Liver cancer is among the leading global healthcare issues associated with high morbidity and mortality. Liver cancer consists of hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), hepatoblastoma (HB), and several other rare tumors. Progression has been witnessed in understanding the interactions between etiological as well as environmental factors and the host in the development of liver cancers. However, the pathogenesis remains poorly understood, hampering the design of rational strategies aiding in preventing liver cancers. Accumulating evidence demonstrates that aberrant activation of the Wnt/β-catenin signaling pathway plays an important role in the initiation and progression of HCC, CCA, and HB. Targeting Wnt/β-catenin signaling potentiates a novel avenue for liver cancer treatment, which may benefit from the development of numerous small-molecule inhibitors and biologic agents in this field. In this review, we discuss the interaction between various etiological factors and components of Wnt/β-catenin signaling early in the precancerous lesion and the acquired mechanisms to further enhance Wnt/β-catenin signaling to promote robust cancer formation at later stages. Additionally, we shed light on current relevant inhibitors tested in liver cancers and provide future perspectives for preclinical and clinical liver cancer studies. Liver cancer is among the leading global healthcare issues associated with high morbidity and mortality. Primary liver cancers consist of hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and hepatoblastoma (HB). Other rare tumors are fibrolamellar carcinoma, hepatocellular adenoma, focal nodular hyperplasia, fibrosarcoma, angiosarcoma, leiomyosarcoma, and lymphoma.HCC is the fifth most frequent cancer worldwide and is closely associated with cancer-related deaths . HCC accCCA is the second most common liver cancer following HCC. CCA is a devastating malignancy with a dismal 5-year overall survival rate of less than 10%. Based on the anatomic location, CCAs are classified into intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) subtypes. iCCA associates better with cirrhosis resulting from HCV than HBV infection. pCCA is closely related to primary sclerosing cholangitis marked by chronic inflammation .HB is the most common pediatric liver malignancy and stems from hepatic progenitor cells that undergo malignant transformation during embryogenesis . AlthougWith the exception of HBs, most liver cancers take decades to facilitate the progression from the precancerous dysplasia of liver cells located in a chronic inflammatory microenvironment towards a malignant phenotype. During this process, the accumulation of aberrant genetic and epigenetic modifications leads to the dysregulation of signaling pathways, which in turn promote the initiation and progression of liver cancers.Aberrantly activated Wnt/β-catenin signaling plays a critical role in precancerous dysplasia as well as the malignant transformation of liver cells and malignant expansion of cancer cells. Here, we summarize the latest advances in our understanding of Wnt/β-catenin signaling in the course of liver cancer development and highlight the clinical implications of Wnt/β-catenin signaling pathway as a rational therapeutic target.The Wnt/β-catenin signaling pathway is evolutionarily conserved and required in both physiological and pathophysiological processes ,6,7. TheWnt/β-catenin signaling is inactive in the normal tissues of adults, except for some stem cell niches . This isOn the contrary, in the active condition Wnt ligands bind at the cysteine rich domain (CRD) of one of the frizzled (FZD) receptors and one of the LRP5/6 co-receptors. Next, the interaction between Wnt ligands and receptors recruits scaffolding proteins disheveled (DVL) and AXIN to the membrane. The AXIN proteins themselves are also under proteolytic control and are rapidly degraded following poly-ADP-ribosylation (PAR) by tankyrase (TNKS) enzymes ,14. The Aberrant activation of Wnt/β-catenin signaling exists at both hepatic precancerous lesions and cancerous foci. Uncovering how the signaling is deregulated in these precancerous lesions will be beneficial to better understand the mechanisms contributing to the progression of liver cancer. The most important event in precancerous lesions is the dysplasia and dysfunction of liver cells infected or affected by the given etiological factors. Thus, it is necessary to elucidate the interaction between these factors and components of Wnt/β-catenin signaling in these cells.HBV infection increases the risk of developing HCC about 100–200-fold . HBV is The hepatitis B surface antigen (HBsAg) regulates the expression of LEF-1, a key transcription factor of β-catenin in the nucleus ,25,26. ALINEs). The HBV genome inserted into a LINE1 element produces an oncogenic HBV-LINE1 chimeric transcript, inducing nuclear localization of β-catenin and triggering target gene expression [The integration of viral DNA into the host genome is a special event during HBV infection that occurs mostly near particular sites, e.g., the long interspersed nuclear elements , the viroporin p7, and the non-structural proteins . Different from HBV, as an RNA virus HCV lacks a DNA intermediate phase during its life cycle. Hence, HCV infection relies on the interaction of its viral proteins with the infected hepatocytes but not the damage to the host genome . CurrentCDH1 gene promoter [As the central component of HCV particles, the core protein is detectable in the cytoplasm, Golgi apparatus, lipid droplets, and nucleus ,32. Partpromoter , leadingAs a component of the HCV RNA replication complex, NS5A enhances the ability of HCV to counteract apoptosis . On the HCV structural E2 protein activates the Src homology region 2 domain-containing phosphatase-2 (SHP-2) , which pHCV enhances Wnt/β-catenin signaling independent of its proteins as well. HCV infection upregulates the expression of microRNA-155 (miR-155), which directly restrains APC expression, one of the major negative regulators in the destruction complex to regulate cytoplasmic β-catenin levels . AdditioFGF18 and FGF20 expression [Apparently, HCV proteins build a network consisting of a plethora of molecular events to stimulate Wnt/β-catenin signaling, which in turn further facilitates HCV infection. Firstly, the combination of Wnt1 and Wnt5a with FZD receptors leads to the release of soluble EGFR ligands , which bpression , which epression .However, whether and how HCV particles regulate Wnt/β-catenin signaling in the HCV-infected cholangiocytes is still unclear.Chronic alcohol abuse leads to alcoholic liver disease, which progresses from fatty liver through alcoholic hepatitis, hepatic fibrosis to cirrhosis, and ultimately HCC. A widely used in vivo model of chronic alcohol abuse is to feed adult male Long Evans rats with 37% ethanol for 8 weeks. In this model, nuclear and cytoplasmic expression of β-catenin was decreased in the liver, indicating that Wnt/β-catenin signaling is disrupted ,54. In lThe paradoxical conclusions in regards to the effect of chronic alcohol abuse on the Wnt/β-catenin signaling question the optimal model for alcoholic liver disease, especially the animal strains, ethanol concentration, as well as exposure time. For humans, it takes years to develop a fatty liver without abnormal physical findings, and decades to alcoholic hepatitis (often with concomitant cirrhosis). Short-term ethanol consumption may recapitulate the early stage of alcoholic liver disease, while the 4-month mouse models may coincide with late stage. Thus, Wnt/β-catenin signaling could play a different role from the early to late stages of alcoholic liver disease, which needs further investigation.Non-alcoholic fatty liver disease (NAFLD), featuring fat accumulation in hepatocytes, ranges from simple steatosis to non-alcoholic steatohepatitis (NASH). The latter is associated with inflammation and fibrosis and is a major risk factor for the onset and progression of HCC . From NAAberrant adipogenesis is the central event for NAFLD, which needs the transcription factor peroxisome proliferator-activated receptor γ (PPARγ). However, Wnt/β-catenin signaling inhibits PPARγ mRNA expression ,62. ThusAXIN2 promoter, and downregulation of microRNAs negatively regulating Wnt/β-catenin signaling [The increased burden of fat in hepatocytes, oxidative stress, and lipid peroxidation induces hepatic inflammation and fibrosis, exacerbating to NASH. During this process, Wnt/β-catenin signaling is restored by the overexpressed aortic carboxypeptidase-like protein (ACLP) that specifically binds FZD8 and LRP6 to form a ternary complex facilitating extracellular signaling transduction . Levels ignaling ,68.Aspergillus fungi, contaminating food supplies worldwide. Aflatoxin-B1 is the most toxic aflatoxin, and it has been well validated to dramatically increase the risk of HCC in humans and animals [TP53 and cooperates with HBV in causing p53 mutations in HCC [Aflatoxins are a class of carcinogenic mycotoxins produced by  animals . Aflatoxs in HCC . Additios in HCC ,72. Howes in HCC . The disAccordingly, most major etiological factors of liver cancers contribute to the activation of Wnt/β-catenin signaling in precancerous lesions through multiple mechanisms, although non-virus factors suppress its activity temporarily in the early stage of pathological damage . The eleCTNNB1 encoding β-catenin are detected in 15–25% of HCCs, producing mutated β-catenin escaping phosphorylation and subsequent degradation [AXIN1 are reported in 10.4% of HCCs, followed by AXIN2 and APC, which are mutated in 3.3% and 1.4%, respectively [CTNNB1 mutations show higher frequencies in HCV-related (26.7%) and non-viral HCCs (21.1%) than HBV-related HCCs (11.6%). Conversely, AXIN1 mutations are more frequent in HBV-related HCCs (18%) compared to HCV-related (14%) and non-viral HCCs (8%) [Around 40–70% of HCCs show β-catenin nuclear accumulation, augmenting Wnt/β-catenin signaling activity ,75,76,77radation ,79,81,82However, β-catenin nuclear accumulation is recently reported to be restricted to late-stage HCC. At earlier stages, β-catenin is primarily located at the plasma membrane in complexes with multiple cadherin family members, where it drives tumor cell survival by enhancing the signaling of growth factor receptors such as EGFR . This stAPC gene leads to the loss of APC protein [In addition, other genetic and epigenetic alterations in relevant genes enhance Wnt/β-catenin signaling in liver cancer cells. Wnt ligands are elevated by excessive secretion from liver cancer cells, infiltrating macrophages as well as other cell types within cancerous foci ,84. In c protein . TogetheCTNNB1 (1.5%), AXIN1 (4%), and APC (2%) [Aberrant activation of Wnt/β-catenin signaling is observed in the majority of CCA, which closely associates with tumor malignancy and patient outcome. Differently from HCC, mutations in related genes are less frequent in CCA, i.e., APC (2%) . ApparenAPC (2%) ,90. Wnt APC (2%) . AdditioAPC (2%) ,93. SimiAPC (2%) ,95. CTNNB1, including point mutation or deletions in exon 3 [APC (20.51%), AXIN1 (1.67%), AXIN2 (3.75%), and LGR6 (12.5%) [Activation of the Wnt/β-catenin signaling pathway is deemed an important hallmark during the development of hepatoblastomas . Around n exon 3 . Other m (12.5%) . TranscrDuring the decades from risk factor exposure to final liver cancer, liver cells acquire activation of Wnt/β-catenin signaling due to the molecular interaction between etiological factors and the host. Then these cells evolve into tumor cells with robust stimulation strengthened by genomic, epigenetic, and transcriptional alterations of the components involved in Wnt/β-catenin signaling, conferring proliferative advantage and thereby contributing to the progression of tumor formation .CTNNB1 mutation in pericentral hepatocytes leads to glutamine-dependent mTORC1 activation and show a notable therapeutic benefit of mTORC1 inhibition in CTNNB1 mutant HCC in vivo. This provides a novel way to specifically target this subset of HCCs [As outlined above, accumulating evidence validates the pivotal role of aberrant activation of Wnt/β-catenin signaling in the pathological process of liver cancers. It highlights potential benefits to targeting components involved in Wnt/β-catenin signaling for liver cancer treatment. During the last decades, a tremendous progression of exploring potent Wnt/β-catenin signaling inhibitors has been witnessed for various tumor types.  