Research On The Expression,Function And Mechanism Of ARHGAP11A In Hepatocellular Carcinoma

Background Hepatocellular carcinoma(HCC),accounting for 90% of primary liver cancer cases,is one of the most prevalent cancers worldwide and exhibits high mortality.The poor prognosis of HCC is mainly associated with the high frequency of late-stage disease,recurrence and metastasis.Therapeutic strategies aiming at HCC survival improvement have not yet achieved a satisfactory outcome.Hence,more elucidation about HCC signal pathways in malignancy regulation may help to identify novel effective molecular targets for HCC treatment.RhoGTPase-activating proteins(RhoGAPs)have been thought to accelerate the intrinsic GTP hydrolysis activity of RhoGTPases,cycling them to their inactive GDP-bound state.RhoGAPs generally link cell migration and proliferation pathways,and are frequently downregulated in cancers.RhoGAPs is emerging as a new class of biomarkers for diverse cancers.DLC-1/2(Deleted in Liver Cancer-1/2)and STARD13 are downregulation in hepatocellular carcinomas,PIK3R1 is downregulation in prostate cancer,β-chimerin is downregulation in breast cancer,ARHGAP8 is downregulation in colorectal and breast cancers,ARHGAP20 is downregulation in leukemia.Hence,the prevailing dogma in the field is that RhoGAPs are tumor suppressors.However,recent evidence has begun to challenge this notion,which demonstrated that ARHGAP11 A expression was upregulated in colon cancer and breast cancerand and acts as oncogene.In colon cancer and breast cancer,ARHGAP11 A plays important role that could promote the ability to metastasize and proliferate in a RhoA-dependent way.However,there is little data on the association of ARHGAP11 A with HCC malignant phenotypes and it is not know whether ARHGAP11 A plays a role in HCC via a RhoA-dependent way.Aims The main purpose of this study was to clarify the expression and clinical significance of ARHGAP11 A in HCC,to investigate the effect of ARHGAP11 A on malignant progression of HCC in vivo and vitro,to elucidate the mechanism of ARHGAP11 A on its function in HCC.Methods 1.We analyzed RNA-Seq data from The Cancer Genome Atlas(TCGA)Project to examine the expression of ARHGAP11 A in HCC.Its correlation with clinicopathological stage and prognosis was also analyzed.We then verified the exactness of the data from TCGA by RT-PCR in 15 paired HCC tissues and pericarcinomatous tissue.We further detected the expression of ARHGAP11 A in 75 HCC tissues with immunohistochemistry and its clinical and prognostic significance.2.To suppress the expression of ARHGAP11 A,sh RNA or NC(negative control)for ARHGAP11 A was transfected into Hep3 B and MHCC97-H cells,respectively.Cell Counting Kit-8(CCK-8)and colony formation assay was used to measure the changes in cell proliferation after transfected with sh RNA or NC.We explored the effect of ARHGAP11 A on cell cycle and apoptosis via Flow cytometry.Transwell and wound healing assay used to measure the changes in cell invation and migration after transfected with sh RNA or NC.EMT associated markers was detected by western blot.3.We further studied the effect of ARHGAP11 A on growth and metastasis of tumor xenografts in vivo via nude and C57 BL mice.4.Gene microarray and Ingenuity Pathway Analysis(IPA)software was used to analyze the role of ARHGAP11 A in tumor and potential downstream genes.RT-PCR and Western-Blot used to verify the exactness of Gene microarray.5.We down-regulated RAC1 B in HCC cells by si RNA to detect the effect on invasion,migration and EMT.Recovery experiment used to verify whether the role of ARHGAP11 A in HCC mediated by RAC1 B.6.To explore the possible mechanisms that RAC1 B regulated by ARHGAP11 A via Co-ip/protein mass spectrometry/GST pull down assay.Results 1.ARHGAP11 A was highly expressed in most HCC tissues compared with normal-like tissues and correlated with the clinical grade,TNM stage and pathological stage of HCC from TCGA.In addition,Kaplan-Meier survival analysis revealed that HCC patients with low ARHGAP11 A expression exhibited prolonged survival.The median overall survival was 2131 and 1149 days in patients with low and high ARHGAP11 A expression,respectively.High expression of ARHGAP11 A was identified to be correlated with tumor size,differentiation,metastasis and TNM stage but not with other clinicopathological characteristics,such as gender,age,and AFP in patients with HCC.2.ARHGAP11 A expression was silenced in two human HCC cell lines,Hep3B(low malignancy)and MHCC97-H(high malignancy)with sh RNA constructs.The effectiveness of ARHGAP11 A silencing was confirmed by RT-PCR and western blot.3.CCK-8 and colony formation assays showed that ARHGAP11 A deletion significantly decreased the proliferation of both HCC cell lines.ARHGAP11 A deficiency increased the percentage of cells in G0/G1 phase and decreased the percentage of cells in S phase.Curiously,no difference was found in the cell apoptosis level.ARHGAP11 A silencing inhibited the invasion capacity of HCC cells,as indicated by a Transwell assay.Migration ability was also confirmed to be restricted following ARHGAP11 A knockdown via a wound healing assay.ARHGAP11A-knockdown recovered HCC cell’s polarity and cell-cell adhesion from a motile,multipolar,and spindle-shaped cell phenotype.Expression of the epithelial marker E-cadherin increased,while expression of the mesenchymal markers N-cadherin and Snail decreased in ARHGAP11A-knockdown cells.4.ARHGAP11 A deletion suppresses in vivo growth and metastasis of tumor xenografts.5.We identified 255 upregulated genes and 179 downregulated genes in Hep3 B cells following ARHGAP11 A knockdown,most genes involved in cell proliferation were downregulated by ARHGAP11 A silencing via Gene microarray.(IPA)software identified the major functionally related gene groups that were differentially expressed in sh ARHGAP11 A cells compared with control cells.Pathways implicated in cellular development,cell growth and proliferation,and cellular movement,among others,were mostly suppressed.6.Rac1 B silencing significantly reduced the capacity of invasion and migration of HCC cells.Meanwhile,Rac1 B knockdown also led to increased E-cadherin expression and decreased N-cadherin and Snail expression.The role of ARHGAP11 A in HCC is mediated by RAC1 B.7.Co-IP assay has confirmed the positive interaction between ARHGAP11 A and Rac1 B.RhoGTPase pulldown showed that ARHGAP11 A knockdown increased RhoA and decreased Rac1 B,but not Rac1 activity in Hep3 B cells.Mass spectrometry(MS)also confirmed ARHGAP11A-Rac1 B interaction,nevertheless,the ubiquitination of Rac1 B was not detected.Conclusion 1.ARHGAP11 A is frequently upregulated in HCC,and associated with clinical prognosis.ARHGAP11 A regulates HCC cell in vitro and in vivo proliferation,migration and invasion,and EMT development.ARHGAP11 A acts as a protooncogene in HCC.2.The GAP domain of ARHGAP11 A can inhibit activity of RhoA,but not Rac1 or Rac1 B in HCC.3.ARHGAP11 A can facilitate HCC malignant progress through Rho-independent and RAC1B-dependent pathway.