGAAGA-3′, GAPDH reverse: 5′-ACTGTGAGGAGGGGAGATTC-3′. All reactions were performed in triplicate. The fold change for each gene relative to the control group was calculated using the 2 − ΔΔCt method.Total protein extracts were separated by Trisglycine polyacrylamide gels and transferred to polyvinylidene fluoride membranes . Membranes were incubated with primary antibodies followed by horseradish peroxidase-labeled secondary antibody. GAPDH was used as a loading control. The antibodies used in the experiments are shown in Additional file The separation of nuclear and cytosolic fractions was carried out using the PARIS Kit (Life Technologies) according to the manufacturer’s instructions. For quantification PCR, GAPDH and U6 were used as used as fractionation indicators. Primer sequence for U6 was: Forward: 5′- CTCGCTTCGGCAGCACA-3′.For in vitro assays, to interfere the expression of lnc-GNAT1-1, siRNA interference sequences targeting Lnc-GNAT1-1 were designed and synthetized , and a final concentration of 50nM were used for transient transfection; to overexpress lnc-GNAT1-1, full-length human lnc-GNAT1-1 cDNA was cloned into the pcDNA3.1 expression vector . Lipofectamine 3000  was used for transfection according to manufacturer’s instructions.For in vivo studies, lnc-GNAT1-1 overexpression cell line was used. The lnc-GNAT1-1 gene was cloned in to a lentivirus vector LV-GFP-Puro, and RKO cells were used for infection. Stable transfection cells were established by puromycin antibiotic selection applied for 3 days, with a concentration of 5ug/ml. The lnc-GNAT1-1 overexpression cells and control cells were named RKO-LV-lnc-GNAT1-1 and RKO-LV-NC, respectively.Cell proliferation assay was performed with Cell Counting Kit-8 (CCK8) according to the manufacturer’s protocol, and detected at 0, 24, 48, 72 and 96 h. Cells in each group were tested for 5 replicates.For colony formation assay, transfected cells were seeded into each well of a 6-well plate on day 0 then incubated for another 12d. Then, the wells were fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet. The form colonies were counted and analyzed by Image J software.Cells were cultured in 6-wells plate until the cell density reached more than 95% confluence. Then, a vertical wound was scratched using a 100ul microtip. The cells were washed with phosphate-buffered saline (PBS) twice to remove the cell debris. Images were captured at 0 and 24 h or 36 h to assess wound closure.For cell cycle analysis, cells were stained with propidium iodide (PI) solution (BD Cycletest™ Plus DNA Kit) and were analyzed by a fluorescence-activated cell sorter (FACS) 48 h after transfection. For apoptosis analysis, 48 h after transfection, cells were stained with BD FITC Annexin V Apoptosis Detection Kit I followed by FACS analysis according to the manufacturer’s protocol.5) in serum-free media were placed into the upper chamber to do migration assays (without Martrigel) and invasion assays . Media containing 20% FBS was placed in the lower chamber as a chemoattractant. After 48 h incubation, the non-invading cells were removed with cotton swabs. Migrated or invasive cells at the bottom of the membrane were stained with methanol 0.1% crystal violet and images were captured under the microscope. The number of the cells were counted by Image J software.Cell invasion assay was performed using Corning Polycarbonate Membrane Insertin transwell chamber . After transfection for 24 h, cells  staining.Stably transfected cells were washed twice and resuspended in 1 × Hank’s buffer at a concentration of 2 × 10t-test (2-tailed). Paired qPCR relative expression results were compared with Wilcoxon Signed Ranks test. The Chi-squared test (χ2 test) was used to evaluate the relationship between the clinicopathological features and lnc-GNAT1-1 expression. Kaplan–Meier analysis and Cox regression analysis was used for survival analysis. For all, P values of <0.05 were considered statistically significant.SPSS 16.0 software  was used to perform the statistical analysis. Data were presented as mean ± SD from at least three separate experiments. Comparison between groups was made using the Student’s unpaired P < 0.001, Fig. As mentioned above, we previously conducted lncRNA microarray and explored the global expression profiles of lncRNAs in colorectal cancer primary tissues and liver metastatic tissues. Among the 98 differentially expressed lncRNAs transcripts, we noticed that four transcripts of lncRNA lnc-GNAT1-1  were remarkably higher in primary CRC tissues than the liver metastatic tissues, with an average fold change of 42.81. To verify this result, we determinate the expression of lnc-GNAT1-1 in 18 paired CRC primary and liver metastasis tissues. Results showed that lnc-GNAT1-1 was significantly decreased in liver metastatic tissues compared with primary tumor Fig. . We furtp = 0.012), depth of tumor invasion (p = 0.028), distant metastasis (p = 0.022), and tumor stage (p = 0.027). While no significant correlation was detected with other clinicopathological features, such as age, gender, tumor size, and differentiation.Based on the real-time PCR results of the tissues, we divided all the 68 patients into high expression group and low expression group according to the median expression level Fig. . As showp = 0.001). In univariate Cox regression analysis of OS, lnc-GNAT1-1 expression, lymphovascular invasion, depth of tumor invasion, lymph node metastasis, distant metastasis and tumor stage were identified as prognostic indicators, while in multivariate Cox regression analysis, only lnc-GNAT1-1 expression (p = 0.046), lymph node metastasis (p = 0.031) and