of HCCs .Investigations exploring mechanisms underlying hepatocarcinogenesis accumulating over the last decades enrich our understanding of liver cancer development. Together with enormous progression in clinical technology and treatment guidelines, these achievements improve early detection and enable a more efficient treatment design for liver cancer patients. Nevertheless, liver cancer remains a major cause of tumor-related deaths worldwide.Liver cancer patients show high phenotypic and molecular heterogeneity, contributing to the lack of a curative therapy. Correlation between phenotype and molecular characteristics is important for clinical practice in HCC patients, especially those diagnosed at an advanced stage depending on conservative treatment using small molecular inhibitors. In parallel to pathological classification, molecular profiles have been explored to categorize HCC. The group led by Jessica Zucman-Rossi proposed a distinct classification comprising six subtypes based on gene expression and have linked these subtypes to clinical and pathological classifications. Importantly, aberrant Wnt/β-catenin signaling activation is an outstanding biological feature in G5–G6, highlighting the predominant tumorigenic function of this pathway in these groups ,145,146.Due to its key role in HCC and other liver diseases, a large number of compounds inhibiting Wnt/β-catenin signaling have been explored. However, only a minority have been tested in liver cancer, as indicated in CTNNB1 or AXIN1 mutations. Apparently, Wnt/β-catenin signaling activity responds to these inhibitors differently depending on specific genetic defects. Despite a reduction in the signaling activity, it is not sufficient to significantly affect cell growth. These results highlight the importance of identifying the molecular features and genomic traits of liver cancer patients as well as the demand for a combined therapy to improve clinical treatment.Moreover, current studies of molecules targeting components involved in Wnt/β-catenin signaling rarely consider genetic mutation or related molecular alterations. In this regard, we tested the PORCN inhibitor  and tankWnt/β-catenin signaling is aberrantly stimulated during the course of liver cancer progression. The underlying mechanisms range from complicated regulation by etiological factors in precancerous lesions at early stages to acquired genomic, epigenetic and transcriptional alterations of the components involved in Wnt/β-catenin signaling in tumor foci at later stages. Therefore, targeting Wnt/β-catenin signaling potentiates a novel avenue to treat liver cancers as well as non-tumor liver diseases. However, application of molecular inhibitors targeting Wnt/β-catenin signaling for liver cancer treatment remains dim. A deeper study to explore the sensitive subgroups to these inhibitors carrying particular molecular features would aid in designing more efficient treatment strategies. Additionally, investigations targeting Wnt/β-catenin signaling in non-tumor liver diseases are still lacking, which demonstrate the need for further studies in this field.


The TOP/ FOP-Flash reporter assay and Western blotting showed SOX17 inhibited the activity of the Wnt/β-catenin signaling pathway in cervical cancer. Further, firefly luciferase reporter assay and quantitative chromatin immunoprecipitation (qChIP) assays confirmed that SOX17 trans-suppressed the expression of β-catenin by directly binding to the specific region of the β-catenin promoter. Together, our data demonstrated that SOX17 restrained the proliferation and tumor formation by down-regulating the activity of the Wnt/β-catenin signaling pathway via trans-suppression of β-catenin in cervical cancer.The SRY-box containing gene 17 (SOX17) is considered as a regulator in stemness maintenance and a suppressor in some malignant tumors. However, the biological function and molecular mechanism of SOX17 in the process of initiation and progression of cervical cancer remain obscure. In this study, immunohistochemistry showed that the expression of SOX17 was high in the normal cervix, moderate in the high-grade squamous intraepithelial lesion, and low in the cervical cancer. SOX17 inhibited the proliferation and viability of cervical cancer cells in vitro as well as tumor formation in vivo. Additionally, SOX17 induced the cell cycle arrest at the transition from the G According to the latest authoritative data, there were estimated 527,600 new cervical cancer cases and 265,700 deaths worldwide in 20122. Although high-risk human papillomavirus (HPV) is well established as the major risk factor for cervical cancer carcinogenesis3, most HPV infections are transient and cleared within months4. Furthermore, the genetic alterations and epigenetic modifications involved in the initiation and progression of cervical cancer have not been clearly elucidated yet5. Recently, extensive studies have shown that some stem cell self-renewal-associated transcription factors, such as SOX26, SOX97, NANOG8, KLF49, LGR510, UTF111, OCT412, and DAX113, are anomaly modulated and functionally alter signaling pathways during cervical cancer carcinogenesis.Cervical cancer is the fourth most common cancer in women and the seventh overall14. SOX17 plays a key role in the generation and maintenance of neonatal hematopoietic stem cells (HSCs)15 as well as in regulating the fate of human primordial germ cells (PGCs)16. In recent studies, SOX17 has been widely studied in cancers, such as breast cancer17, colorectal cancer18, hepatocellular carcinoma19, gastric cancer20, esophageal cancer21, cholangiocarcinoma22, endometrial cancer23 and cervical cancer24. However, the majority of these studies are mainly focused on the epigenetic alterations, implying that promoter hypermethylation of SOX17 may contribute to aberrant activation of Wnt/β-catenin signaling pathway27. As a transcription factor, the regulatory function of SOX17 on target genes at the transcriptional level contributing to tumorigenesis is insufficiently understood. Furthermore, the molecular mechanisms of SOX17 in cervical carcinoma initiation and progression are largely unknown.As a member of the SOX transcription factor family, SOX17 (SRY-box containing gene 17) has been considered a well-known endoderm markerThe present study demonstrated that SOX17 was down-regulated during the progression of cervical cancer and that SOX17 expression inhibited the proliferation, tumor formation and activity of the Wnt/β-catenin signaling pathway by directly binding to the promoter region of β-catenin in cervical cancer cells.2 in the specified media supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin.Five human cervical carcinoma cell lines  and SW480 (human colon cancer cell line) were purchased from the American Type Culture Collection . HeLa, SiHa and C-33A cells were cultured in high-glucose Dulbecco Modified Eagle Medium . CaSki and SW480 cells were cultured in RPMI1640 Medium . HT-3 cells were cultured in McCoy’s 5A Medium . All the cell lines were cultured at 37 °C in 5% COSurgical resection of 67 tumor samples from primary cervical cancer (CC) patients, 20 high-grade squamous intraepithelial lesion (HSIL) and 31 normal cervix (NC) samples obtained from the First Affiliated Hospital of Xi’an Jiaotong University between January 2008 and December 2016 were chosen for immunohistochemistry (IHC). The histology of all CC tissue samples was verified by surgical pathologists. The histological subtype and stage of the tumors were categorized according to the International Federation of Gynecology and Obstetrics (FIGO) classification. Eight normal cervix fresh tissues and eight cervical cancer fresh tissues were collected from the First Affiliated Hospital of Xi’an Jiaotong University for Western blot analysis.Immunostaining of formalin-fixed and paraffin‐embedded tissue was performed on 4 μm paraffin sections using antigen retrieval for 2 min in boiling 10 mM citrate buffer (pH 6.0). Cultured cells were seeded onto cover slips for 48 h and fixed in 4% paraformaldehyde (pH 7.4) at room temperature (RT) for 20 min. After washing three times in PBS, cells were permeabilized with 0.1% Triton X-100. Subsequently, cells or sections were exposed to the blocking solution (PBS/3% hydrogen peroxide) and incubated with primary antibodies overnight at 4 °C. After three washes in PBS, cells or sections were incubated with secondary HRP‐conjugated antibodies for 30 min at RT and counterstained with hematoxylin. As a negative control, PBS was used to replace the primary antibody. For immunostaining analysis, primary goat polyclonal anti-SOX17  and Ki67  were used.28. An overall score of 0 was defined as negative. A score of 1–4 was defined as weak positive, and a score of ≥4 was defined as strong positive. The examination and scoring of all tissues were performed by two investigators independently.A positive reaction was defined as the observation of a reddish-brown precipitate in the nucleus. The immunoreactivity of immuno-positive sample was determined by multiplying the intensity of the staining  and percentage of stained area Cultured cells were lysed in RIPA buffer , which was supplemented with a protease inhibitor. After incubation, lysates were cleared by centrifugation and then subjected to protein concentration assay . Tissue samples were homogenized in RIPA buffer supplemented with protease inhibitors using a blender. Lysates were subjected to SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk and then incubated with primary antibodies overnight at 4 °C. Immunoreactive bands were incubated with HRP‐conjugated secondary antibodies  for 1 h at RT and visualized with an enhanced chemiluminescence reagent . The membrane was then scanned with a protein-imprinting imaging system . The SOX17 Western blot results were normalized to those of GAPDH for quantification.Primary antibodies used were anti-SOX17 , anti-GAPDH , p-GSKβ , β-catenin , c-Myc , and cyclin D1 . Secondary antibodies used were either horseradish peroxidase-conjugated anti-goat or anti-mouse immunoglobulin G .4) were seeded in 6-well plates in triplicate and counted every 2 days for 1 week using a hemocytometer. Cell growth curves were generated to assess cell proliferation. For cell viability assays, cells were plated at a density of 2,000 cells per well and evaluated by 3--2,5-diphenyl tetrazolium bromide  dye for 7 days according to standard protocol. The number of viable cells was detected by measuring the absorbance at 490 nm. The experiment was repeated in three independent experiments.Cells (2 × 106) in the exponential growth phase were harvested and injected (100 μL per site) into the subcutis on the dorsum of each mouse. Tumors were measured in two dimensions by using manual calipers. Tumor volume was calculated using the following formula: V = 0.5 × length × width × width. Tumor volume was measured every 2–3 days.All animal experiments were conducted in accordance with the animal use guidelines from the Animal Care and Use Committee of the Medical School of Xi’an Jiaotong University. 6–7-week-old female BALB/c-nude mice  were housed in a specific pathogen free (SPF) room with constant temperature (22–25 °C) and humidity (40–50%). Cervical carcinoma cells (1 × 106) were harvested by trypsinization and fixed by 70% ethanol (−20 °C). Cells were then stained with solutions containing propidium iodide  and DNase-free RNase A . After 30 min of incubation, samples were washed and re-suspended in PBS. The samples were run on a FACS Calibur flow cytometer  using CellQuest software. All data were analyzed with FlowJo_V10 cytometer software.Cells  and stored at −20 °C before use. DNA was modified with EpiTect® Bisulfite Kit  according to the instructions. Briefly, 0.5 μg denatured DNA was treated with sodium bisulfite at 95 °C for 5 min, 60 °C for 25 min, 95  °C for 5 min, 60 °C for 85 min, 95 °C for 5 min and then 60 °C for 175 min. Samples were then applied to supplied columns, subjected to column clean up.Total RNA was isolated from cell lines by TRIzol reagent , and cDNA was synthesized using PrimeScript RT reagent Kit . Total cDNA was used as a template for PCR amplification with GAPDH as an internal control. Real-time quantitative PCR was performed in triplicate for each primer set and each cell sample using an Mx3000P QPCR System  and the SYBR Premix ExTaq II . Results were analyzed via the ∆∆Ct method using GAPDH as the housekeeping gene. The specific primers are listed in Table 18 and negative controls, respectively. The specific primers are selected from a previous reference29 and listed in Table TaKaRa EpiTaq™ HS DNA polymerase was used for methylation specific PCR. Thermocycling conditions used were one cycle at 94 for 5 min; 40 cycles at 98 for 10 s, 61 for 30 s, and 72 for 30 s; and final extension at 72 for 5 min. PCR products were loaded with 2% agarose gels followed by staining with ethidium bromide and directly visualized under UV illumination. SW480 methylated DNA and ultra-pure water were used as positiveFull-length SOX17 and β-catenin cDNA was amplified  with the vector bone of pGPU6/GFP/Neo was purchased from GenePharma Co., Ltd . The primers were described in Table Three HeLa-GFP cells and three HeLa-SOX17 cells were used for microarrays. Total RNA was extracted using TRIzol reagent . An Affymetrix PrimeView Human Gene Expression Array was used to investigate the changes in transcriptional profiles. The experiment was performed based on the manufacturer’s standard protocols. Genes with ≥1.5-fold change between two groups were identified as differentially expressed genes.Fragments of the β-catenin promoter (predicted from position −2000 bp to +44 bp), cyclin D1 promoter (predicted from position −2024 bp to +200 bp) and c-Myc promoter (predicted from position −1975 bp to +249 bp) were cloned into the pGL3-Basic Vector  to generate β-catenin, cyclin D1 and c-Myc promoter reporter constructs, respectively. All constructs were verified by sequencing. Cells were seeded in 24-well plates and transiently transfected with plasmids containing firefly luciferase reporters and recombinant promoter reporter constructs. The luciferase activity was measured after incubation for 48 h using the Dual Luciferase Assay kit . All experiments were performed as three independent experiments. The transfection efficiency was normalized with Renilla luciferase activity. The specific promoter activity was presented as the change in the experimental group vs. the control group. The primers and oligonucleotides are listed in Table g at 4°C to pellet cells. Cell pellets were sonicated to shear the chromatin to a manageable size. After sonication, chromatin–protein complexes were immunoprecipitated with 5 μg of anti-SOX17 antibodies  and 20 μL of fully resuspended protein A/G magnetic beads. For the negative control, 1 μg of normal mouse IgG was used. Beads were then washed two times with sonication buffer, and DNA was eluted in elution buffer. Cross-links were reversed overnight. RNA and protein were digested using RNase and Proteinase K, respectively, and DNA was purified with phenol chloroform extraction and ethanol precipitation. Real-time PCR was performed to amplify the regions of interest or internal negative control regions. Each sample was assayed in triplicate, and the fold enrichment ratio was calculated as the value of the ChIP sample vs. the corresponding input sample. Samples that yielded a two-fold enrichment or better were considered positive targets. The primers used for these studies are listed in Table Cervical carcinoma cells were subjected to ChIP using the EZ-Magna ChIP Assay kit . Cells were fixed in 1% formaldehyde solution and incubated at room temperature for 10 min followed by the addition of 2 mL of 10X glycine to each dish to quench the unused formaldehyde. Cells were washed twice with PBS and scraped into a microcentrifuge tube, and cells were then centrifuged at 800xTM database . Four datasets33 were identified using the search parameters “Gene: SOX17”, “Cancer Type: Cervical Cancer”, “Analysis Type: Cancer vs. Normal Analysis” and “Data Type: mRNA”. Then, the search parameters “Gene: SOX17”, “Analysis Type: Cancer vs. Normal Analysis”, “Data Type: mRNA” and “Dataset Size: 151+ samples” were used and the following studies were included: Lee Bladder34, The Cancer Genome Atlas (TCGA) Brain, TCGA Breast, TCGA Colorectal, Cui Gastric35, Chen Liver36, Hou Lung37, Agnelli Myeloma38, TCGA Ovarian, and Barretina Sarcoma39. A summary of SOX17 expression in multiple cancer types was generated.Meta-analysis of SOX17 in multiple cancers was performed using The Oncominet-test (two-tailed) and the Mann–Whitney U-test. The expression variance analysis of CESC was based on the Wilcox test. Meta-analyses were performed as described40. A p value of <0.05 was considered statistically significant. For comparison among groups, the Chi-square test or one-way ANOVA was performed.Statistical analysis was performed with SPSS 19.0 software . All data are expressed as the group means ± standard deviation of the mean (SD). The two-tailed Chi-square test or Fisher’s exact test was used to determine the significance of the differences between the covariates. A univariate analysis was analyzed by Student’s p > 0.05; NC vs. CC, p < 0.05; HSIL vs. CC, p < 0.05). Moreover, immunoreactivity score (IRS) of SOX17 staining revealed that SOX17 expression was significantly decreased from NC (5.16 ± 3.34) to HSIL (3.95 ± 3.79) and finally to CC (1.63 ± 2.50) . To further confirm the SOX17 expression results in cervical carcinogenesis, western blot analysis was performed to detect the SOX17 expression in eight randomly selected NC and eight CC fresh specimens . All the results indicated that SOX17 expression is negatively related to cervical carcinogenesis.The expression status of SOX17 was first evaluated in normal human cervix (NC), high-grade squamous intraepithelial lesion (HSIL) and cervical cancer (CC) samples by immunohistochemistry (IHC). Representative SOX17 staining was observed in the nucleus and/or cytoplasm of positive cells in various cervical tissues Fig. . It showens Fig. . The aveImmunocytochemical assays, RT-PCR and western blot analyses were performed in cervical cancer cell lines. It is revealed that SOX17 expression was found in cervical cancer cell lines Fig.  and S1A.p < 0.05) and lighter  after SOX17 overexpression. Similar results were found in the SiHa-SOX17 cells . Conversely, the tumors grew faster, larger  and heavier  after SOX17 knockdown in HeLa cells. Similar results were found in the SiHa-shSOX17 cells . All these results demonstrated that the SOX17 protein suppresses tumor formation of cervical cancer cells in vivo.To identify the effect of SOX17 on tumor formation of cervical cancer cells in vivo, SOX17-modified cells (left side) and control cells (right side) were subcutaneously inoculated into each posterior flank of the same female nude mouse at the same time. The palpable tumors formed by HeLa cells grew more slowly, smaller . In contrast, SOX17 knockdown induced the Ki67 staining and IRS of tumor tissues weaker and lower, respectively, . Similar results were found SiHa SOX17-overexpressing and knockdown cells . All these results indicated that SOX17 suppresses the tumor formation of cervical cancer cells in vivo by inhibiting the cell proliferative ability.To explore if  SOX17 suppresses the tumor formation of cervical cancer cells by inhibiting cell proliferation, the expression of Ki67 was evaluated in the tumor xenograft tissues by IHC. After SOX17 overexpression in HeLa cells, the tumors displayed a weaker Ki67 staining and lower IRS . In addition, MTT assays showed that SOX17 overexpression resulted in a significant decrease of cell viability in HeLa, C-33A and SiHa cells , and knockdown of SOX17 led to a marked increase of cell viability in HeLa and SiHa cells . These data suggested that SOX17 also suppresses the proliferation and viability of cervical cancer cells in vitro. Collectively, all these results indicated that SOX17 suppressed the tumor formation of cervical cancer cells by inhibiting the cell proliferative ability in vivo and in vitro.To verify if SOX17 inhibits proliferation of cervical cancer cells in vitro, cell growth curve assays and MTT assays were performed in vitro. As shown in Fig. 0/G1 phase  and a great decrease of S/G2/M phase  in the percentage of HeLa cells. Similar results were observed in C-33A  and SiHa cells . Conversely, SOX17 knockdown resulted in a marked decrease in the percentage of HeLa cells  and SiHa cells  in G0/G1 phase as well as an increase in the percentage of cells in S/G2/M phase . Collectively, these results indicated that SOX17 suppressed the proliferation of cervical cancer cells through arrest at the cell transition from G0/G1 phase to the S phase.To further investigate the mechanism by which SOX17 inhibits the proliferation of cervical cancer