distant metastasis (p = 0.044) could be served as independent prognostic factors  of CRC patients. The patients in the high lnc-GNAT1-1 expression group has a significant longer OS than those in the low expression group , with an average fold change of 2.25. Especially, after stratification by TNM stages, plasma lnc-GNAT1-1 level showed a decreased trend with the stages advanced. In our analysis, stage I and stage II cases were grouped together because of limited Stage I cases. The mean relative expression of plasma lnc-GNAT1-1 level in stage I+ II, stage III and stage IV patients were 0.56, 0.25, 0.10, respectively  CRC patients and 94.59% (35/37) healthy controls. The plasma level of lnc-GNAT1-1 was significantly higher in healthy controls than in CRC patients  and accelerated cell cycle  when compared with si-NC cells  was 88.0% Fig. . In vitrlls Fig. . Wound hlls Fig. . Finallylls Fig. .Fig. 3SuP < 0.001, Fig. P < 0.001, Fig. To verify the above results, we further overexpressed lnc-GNAT1-1 in LoVo cells using the pcDNA3.1 plasmid vector. Our results showed that when lnc-GNAT1-1 was overexpressed, the proliferation, colony formation and wound healing abilities of LoVo cells were inhibited Fig. , and f. Next, we evaluated the effect of lnc-GNAT1-1 on CRC liver metastasis process in vivo through injecting lnc-GNAT1-1 overexpression cells (RKO-LV-lnc-GNAT1-1) as well as negative control cells (RKO-LV-NC) into spleens of female nude mice. Seven weeks after injection, the mice were sacrificed, and the formed metastatic tumors on the liver were examined. In LV-lnc-GNAT1-1 group, all four mice formed primary tumors in spleens, as well as metastatic tumors in livers. In contrast, in the control group, only two mice formed tumors in the spleen, and liver metastasis was detected only in one of the above two mice Fig. . The resP value <0.05. A total of 22 genes were identified to be significantly co-expressed with lnc-GNAT1-1, of which, RKIP gene has the highest correlation coefficient (0.9977). Previous studies have shown that RKIP played a tumor suppressor role in cancers [R = 0.645, P < 0.001, Fig. We further explored the underlying mechanism of the role lnc-GNAT1-1 played in CRC cancer biology. Based on our microarray data, the mRNA and lncRNA co-expression analysis was performed, and significant correlation was defined as correlation > 0.99 or correlation < −0.99, and  cancers –8, inclu cancers , through cancers –13. So iLncRNAs have attracted immense research interests from researchers worldwide in recent years. Based upon our previous lncRNA microarray data, we identified a novel lncRNA, lnc-GNAT1-1, which was significantly low expressed in liver metastatic tissues than the primary CRC tumors. Therefore, we explored the role of lnc-GNAT1-1 in CRC in this study. We found that decreased expression of lnc-GNAT1-1 in cancer tissues was associated with lymphovascular invasion, depth of tumor invasion, distant metastasis, tumor stage, as well as OS of the patients; it could be served as an independent prognostic factor for CRC patients. What’s more, our in vitro and in vivo experiments showed that inhibition of lnc-GNAT1-1 could promote the malignant phenotypes of CRC cells, while overexpression of lnc-GNAT1-1 generate an opposite effect. Thus, our data confirmed the hypothesis that as a novel lncRNA, lnc-GNAT1-1 exerts tumor suppressive activity to inhibit cell proliferation and metastasis in colorectal cancer. The underlying molecular mechanisms might be attributed to its regulation on the RKIP -NF-κB-Snail circuit, which had been demonstrated in this study. Especially, as one of the most investigated transcription factors, NF-κB has been proved to regulate multiple cellular processes in cancer, including proliferation, invasion, metastasis, angiogenesis, and chemoresistance , 15.The role lncRNA played in biology of CRC has been studied in recent years, while studies that focused on CRC liver metastasis were limited. To our knowledge, only one previous study was designed to explore the lncRNAs related with liver metastasis of CRC. Ye and colleagues  performeAnother important finding of this study is that lnc-GNAT1-1 is decreased in plasma of CRC patients than in healthy controls, with an AUC of 0.720 in the ROC analysis, which indicates that lnc-GNAT1-1 has a moderate to well efficiency for the diagnosis of CRC. Recently, many studies have focused on lncRNAs as potential tumor markers for cancer diagnosis and prognosis for their stability and non-invasiveness. Zhou et al. evaluated the potential of 8 lncRNAs as diagnostic markers for gastric cancer, and finally confirmed that plasma H19 could serve as potential diagnostic biomarker for gastric cancer, in particular for early stage patients . In addiAlthough the detailed mechanisms that lncRNAs participate in cancer biology have yet to be elucidated, current studies have shown that the gene regulation role lncRNAs played is mainly through its interacting with proteins, especially nuclear proteins, because lncRNAs (including lnc-GNAT1-1), were predominantly located at the cell nucleus. Previous studies have shown a variety of interactions between lncRNAs and proteins, for instance, LOC389641 with E-cadherin , SNHG5 wIn conclusion, we have identified a novel lncRNA, lnc-GNAT1-1, which is decreased in CRC tissues and plasma and associated with higher overall survival of CRC patients. It could inhibit the aggressive phenotypes of CRC cells both in vitro and in vivo. We also showed in this study that lnc-GNAT1-1 could regulate the RKIP -NF-κB-Snail circuit in CRC cells. Our results have provide a new therapeutic target for CRC treatment.