cells, FACS was performed for cell cycle analysis on SOX17-modified cervical cancer cells and control cells. As shown in Fig. 54. It showed that the mRNA levels of CTNNB, CCND1, and MYC  were significantly decreased in SOX17 overexpression cells and increased in SOX17 knockdown cells . These phenomena were also confirmed in the SOX17-modified SW480 cell lines , whereas SOX17 knockdown dramatically increased the activity in HeLa and SiHa cells . Also, these phenomena were observed in SW480 cell . All these data suggested that the activity of the Wnt/β-catenin signaling pathway is attenuated by SOX17 in cervical cancer cells.Although β-catenin, cyclin D1, and c-Myc were markedly inhibited by SOX17, p-GSK3β failed to show significant expression change. These data are insufficient to conclude that SOX17 suppresses the activity of the Wnt/β-catenin signaling pathway. The TOP/FOP Flash luciferase reporter assay was conducted to detect Wnt/β-catenin signaling activity. Compared to control cells, SOX17 overexpression led to a significant inhibition of TOP-Flash reporter activity in HeLa, SiHa and C-33A cells  and mRNA level . Simultaneously, SOX17-overexpressing cells transfected with pIRES2-AcGFP-CTNNB1 showed higher rate of growth and viability than cells transfected with empty vectors . As shown in Figure 0/G1 phase and increased the percentage in S/G2/M phase . These results suggested that β-catenin might be the key molecule by which SOX17 suppressed the proliferation of cervical cancer cells by inhibiting the activity of the Wnt/β-catenin signaling pathway.All of these three molecules, β-catenin, cyclin D1 and c-Myc, were down-regulated by SOX17 in cervical cancer cells, but β-catenin is the upstream molecule of cyclin D1 and c-Myc in Wnt/β-catenin signaling pathway. Thus, the recombinant β-catenin plasmid was transiently transfected into the SOX17-overexpressing cells to detect if the proliferation suppression of cervical cancer cells by SOX17 could be rescued. After transfection of pIRES2-AcGFP-CTNNB1, β-catenin, cyclin D1 and c-Myc expression levels were elevated in SOX17-overexpressing cells compared to controls at both protein level . These results supported that SOX17 acted as a negative regulator of β-catenin in clinical cervical cancer tissues.To validate the correlation between the expression of SOX17 and β-catenin in clinical cervical cancer specimens, their expression levels were detected by western blot Figure . The resp > 0.05; Figure S4B, p > 0.05). Next, six luciferase reporters were constructed containing β-catenin promoter fragments with different deletions between −2000 and +44 upstream of transcriptional start site. As shown in Fig. p < 0.05, p < 0.01; Fig. p < 0.001, p < 0.001). In the other promoter regions, including P3 (−1472 to +44), P4 (−1188 to +44), P5 (−888 to +44) and P6 (−484 to +44), the luciferase activities failed to show significant differences by SOX17 overexpression (p > 0.05). Simultaneously, the similar results were observed in SW480 cell . These results showed that the sequence between the nucleotides −1756 and −1473 in the β-catenin promoter may contain the SOX17-binding site.Our results above demonstrated that SOX17 inhibited the activity of Wnt/β-catenin signaling pathway through trans-suppressing the key molecule, β-catenin. Therefore, a dual-luciferase reporter assay was performed to identify if SOX17 directly bind the promoter of β-catenin in cervical cancer cells. The results showed that the transcriptional activity of cyclin D1 and c-Myc were not modulated in SOX17-overexpressing HeLa, SiHa and C-33A cell lines . The increased occupancy of SOX17 on S1 and S2 region of β-catenin promoter were also observed in the SW480 cell .Next, quantitative ChIP (qChIP) assays were performed to identify the chromatin occupancy of SOX17 on β-catenin promoter at the specific region (−1756 and −1473) in SOX17-overexpressing cells in vivo. RT- PCR using specific S1(−1756 bp to −1610 bp) and S2 (−1614 bp to −1463 bp) primers was performed to amplify the binding region. As shown in Fig. http://jaspar.genereg.net/). 5′-ATTGT-3′ was located between the nucleotides −1632 and −1618, in the S1 region of  P2 promoter. However, the enrichment of SOX17 was higher in S2 than S1 . A meta-analysis was performed across four studies of cervical cancer in ONCOMINETM database . Table p = 0.005). Moreover, a total of 365 studies on various cancer types were retrieved from the OncomineTM database . A systematic down-expression of SOX17 in multiple cancer types was observed in across the ten datasets interrogated .To identify the clinical implications of SOX17, the expression of SOX17 was analyzed in TCGA database. The results showed that SOX17 expression was significantly reduced in cervical cancer (Fig. e Figure , of whicTaken together, our findings suggested that SOX17 inhibited the tumor formation, proliferation and activity of the Wnt/β-catenin signaling pathway by directly binding to the region between the −1756 and −1473 nucleotides in the β-catenin promoter in cervical cancer cells.43, hematopoietic stem cells maintenance15, human primordial germ cell fate regulation16 and tumorigenesis22. In pancreatic tumors and glioblastomas, SOX17 has been established as a key regulator of inducing tumorigenesis and promoting tumor angiogenesis45. However, in colorectal cancer18, hepatocellular carcinoma19, gastric cancer20, cholangiocarcinoma22, breast cancer17, and endometrial cancer23, SOX17 acts as a tumor suppressor. In cervical cancer, five studies have reported the function of SOX1748. However, these studies mainly reported the phenomenon of high methylation in SOX17 promoter. Whether SOX17 participates in the process of initiation and progression of cervical cancer and how it contributes to the cervical carcinogenesis remain obscure. In the present study, IHC and western blot analysis revealed low expression of SOX17 expression in cervical cancer and the MSP assays showed high methylation of SOX17 promoter in cervical cancer cell lines, indicating that SOX17 might suppress the progression of cervical cancer. These results were in consistence with other studies in cervical cancer tissue48. Next, SOX17 was found to suppress tumor formation by inhibiting cell proliferation in vivo and in vitro through tumor xenograft, cell growth and cell viability assays. Furthermore, cell cycle analysis determined SOX17 induced cell cycle arrest at the cell transition from G0/G1 phase to S phase. This is the first study identified the SOX17 inhibited the proliferation and tumor formation by arresting the cells at cell cycle transition from G0/G1 phase to S phase in cervical cancer, in consistence with the results of another study in human oligodendroglioma49.SOX17 has been reported to be involved in definitive endoderm formation54. However, the majority of the studies did not elucidate the mechanism of SOX17 suppressing activity of Wnt/β-catenin signaling pathway51. Sinner et al55. and Chen et al49. reported the specific mechanism of SOX17 repressing the Wnt/β-catenin signaling pathway at the protein level in colon carcinoma and oligodendroglioma, respectively. They found that SOX17 interacted with both TCF4 and β-catenin proteins and formed a complex, mediating degradation of β-catenin and TCF4 by the proteasome independent of GSK3 activity.Several studies demonstrated that SOX17 inhibited the carcinogenesis by suppressing activity of the Wnt/β-catenin signaling pathway24. and van der Meide et al46. found that SOX17, together with some antagonists of WNT/β-catenin signaling pathway were highly methylated, implying an antagonism function of SOX17 in WNT/β-catenin signaling pathway. In the present study, the TOP/FOP flash luciferase reporter assay revealed that the Wnt/β-catenin signaling activity was attenuated by SOX17 in cervical cancer. Moreover, the expression of β-catenin, cyclin D1 and c-Myc, the key molecules of the Wnt/β-catenin pathway were attenuated by SOX17 at both mRNA and protein level. It seems the expression of β-catenin in microarray and RT-PCR analysis lacks concordance. It might be because the gene expression is affected by some factors, such as external environment, processing method, sampling time, etc. Also, the gene probes of microarray may detect a different region from that real-time PCR primers amplified. Next, the recombinant β-catenin plasmid was transiently transfected in the SOX17-overexpressing cells and the results showed that the proliferation suppression and cell cycle arrest of cervical cancer cells by SOX17 were rescued. Moreover, the mRNA and protein expression of cyclin D1 and c-Myc repressed by SOX17 were also elevated. Moreover, the negative correlation of SOX17 and β-catenin expression was observed in the clinical cervical cancer specimen. These results revealed that β-catenin might be the key molecule, by which SOX17 attenuate the activity of Wnt/β-catenin signaling pathway.In cervical cancer, Chen et alTo further explore if SOX17 could directly trans-suppress the transcription of β-catenin, the dual-luciferase assays were conducted and revealed that SOX17 directly bind to the promoter of β-catenin, instead of cyclin D1 or c-Myc, thus deactivate the β-catenin promoter. The qCHIP assays further confirmed SOX17 directly bind to the region between −1756 and −1473 of the β-catenin promoter. The predicted binding site of 5′-ATGCG-3′ was located between the nucleotides −1632 and −1618, in the S1 region of P2 promoter. However, there was also a higher enrichment of SOX17 in S2 region, indicating that there might be another site that SOX17 recognize and bind. Moreover, these phenomena were also validated in human colon cancer cell. Our study is the first study that elucidated the specific region of β-catenin promoter, to which SOX17 directly bind to trans-suppress the β-catenin, resulting in inactivation of the Wnt/β-catenin signaling pathway.Above all, we demonstrated that SOX17 directly bind to the β-catenin promoter at the region between −1756 and −1473 and trans-suppressed β-catenin, leading to the down-regulation of β-catenin target genes, such as cyclin D1 and c-Myc. Consequently, the activity of Wnt/β-catenin signaling pathway was attenuated, resulting in the inhibition of cell proliferation and tumor formation in cervical cancer cells (Fig. In conclusion, our findings firstly demonstrated that SOX17 expression was down-regulated in cervical cancer tissues, SOX17 inhibited the tumor formation, proliferation and suppressed the activity of Wnt/β-catenin signaling pathway by directly binding to the β-catenin promoter and trans-suppressing β-catenin in cervical cancer cells.Figure S1Figure S2Figure S3Figure S4Table S1Table S2Table S3Table S4Supplementary figure legends


The emerging evidence reveals that protein arginine methyltransferase 5 (PRMT5) is involved in regulation of tumour cell proliferation and cancer development. Nevertheless, the exact role of PRMT5 in human lung cancer cell proliferation and the underlying molecular mechanism remains largely obscure. Here, we showed that PRMT5 was highly expressed in human lung cancer cells and lung cancer tissues. Furthermore, we generated PRMT5 stable knockdown cell lines (A549 and H1299 cells) and explored the functions of PRMT5 in lung cancer cell proliferation. We found that the down‐regulation of PRMT5 by shRNA or the inhibition of PRMT5 by specific inhibitor GSK591 dramatically suppressed cyclin E1 and cyclin D1 expression and cell proliferation. Moreover, we uncovered that PRMT5 promoted lung cancer cell proliferation via regulation of Akt activation. PRMT5 was directly co‐localized and interacted with Akt, but not PTEN and mTOR. Down‐regulation or inhibition of PRMT5 markedly reduced Akt phosphorylation at Thr308 and Ser473, whereas the expression of PTEN and mTOR phosphorylation was unchanged, indicating that PRMT5 was an important upstream regulator of Akt and induced lung cancer cell proliferation. Altogether, our results indicate that PRMT5 promotes human lung cancer cell proliferation through direct interaction with Akt and regulation of Akt activity. Our findings also suggest that targeting PRMT5 may have therapeutic potential for treatment of human lung cancer. The NSCLC is the first leading cause of cancer death and the most frequently diagnosed cancer in China. Surgery, chemotherapy, radiotherapy and combination therapy are the main treatment options for lung cancer patients. Nevertheless, the therapeutic effects of those treatments are still poor and the mortality of NSCLC patients remains unacceptably high.Protein arginine methyltransferase 5 (PRMT5) is the type II arginine methyltransferase that catalyses the symmetrical dimethylation of protein substrates at the arginine residues. Recently, accumulating evidence has shown that PRMT5 is involved in a variety of biological processes via different signalling axis, such as modification of histones and gene expression, chromatin reconstruction, protein modification, cell cycle progression, cell invasion, cell metabolism and development.In this study, our results revealed that PRMT5 was overexpressed in human lung cancer cell lines and human lung cancer tissues. Stable knockdown of PRMT5 or treatment of PRMT5 specific inhibitor in A549 and H1299 cells dramatically suppressed cell proliferation and decreased cyclin E1 and D1 expression. Additionally, PRMT5 promoted lung cancer cell proliferation through direct interaction with Akt and regulation of Akt activity, but not interaction with PTEN and mTOR. Our findings suggest that PRMT5 is a key regulator for human lung cancer cell proliferation and identifying PRMT5 downstream targets will aid in illustrating the role of PRMT5 in human lung cancer development.22.12% and 95% air. PRMT5 specific inhibitor GSK591 (cat no. SML‐1751) was purchased from Sigma.Primary human foetal lung fibroblast cells  were cultured in RPMI1640 medium with 10% (v/v) foetal bovine serum . Human lung adenocarcinoma cells A549, H1299, H322, H441 and PC14 were used in this study. A549 cells were cultured in F12K medium/Dulbecco’s Modified Eagle’s Medium (1:1)  supplemented with 10% (v/v) FBS (Sigma) and the other cells were cultured in RPMI 1640 medium  with 10% FBS. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.2Human PRMT5 and Akt shRNA knockdown lentiviral constructs were generated using pLVTHM vector, and lentivirus were generated by co‐transfection along with packaging plasmid MD2G (Addgene) and helper plasmid PAX2 (Addgene) into HEK293T cells. After 48 hours, the medium was collected and viral titers were pre‐determined. In all indicated experiments, the same number of viral particles was used. Human PRMT5 knockdown targeting sequences are: shRNA‐1,5’‐GGATAAAGCTGTATGCTGT‐3’; shRNA2: 5’‐GCCATCTATAAATGTCTGCTA‐3’. Human Akt knockdown targeting sequences is: 5’‐CCCGAGGTGCTGGAGGACA‐3’.2.3A549 and H1299 cells were seeded in 6‐well plate for 24 hours before transduction. The cells were infected with lentivirus containing human PRMT5 shRNA or scramble shRNA for 48 hours and then the cells were selected with puromycin  and the non‐infected cells were killed. The stable depletion of PRMT5 cell lines was subjected to next experiments.2.4Total RNA was isolated from A549 and H1299 cells or human lung cancer tissues using TRIzol reagent  according to the manufacturer’s recommendation, and the equal amount of RNA was subjected to reverse transcription. Quantitative real‐time PCR (qRT‐PCR) was performed with SYBR green fluorescent Dye  with an ABI7800 PCR machine (Applied Biosystems), and GAPGH was used as an internal control. Relative mRNA expression was determined by the ΔΔ−Ct method. Primer sequences are: human PRMT5 forward: 5’‐CCTGTGGAGGTGAACACAGT‐3’ and revise: 5’‐AGAGGATGGGAAACCATGAG‐3’; GAPDH, forward: 5’‐GAAGGTGAAGGTCGGAGTCAACG‐3’ and revise: 5’‐TGCCATGGGTGGAATCATATTGG‐3’.2.5For cell proliferation assay, the A549 and H1299 cells were infected with lentivirus containing PRMT5 shRNA or scramble shRNA and seeded into 24‐well plates (5000 cells/plate). Cell proliferation was assessed by cell counting kit‐8  at the indicated time points according to the manufacturer’s protocol. Cell proliferation detection was determined by absorbance at 450 nm using an Infinite 200 plate reader . To evaluate the effect of PRMT5 inhibitor GSK591 on cell proliferation, A549 and H1299 cells were seeded into 96‐well plates (3000 cells/plate) and treated with vehicle and GSK591 at the indicated concentration and the cell proliferation was measured by CCK‐8.2.6Proteins were extracted from A549, H1299, H322, H441 PC14 and IMR90 cells and human lung cancer tissues using lysis buffer  and the extracted proteins were separated in SDS‐polyacrylamide gels and transferred to PVDF membranes. The membranes were washed twice for 5 minutes with PBST and incubated with the PRMT5 , cyclin E1 , cyclin D1 , Akt , p‐Thr308‐Akt , p‐Ser473‐Akt , GSK3β , p‐Ser9‐GSK3β , GSK3α , p‐Ser21‐GSKα , PTEN , p‐mTOR‐Ser2448 , total mTOR  and Tubulin  antibodies at 4°C overnight. After incubation, the membranes were labelled with goat anti‐mouse conjugated to HRP or goat anti‐rabbit conjugated to HRP secondary antibodies . Data were analysed using LI‐COR Image Studio Software .2.7To examine the endogenous interaction between PRMT5 and Akt, A549 and H1299 cells were seeded into 10 cm plates and infected with lentivirus containing PRMT5, Akt or scramble shRNA. Proteins were extracted from these cells using lysis buffer . The cell lysates were pre‐cleared with protein A or protein G beads at 4°C for 1 hour. Next, 5 µg IgG, PRMT5 and Akt antibody were added into the cell lysates and incubated at 4°C overnight. Subsequently, protein A or protein G beads were added into the cell lysates and incubated at 4°C for 3 hours and then the beads were washed three times with wash buffer . Immunoprecipitates were detected by Western blotting.2.8A549 and H1299 cells were cultured on glass cover slips and incubated overnight to establish adherence. The cells were fixed with 4% paraformaldehyde for 20 minutes at 37°C, followed by permeabilization in ice‐cold methanol for 10 minutes at −20°C. The cells were incubated in blocking buffer  for 1 hour at room temperature, followed by incubation with anti‐PRMT5 and anti‐p‐ser473‐Akt antibody (diluted 1:100 in blocking buffer) at 4°C overnight. Cells were washed twice for 5 minutes in PBS, and then incubated for 2 hours with Alexa Fluor 488‐conjugated goat anti‐rabbit secondary antibody  for p‐ser473‐Akt and Alexa Fluor 568‐conjugated goat anti‐mouse secondary antibody  for PRMT5 (diluted 1:500 in blocking buffer) at room temperature. Nuclei were stained with DAPI  for 10 minutes at room temperature before observation. The images were captured with Nikon Eclipse E600 fluorescence microscope.2.9A549 and H1299 cells were seeded into 10 cm culture dish and infected with lentivirus containing PRMT5 shRNA or scramble shRNA. After 48 hours, the cells were selected with puromycin and the stable depletion of PRMT5 cells were subjected to Akt kinase activity assay by Western blot analysis. The Akt kinase activity was detected by an Akt kinase assay kit (non‐radioactive) according to the manufacturer’s instructions .2.10t test. Difference with P < 0.05 was considered statistically significant.All experiments were carried out in triplicate under identical conditions and data were represented as means ± SEM Differences between two groups were analysed by unpaired two‐tailed Student’s 33.1To investigate the functions of PRMT5 in human lung cancer, we firstly examined the PRMT5 protein expression level in different human lung cancer cell lines. As shown in Figure 3.2To investigate whether PRMT5 is implicated in lung cancer cell proliferation, we delivered the PRMT5 and scramble shRNA into A549 and H1299 cells by lentivirus and generated PRMT5 stable knockdown cells. As shown in Figure Subsequently, cell proliferation rate was measured in these PRMT5 stable knockdown cells. As shown in Figure 3.3To further investigate the role of PRMT5 in lung cancer cell proliferation, we used PRTM5 specific inhibitor, GSK591, to black PRMT5 activity and cell proliferation was evaluated. We found that proliferation of A549 and H1299 cells was markedly reduced in a dose dependent manner, whereas proliferation of IMR90 cells was almost no change Figure A,B, indi3.4PRMT5 plays an important role in cancer cell growth and proliferation, especially in human lung cancer cells. However, the underlying molecular mechanism is completely unknown. Previous study has reported that PRMT5 up‐regulated cyclin‐dependent kinase and PI3K/Akt signalling pathway.3.5PI3K/Akt signalling pathway is essential for cell proliferation, especially in many human cancer types.Finally, we evaluated the PRMT5 expression level and Akt activity in human lung cancer tissues and adjacent normal tissues. As shown in Figure 4To date, it is clear that PRMT5 is an oncoprotein and plays a pivotal role in various human cancer progressions through the regulation of different signalling cascades. However, it is still unclear whether PRMT5 interacts with Akt, another important oncoprotein in human cancers, and regulates Akt activity in human lung cancer. The findings in this study showed that PRMT5 was overexpressed in human lung cancer cells Figure . This phPrevious study has reported that silencing PRMT5 arrested cell proliferation at G1 phase.Protein kinase B  is required for different cellular processes, from the regulation of cell cycle, survival and growth to control of cell metabolism.In summary, our results revealed that PRMT5 was frequently overexpressed in human lung cancer cells and tissues. Silencing PRMT5 or blocking PRMT5 activity prevented lung cancer cell growth and proliferation through induction of cell cycle arrest. Moreover, PRMT5 directly co‐localized and interacted with Akt but not PTEN or mTOR and down‐regulation of PRMT5 decreased Akt phosphorylation and kinase activity. These results strongly imply that PRMT5 may serve as a new therapeutic target for the treatment of human lung cancer. More importantly, the new insights into the activation of Akt medicated by PRMT5 provide much new information for the mechanisms of carcinogenic effect of PRMT5 in human lung cancer.All the authors declare that they have no conflict of interest.


The results showed that eCG up-regulated ovarian OCT4 expression. Meanwhile, OCT4 expression in granulosa cells was also up-regulated by FSH, and knockdown of OCT4 by siRNA significantly decreased FSH-induced cellular viability. Moreover, gonadotropin increased p-GSK3β (Glycogen synthase kinase 3-beta) level, β-catenin expression and its translocation to nuclear in ovarian cells. In addition, the inhibition of GSK3β activity by CT99021 significantly increased the expression of β-catenin and OCT4 in granulosa cells. And knockdown β-catenin by siRNA dramatically abolished FSH-induced OCT4 expression and cellular development. Furthermore, FSH-induced the phosphorylation of GSK3β, expression of β-catenin and OCT4, and translocation of β-catenin were mediated by the PI3K/Akt pathway. Taken together, the present study demonstrates that FSH regulated OCT4 expression via GSK3β/β-catenin pathway, which was mediated by the PI3K/Akt pathway. And these regulations are involved in ovarian cell development.As a member of the POU (Pit-Oct-Unc) transcription factor family, OCT4 (Octamer-binding transcription factor 4) is associated with the cellular proliferative. However, the roles of OCT4 in regulating the transition from preantral follicle to early antral follicle are still remains unclear. To evaluate the effect of OCT4 on cellular development in ovary, mice were injected with eCG  OCT4in vivo , unless otherwise indicated. M199 was purchased from Gibco Bethesda Research Laboratories . PI3K inhibitor (LY294002) and Akt inhibitor (API-2) were purchased from Selleck . The Cell Counting Kit-8 (CCK-8) was used to analyze cell viability, which was purchased from Dojindo . Lipofectamine 3000 was purchased from Invitrogen . Rabbit polyclonal anti-OCT4 (ab19857), rabbit monoclonal anti-β-catenin antibody (ab32572), rabbit monoclonal anti-GSK3β antibody (ab3291), rabbit monoclonal anti-Phospho-GSK3β(Ser9) antibody (D3A4), and rabbit polyclonal anti-GAPDH (ab9485) were purchased from Abcam . Rabbit monoclonal anti-Phospho-GSK3β (Ser9) antibody (D3A4), rabbit polyclonal anti-phospho-Akt antibody (#9271) and rabbit polyclonal anti-Akt antibody (#9272) were from Cell Signaling Technology . Horse radish peroxidase (HRP)-conjugated anti-rabbit IgG was from Santa Cruz Biotechnology, Inc. . Revert Aid First Strand cDNA Synthesis Kit, TRIzol Reagent were obtained from Thermo Fisher Scientific Inc. , SYBR Green PCR kit was purchased from Bio-Rad . PCR primers for OCT4 and 18S rRNA were from Sunbiotech Inc. .Kunming White female mice  were purchased from the Beijing Vital Laboratory Animal Technology Co. . Mice were maintained under constant conditions of temperature (24–26°C) and humidity (60 ± 2%) with a 12/12-h light/dark cycle and received pathogen-free water and food for maintenance. All animal treatment procedures were in accordance with the Principles of the Care and Use of Laboratory Animals and China Council on Animal Care and were approved by the Institutional Animal Care and Use Committee of Capital Normal University. Mice were injected subcutaneously (s.c.) with 10 international units (IU) of eCG in 100 μL of phosphate buffered saline (PBS) containing 0.2% (w/v) bovine serum albumin (BSA) or PBS alone. In some experiment, mice were injected subcutaneously with DES , and ovaries were collected at 72 h after euthanized by cervical dislocation.5 per well in six well plate) were plated with 2 ml of M199 medium  containing fetal bovine serum  under a humidified atmosphere of 95% air and 5% CO2. The cells were treated with FSH (100 ng/ml) for different duration. In some experiments, cells were treated with OCT4 activator O4I2 (12.5 μM) for 24 h or pretreated with PI3K inhibitor LY294002 (10 μM), Akt inhibitor API-2 (10 μM) 1 h or GSK3β inhibitor CT99021 (15 μM) 12 h before hormone treatment, respectively.Granulosa cells were collected by follicular puncture with a 26.5-gauge needle. And cell number and viability were estimated by Trypan blue dye-exclusion test. Granulosa cells (9 ×10The siRNA was transfected into granulosa cells according to the instruction of the manufacturer's protocol. Granulosa cells were transfected (48 h) with OCT4 or β-catenin siRNA (GenePharma) and scrambled sequence control (GenePharma), using Lipofectamine 3000 (Invitrogen) according to manufacturer's instructions.Western blotting analysis was performed as described previously , 33. Bri−ΔΔCT method . And the mRNA abundance of target genes was analyzed by real-time PCR and normalized to 18S rRNA. Specific primer pairs used in the experiments are listed in T method .The cell viability was measured by Cell Counting Kit-8  . BrieflyGranulosa cells were cultured in poly-D-lysine  coated 8-well glass culture slides  for immunofluorescence analysis. After treatment, cells were fixed in 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and then blocked with 5% BSA in PBS for 30 min. And then, cells were incubated with monoclonal anti-β-catenin (1:200 dilution in blocking solution) at 4°C for overnight. After washing three times with PBS, cells were then incubated with Alexa Fluor 488-conjugated secondary antibody  for 1.5 h at room temperature. After incubating with DAPI, the coverslips were mounted on object slides using fluorescentmounting medium. Immunofluorescence was visualized using an immunofluorescence microscope (Olympus BX51), and images were recorded by using Laser Scanning Microscope LSM 780 .t-test, one-way or two-way (repeated-measure) ANOVA . When significant differences were found, means were compared by the Bonferroni post-test. P < 0.05 were considered to be statistically significant.All the results are expressed as means ± SEM of at least three independent experiments. The statistical differences between treatments were calculated with unpaired or p < 0.05), respectively.To investigate the effect of eCG on the expression of p-GSK3β, β-catenin, and OCT4 protein in mice ovary, eCG were injected for 24 or 48 h, respectively, and determined ovarian OCT4 content using western blotting analysis. As shown in p < 0.05) after eCG treatment, while there is no significant difference at 48 h (p < 0.05), respectively. In addition, eCG significantly increased β-catenin translocation from the cytoplasmic to the nuclear at 24 h (p < 0.001).Meanwhile, we also detected the p-GSK3β (Ser9) level after eCG treatment. The results showed that the level of p-GSK3β (Ser9) was significantly increased 1.82-fold at 24 h . Moreover, the expression of OCT4 mRNA was also significantly changed after FSH treatment for 48 h. These results suggest that the regulation of OCT4 expression in granulosa cells by FSH occurs at the transcription level or via translational processing.To determine whether FSH regulates the expression of OCT4, we cultured granulosa cells with FSH for 24 or 48 h. As shown in p < 0.05). Meanwhile, O4I2 significantly induced cellular viability compared that with the control group (p < 0.05). In contrast, cellular viability was significantly attenuated by OCT4 knockdown (p < 0.05).To investigate the function of OCT4 in granulosa cell development, we cultured cells with OCT4 activator (O4I2) or siRNA with and without FSH. As shown in p < 0.05). Moreover, FSH also significantly increased β-catenin translocation from the plasma to nucleus (p < 0.01). These results were also confirmed by immunofluorescence cell staining  to 1.80 (12 h)-fold (staining .p < 0.05).To further determine whether β-catenin is essential for FSH-induced OCT4 expression, we applied siRNA to knockdown β-catenin expression. While OCT4 protein content was up-regulated by the presence of FSH, this effect was significantly attenuated 1.79-fold by β-catenin knockdown (p < 0.05).To verify the hypothesis that regulations of β-catenin and OCT4 by hormone were mediated by phosphorylating GSK3β (Ser9), we cultured granulosa cells with FSH, and the content of p-GSK3β were detected by western blotting. As shown in p < 0.05) and OCT4 (p < 0.05) induced by FSH were significantly enhanced by CT99021. Moreover, CT99021 also increased FSH-induced translocation of β-catenin by 1.65-fold, which was also confirmed by immunofluorescence results .To determine the role of GSK3β in hormone-induced β-catenin and OCT4 expression, granulosa cells were pretreated with CT99021 (GSK3β activity inhibitor) for 12 h, and then treated with FSH for another 18 or 24 h. The results showed that the expression of β-catenin . The expression of β-catenin or GSK3β after hormones treatment for 18 or 12 h were also detected, respectively. The same effects were also showed on β-catenin and p-GSK3β expression (p < 0.05).Previous studied had showed that FSH promoted granulosa cells development through PI3K/Akt pathway , 9. To dIn the present study, we have demonstrated that FSH-induced granulosa cells development through up-regulating OCT4 expression in mice. These responses are mediated by p-GSK3β (Ser9), β-catenin expression, and translocation. Moreover, the activated PI3K/Akt pathway is also involved in these regulations. To the best of our knowledge, it is the first study to prove that OCT4 plays a crucial role in FSH-induced development in granulosa cells at the preantral to early antral transition stage of follicles.It is well-known that gonadotropin is important for follicle development, which regulates OCT4 expression and functions in ovary . As a trin vivo. The results in granulosa cells in vitro confirm the hypothesis that FSH increased β-catenin expression and translocation to nuclear, which are essential to OCT4 expression. Moreover, the regulations on follicular development by β-catenin may be also mediated by regulating estrogen biosynthesis since β-catenin is a transcriptional regulator of CYP19A1 (The previous reports show that β-catenin is very important to the stem cell proliferation  and hair CYP19A1 .in vivo, which was accompanied by the increased β-catenin expression. Moreover, FSH increased p-GSK3β level and β-catenin expression in granulosa cells in vitro. It is possible that the increased intracellular β-catenin-induced by gonadotropin is due to the phosphorylation of GSK3β at Ser9 as the inactive form in the present study. Moreover, gonadotropin also increased the translocation of β-catenin from the cytoplasm to the nucleus. In addition, FSH-induced OCT4 expression was attenuated by LY294002 and API-2, respectively. The results indicate that the regulation of hormone on OCT4 is mediated by the PI3K/Akt pathway. It is consistent with the previous reports that the activated Akt inactivates GSK3β and inhibits β-catenin degradation by phosphorylating GSK3β at Ser9 (It is also well-known that β-catenin is degraded after forming a complex with the activated GSK-3β . GSK3β,  at Ser9 –31, 42.In conclusion, our findings demonstrate that OCT4 is a novel positive regulator for the granulosa cell development in the early stage of follicles. As indicated by the model , FSH indAll datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Capital Normal University.DH and QW performed the experiments and analyzed the data. CZ, XM, and DH interpreted the results of the experiments. XW and KX prepared the figures. YT, WL, KX, and XH contributed to the discussion. CZ designed the research and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


LRRK2 gene is associated with Parkinson's disease (PD) as a number of mutations within the gene have been shown to be susceptibility factors. Studies on various global populations have determined that mutations such as G2019S, G2385R, and R1628P in LRRK2 increase the risk of developing PD while the N551K‐R1398H haplotype is associated with conferring protection against developing PD. Here we report a study looking at the N551K and R1398H variants for the first time in the Malaysian population.TheCases (523) which conformed to the United Kingdom PD Brain Bank Criteria for PD were recruited through trained neurologists and age‐ and ethnically matched controls (491) were individuals free of any neurological disorder. The N551K and R1398H mutations were genotyped using the Taqman SNP genotyping assay.A significant protective association for N551K was found in those of Malay ancestry, with a protective trend seen for R1398H. A meta‐analysis of Chinese individuals in this cohort with other published cohorts of Chinese ancestry indicated a significant protective role for N551K and R1398H.This study reports that the N551K‐R1398H haplotype is also relevant to the Malaysian population, with a significant protective effect found in those of Malay and Chinese ancestries. The loss of the dopaminergic neurons leads to a range of movement problems including rigidity, bradykinesia, and tremors.LRRK2) gene has been extensively studied in relation to both familial and sporadic forms of Parkinson's disease. The PD mutation database reports 127 mutations in this gene, with the G2019S mutation accounting for 40% of North African Arab and 20% of Ashkenazi Jewish PD cases  on a 7,500 Fast Real‐Time PCR machine. Genotypes were confirmed by sequencing in a subset of 20 individuals to confirm the genotypes and determine the error rate.2I statistics  while Review Manager 5 (RevMan 5) . Sixty per cent of PD patients and 51% of controls were male. The cohort consisted of 168 Malay PD cases, 133 Malay controls, 279 Chinese PD cases, 269 Chinese controls, 76 Indian PD cases, and 89 Indian controls.The mean age at PD diagnosis was 57.4 ± 11.8 years and the mean age of controls was 59.3 ± 9.4 years (r2 = 0.906), similar to what has been reported by Tan et al., Linkage analysis (Haploview 4.2) indicated that N551K and R1398H are in linkage disequilibrium  was obtained for N551K, and an OR of 0.699  was obtained for R1398H, suggesting a reduced risk of developing PD in carriers  of 0.623 . No significant difference was found between the Chinese case and controls . The Indian subgroup showed a similar, albeit nonsignificant trend , likely due to the small sample size. When we performed a meta‐analysis for N551K on the combined Chinese datasets from this study, and those by Tan et al. and Wu et al., the analysis showed a significant protective effect with an OR of 0.79 . Meta‐analysis on Malay and Indian samples was not performed as there are no other published studies on these populations for N551K.When analyzed according to ethnicities, a protective association for N551K was detected in Malays , but the results were not significant in the Chinese  and Indians . We performed a meta‐analysis of R1398H  and this showed an OR of 0.81 , .When analyzing R1398H according to ethnicities, the protective association was detectable with borderline significance in Malays  underlying the protective effects of the N551K‐R1398H haplotype are not clear at present. The N551K variant is not within any domain of the LRRK2 protein, but is in linkage disequilibrium with the R1398H variant, which lies within the Ras‐of‐complex (ROC) GTPase domain, and enables the binding of guanine nucleotides via a phosphate‐binding motif.LRRK2 such as G2019S elevate the level of kinase activity , a condition now regarded as a prodromal symptom of synucleinopathies, most commonly PD. In a case–control study involving 350 RBD patients and 869 controls, the N551K‐R1398H haplotype was significantly associated with a reduced risk of developing RBD (Bencheikh et al., In conclusion, we show that consistent with other published reports on the protective effect of N551K and R1398H, these variants are also protective in the Malaysian Malay and Chinese ethnicities. Further studies will need to be done to determine the cellular mechanism of how this protective effect is mediated.The authors have no conflicts of interest to declare.


State-of-the-art ultra-sensitive blood glucose-monitoring biosensors, based on glucose oxidase (GOx) covalently linked to a single layer graphene (SLG), will be a valuable next generation diagnostic tool for personal glycemic level management. We report here our observations of sensor matrix structure obtained using a multi-physics approach towards analysis of small-angle neutron scattering (SANS) on graphene-based biosensor functionalized with GOx under different pH conditions for various hierarchical GOx assemblies within SLG. We developed a methodology to separately extract the average shape of GOx molecules within the hierarchical assemblies. The modeling is able to resolve differences in the average GOx dimer structure and shows that treatment under different pH conditions lead to differences within the GOx at the dimer contact region with SLG. The coupling of different analysis methods and modeling approaches we developed in this study provides a universal approach to obtain detailed structural quantifications, for establishing robust structure-property relationships. This is an essential step to obtain an insight into the structure and function of the GOx-SLG interface for optimizing sensor performance. These complex materials often strive to emulate hierarchical architectures that are typical of natural biological systems4. It is challenging to precisely manufacture such detailed structures, and one major limitation lies in the availability of imaging methods that are needed for determining the detailed nanoscale organization of a manufactured biohybrid materials and then using this information for optimal manufacturing of materials with required parameters. Here, we present our investigation into a new generation of graphene-based glucose biosensors where accuracy of structural organization is of prime concern for the sensor functionality. We relate the performance of this sensor to detail the nano-scale structure. We also describe a new approach for extracting the structural details from small-angle neutron scattering data.Biohybrid composites that integrate biological functionality with the nanoscale organization of inorganic compounds hold great promise for designing materials that take inspiration from nature to combine biological or biomimetic functional components with man-made synthetics8. Graphene, a two dimensional single-atom-thick crystalline nanomaterial, has engaged a wide range of markets due to its superlative qualities9. Unlike semiconductors that are reaching fundamental limitations for device fabrication, graphene based electronic devices hold an immense potential for future electronics10. Proteins confer exceptional stereospecificity to graphene when covalently attached in the design of biosensors. Glucose oxidase (GOx), a redox enzyme that increases the rate of glucose oxidation, has been extensively employed in biosensors to determine the concentration of glucose in biofluids by estimating the number of electrons released during the redox reaction15. GOx in conjunction with graphene interface acts as an efficient conveyor belt, receiving the electrons from flavin adenine dinucleotide (FAD) cofactors in GOx and ballistically transporting them as output, to give a direct quantitative measure of glucose in biofluids8. The graphene-protein interface is the gateway through which electrons shuttle from the FAD centers in GOx4.The graphene-based sensors are expected to aid direct and effective electron transfers by eliminating the use of redox mediators and oxidation overpotential4, small-angle scattering, and 3D NMR imaging. Small-angle neutron scattering (SANS) probes structural features in the region from 1 nanometer to hundreds of nanometers. The prime objective of the present SANS study is to establish a detailed understanding of the GOx matrix structure deposited on graphene and relating the structural modifications to different sample conditions, and in future relating this information with the functionality of sensors in different applications.Structural details of immobilized GOx on pristine graphene are hard to derive, and at present, the repertoire of structural methods are available to investigate interfaces which are largely limited to in silico molecular dynamics17. SANS provides unique capabilities to determine the organization and hierarchical structures of different characteristic length scales in a single measurement18. In the present study we have applied a novel scaling approach to Unified Fit19 based analytical method to simultaneously extract the structure of individual GOx macromolecules in the biosensor and the architecture of assemblies that it forms within the sensor material. We use the Unified Fit in combination with ab initio methods provided by tools in the openly available ATSAS software suite and rigid body docking to obtain biologically relevant structural models for different structural levels21. The scaling approach overcomes the limitations of ab initio models in order to evaluate average models of the hierarchical assemblies. The results of this study demonstrate the ability of SANS to differentiate between changes in hierarchical structures in sensor matrices, which correlates well with the individual manufacturing processes.Recent enhancements in capabilities of small-angle scattering techniques for biological samples have occurred in parallel with new methods for analyzing and modeling of dataab-initio models, that are compared with the electrostatic potential on the GOx crystal structure evaluated using continuum electrostatics calculations.Figure 19. This analysis approach yields a set of parameters that describe the average topological arrangement at each level of the hierarchical structure. The parameters extracted from these fits are given in Table We have investigated the structural details of GOx-graphene sensor material on a length scale spanning 1–200 nm by small-angle neutron scattering (SANS). Figure Rg of 25 to 26 Å is observed in both samples at the largest measured scattering angles . This size matches with the length scale of individual GOx enzymes and interpretation of this feature as stemming from GOx enzymes is therefore very plausible. The overall envelope shape of individual enzyme molecules in solution can successfully be obtained from small-angle scattering data21. For a dense packing of enzymes as encountered in the GOx sensor material, such a detailed analysis is obstructed by the additional scattering features that arise from the higher-levels of structure. In order to overcome this limitation, we have subtracted the higher-level scattering features, which were determined by their corresponding 3-level fit parameters. The residual scattering was then used for generating shape models of GOx enzyme by applying tools from the ATSAS suite as described in detail in the methods section23.A size parameter in the form of apparent radius of gyration, PyMOL24. Apparently, the GOx dimer high-resolution structure, which was obtained through crystallizing GOx from a dilute solution, describes the conformation of GOx in the graphene sensor material rather well, especially for the sensor material prepared at neutral pH 7. Intriguingly, the sensor material that was prepared at basic pH 9 shows a somewhat increased separation between the subunits—note that some residues on the left side of Fig. The final DAMAVER density maps for GOx enzymes in the sensor matrix of sample 1 and 2 are shown as blue surface in the central column of Fig. PBD2PQR on a linear red-green-blue colour scale representing EPs of −7.5−0KBT/e. The EP over the structure differs markedly in the neck region of the dimer structure. The marked differences in the electrostatic potential distribution in the neck region may alter the electrostatic interactions at the dimer interface, and hence, induce greater separation of the dimer subunits at the elevated pH level25. Importantly, changes of the electrostatic potential may lead to altered glucose recognition and loss of its catalytic abilities. We note that this spatial separation is permanently retained in the final sensor material, since the SANS data was acquired after washing the material with neutral buffered saline solutions after functionalization at higher pH. The graphene functionalized GOx sensor material is organized into nano- to mesoscale clusters with an average diameter of just above 1000 Å. This is evident from the observed gyration radii at low scattering angles (q < 0.01 Å). The Unified Fit here yields overall size parameters Rg of 590 Å and 550 Å for sample 1 and 2 respectively. The steep drop-off in intensity, which was fit by a q−4 power-law supports the notion of 3-dimensionally extended dense clusters that are bound by a well-defined surface. The inferred dimension and nature of the clusters compare well with the reported ESEM micrographs of surface morphology of immobilized GOx on chitosan supported mesoporous carbon26 and cross-linked enzyme aggregates of horseradish peroxidase and GOx reported previously27. The limitations of the surface morphology information from the ESEM micrographs are testimony to the utility of the present work where (submicron) cluster morphology is deciphered using SANS.Since the differences in sample preparation are in the pH environment, the pronounced neck region for sample 2 can be further examined using electrostatic potential (EP) evaluation employing continuum electrostatics calculations, shown in Fig. q-axis to match the overall size of the GOx dimer using Eq.  where the shape was calculated from the individual level scattering contributions obtained from the 3-level Unified Fit using Eqs –8. As shsing Eq. . This stThe dummy ‘atom’ models obtained for the clusters look similar for both samples as shown in envelope structures in Fig. g scaling. Nonetheless, these results suggest that GOx is packed quite densely in the sensor material prepared at neutral pH while preparation at higher pH leads to a somewhat less dense sensor material at the nanoscale. A lower packing density in the material prepared at pH 9 is consistent with repulsive surface charges on the GOx discussed above. Lower packing density of the sensor material provides a second plausible explanation for the lower performance observed in sensors prepared at higher pH.Independent of any models, the SANS data allows in estimating additional quantities of interest: The ratio of absolute scattering intensity of the cluster to that of the individual GOx enzyme gives an estimate of the number of GOx dimers in the cluster. This number is about 9500 in sample 1 and 4600 in sample 2. Moreover, from the comparison of the size parameters of the cluster and GOx dimer we can estimate that the cluster volume is approximately 12400 times larger than the GOx volume in sample 1 and 9300 times larger than in sample 2. From this we can estimate a packing density of GOx enzymes of 0.76 in sample 1 and 0.50 in sample 2. These are rough estimates as uncertainties accumulate in the combination of fit parameters and anisometric shape of particles was not taken explicitly into account when estimating volume scaling from Rza, evaluated from the absolute scattering intensity to be 10 and 17 for sample 1 and sample 2 respectively. On the other hand, if we assume that volume scales as Additional details of the spatial arrangement of GOx molecules inside clusters of graphene sensor material can be extracted from the central region of the scattering curves zc, Table q-range that corresponds to length scales larger than their thickness but smaller than their radius28. The unified fit yields power law exponents On the other hand, the comparison of size parameters of aggregates and the larger clusters yields large numbers of several hundred aggregates within a cluster  was cloned into a replicative plasmid vector and expressed in Kluyveromyces marxianus var. marxianus CBS 6556 strain as described previously36. A partially purified enzyme (Medium grade GOx) obtained from cell-free supernatant prepared by culture centrifugation  was concentrated and exchanged by Prep/Scale Spiral Wound Ultrafiltration with a pore size of 10 kDa (Millipore) in 20 mM Tris buffer (pH 7.4) with 100 mM NaCl. The pure enzyme (High grade GOx) was subjected to a second purification step with Q-Sepharose F.F. column (strong anion exchanger) equilibrated with the same buffer, and a linear gradient of 0–0.5 mM NaCl in 20 mM Tris buffer (pH 7.4) was used to elute the column at a flow rate of 0.5 ml.min−1. Enzyme activity recovery and purification factor were 89% and 1.5 for the Medium grade GOx, and 17.3% and 17 for the High grade GOx. Both Medium grade and High grade GOx were lyophilized to achieve higher stability at room temperature. All steps of purification were assayed for their enzyme activity and protein content. GOx’s activity was determined by the o-dianisidine reduction method using a commercial glucose oxidase from Aspergillus Niger  as standard at 37 °C and pH 4.537. The homogeneity of the partially and highly purified protein was confirmed by SDS–PAGE analysis . The Cu foil was then heated to 950 °C in Ar/H2 atmosphere. When the temperature becomes 950 °C, Ar/H2 flow was stopped and hexane vapor is passed through the quartz tube maintaining the tube pressure of 500 mTorr for 4 min. The flow rate of hexane was maintained at ~ 4 mL/h (~2–3 sccm). The furnace was then suddenly cooled to room temperature. The graphene growth has taken place over Cu foil and this film was further processed for PMMA based transfer process.Single layer graphene film was synthesized on a 25 μm thick clean copper (Cu) foil by the chemical vapor deposition technique using hexane as the carbon source2CO3-NaHCO3 buffer solution (pH 7.0 for Sample 1 and pH 9.0 for Sample 2) overnight at 4 °C followed by rinsing with DI water and phosphate buffered saline solution . To deactivate and block the excess reactive groups remaining on the surface, the device was further incubated with 0.1 M ethanolamine solution (pH 7.0 for Sample 1 and pH 9.0 for Sample 2) for 30 min and then rinsed with DI water. Raman spectrum of GOx immobilized on single layer graphene (SLG) collected at ~ pH 7 (blue), after subtraction of SLG, is shown in Fig. 2 carbon is an indication of reconstruction of the carbon network after immobilization of GOx on the graphene surface26.A graphene device (CVD Graphene or Graphene Foam) was incubated with a 5 mM linker molecule  in dimethylformamide (DMF) for 2 hours at room temperature and washed with pure DMF and deionized (DI) water for three times. The linker-modified graphene was then incubated with 10 mg/mL of recombinant GOx in NaThe device has a stack of single layer graphene sandwiched between two gold pads deposited on top of Silicon Dioxide layer on a silicon wafer. A few microns thick layer constituting of an estimated 600–1,000 layers of functionalized GOx deposited over the single layer graphene layer constitutes the activated matrix investigated using SANS. A schematic of the active matrix under investigation is shown in Fig. 41. The instrument schematic is shown in Fig. −1 < q < 0.5 Å−1), at a wavelength of 6 Å with a wavelength spread, Δλ/λ of 0.15. The data acquisition time was 5 hours for each sample at each SDD configuration.SANS experiments were performed at the Bio-SANS instrument at the High-Flux Isotope Reactor in Oak Ridge National Laboratory2 and gold depositions as background.The SANS −1 is plotted against a reduced angle, called the scattering wave vector q. λ is the wavelength and 2θ is the scattering angle. SANS data can be used to quantify topological features in complex materials42. SANS from a hierarchical system displays multiple structural levels which can be evaluated using scattering laws for mass/size and surface/density properties44. In each structural level, a Guinier’s law, , and a power-law, q-regions respectively, where G, Rg, B and df are the Guinier’s law prefactor, radius of gyration, power-law prefactor and fractal dimension respectively45. A Guinier-law is an exponentially decaying function, which decays near qRg = 1 region and the actual shape of the decay, also known as the Guinier knee, depends on the shape and size of a particle. On the other hand, the topological features of a particle can be inferred from the surface scattering via Power-law with two variables, Bi and df, at higher q. Bi is directly proportional to the surface area of the particle while df provides a measure of mass to size scaling or density of an average particle. These local scattering laws can be combined using the Unified Function to describe multiple levels of structures. The choice of the number of the levels used for the data interpretation depends on the features in the SANS curve. Owing to the presence of three distinct bend/hump in the SANS curve , the GOx dimer system is fitted with a three level Unified Fit, as:49erf is the error function. The subscripts c, a and G indicate different levels of GOx organizations namely the clusters, aggregates and GOx dimer levels, respectively. The power–law prefactor50,ni and Si are the number and surface area of particles in a structural level while Δρ is the contrast factor. The power law exponent, more widely called the fractal dimension, is the mass to size scaling parameter. It is a direct measure of density whence a higher fractal dimension translates to a higher mass density. The Guinier prefactors50ρ, remains the same across all the levels, the relation47,In isotropic small-angle scattering as in present case, an azimuthally averaged intensity in units of cmThe ab initio modeling approaches such as the DAMMIN program in the ATSAS suite, developed by Svergun’s group21. The program generates dummy bead coordinates of structures consistent with isotropic SANS experimental data51 using finite volume ensembles with the help of parameterized envelope functions. The algorithm used to generate the individual volume elements is bound by constraints of compactness, which takes size and distribution inputs from GNOM fits of SANS data52. The GNOM program can be conveniently used to fit single structural levels but presently has limitations with respect to hierarchical topologies.Small-angle scattering techniques such as SANS are widely used to evaluate three dimensional protein structures by implementing In the present study, the total scattering intensity has been separated into a set of three distinct intensities such that,The GOx enzyme level scattering can therefore be obtained using the contribution from GOx level using ab initio modeling, the size ranges of the larger hierarchical structures were scaled to that of the GOx dimer in order to limit the search volume to generate appropriate dummy atom models using the GNOM fits. This was achieved by scaling the q-range of cluster and aggregate structures as,For p(r), that may be calculated from the intensity using a Fourier transform,The result of application of Eqs , 11 are r = 0 to a maximum distance Dmax is selected in the range of 2.5–3 times Rg, depending on the feasibility of shape of the distribution function. We started with a Dmax of 70 and adjusted thereafter as required. The P(r) plots for the two samples for the GOx, aggregate and cluster levels are shown in the Fig. Dmax are in the Table 52 output file is used to generate 20 low-resolution ab initio models using the DAMMIN program21. Thereafter, these set of 20 ab initio models were sorted, aligned and averaged using the DAMAVER program suite to generate final models53. Table ab-initio modeling using ATSAS package including the normalized spatial discrepancy (NSD), the number of reconstruction included in the final model generated by DAMAVER and Dmax used for generating GNOM fits.In practice, the integration is over 49.The ability of differentiating and effectively outlining the thermodynamics and topologies in fractal and hierarchical structure using Unified Fit on SANS is well investigated54. Subsequently a rigid-body docking approach was employed to perform exhaustive six-dimension conformational searches (3 rotations and 3 translations) to fit the high-resolution crystallographic model (PDB ID: 1GPE55 at 1.8 Å resolution and a Diffraction Precision Index of 0.1 Å57) into the modeled low-resolution averaged molecular envelopes. The entire rigid-body docking procedure was automated using the CoLoRes54 module of Situs. This cross-correlation function-based module implements fast Fourier transform algorithms and Laplacian filter for an effective computation of the degrees of freedom of the crystallographic model corresponding to the fixed molecular envelope. The docking was performed with an angular step of 20, followed by Powell minimization.To effectively characterize the structure of immobilized GOx, a three-dimensional volumetric map was generated from the averaged model using the pdb2vol module of the Situs software package58. The high resolution crystallographic model (PDB ID: 1GPE55) was used with PARSE forcefield optimized for examining models under implicit solvent conditions of pH 7 and pH 958. The APBS solves a linearized form of Poisson-Boltzmann equation to evaluate the EP, which have been visualized using Jmol PBD2PQR on a linear red-green-blue colour scale representing EPs of −7.5−0KBT/e as shown in Fig. The electrostatic potentials (EPs) were evaluated using Adaptive Poison-Boltzmann Solver (APBS) and PBD2PQR software package which utilizes the continuum electrostatics calculationsSupporting Information: Structural determination of Enzyme-Graphene Nanocomposite Sensor Material