The Preliminary Study On The Role And Mechanism Of HOXA9 Gene In The Development And Progression Of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is one of most lethal cancers worldwide,especially in East Asia and sub-Saharan Africa.In China,HCC is now the third and sixth most frequently occurring cancer in men and women,with about 466,100 cases of newly diagnosed HCC patients in China in 2015.What's more,because most patients are diagnosed at an advanced stage,the overall survival of HCC is low.Now HCC has been the third cause of cancer deaths in China.Thus,the burden and cost of HCC in China is great.Virus infection,including HBV and HCV,exposure to aspergillus-derived aflatoxin,and heritable diseases such as alpha-1 antitrypsin deficiency and Wilson disease are possible risk factors for HCC.The molecular mechanism of HCC is heterogeneous and extremely complex.In recent years,with the development of sequencing technologies and the completion of human genome project,some genetic markers of HCC have been reported,but the specificity and sensitivity is relatively low.Therefore,to improve the diagnosis and prognosis of HCC,more new and specific biomarkers are needed.Homeobox(HOX)gene was first detected in drosophila and later confirmed to be present in vertebrates containing humans.Their coding products are a transcriptional factor family and perform functions in embryonic development and cellular differentiation.In mammals,HOX gene family includes 39 members,and abnormal expression of these genes is closely associated with the body dysplasia and a wide variety of tumors.HOXA9 gene is one of the HOX gene families,which is of great importance to hematopoiesis.Recent studies have found that HOXA9 gene is increased in almost fifty percent of acute myeloid leukemia patients,which is related to its poor prognosis.Besides,disordered expression of HOXA9 is discovered in breast cancer,ovarian cancer,malignant glioma and non-small cell lung cancer.However,no literature had been reported about the role of HOXA9 in hepatocellular carcinoma.Epithelial-to-mesenchymal transition(EMT)is one of the major mechanisms of cancer metastasis.The signal pathways regulated EMT are complex and various,of which the TGF-? pathway plays a significant role.In TGF-?-induced EMT,smads induce gene reprogramming by directly activating the expression of EMT related transcription factors,and then cooperating with them in the control of target genes.These transcription factors include Snail1,Snail2,ZEB1,ZEB2,and Twist.Our previous studies found that HOXA9 gene had a stop-gain mutation in HCC by using whole genome exon sequencing technique.On the basis of this,we found that the expression of HOXA9 gene in HCC tissues and cells were relatively decreased,and HOXA9 could inhibit the proliferation,migration and invasion abilities of HCC.By studying the relationship between HOXA9 gene and EMT pathways,we found that TGF-?1 could decrease the expression of HOXA9,and HOXA9 inhibits EMT pathway and is related to migration and invasion of HCC.Objective 1.To detect the expression of HOXA9 gene in HCC tissues and cells,and analyze its relationship with clinicopathological parameters.2.To study the effect of HOXA9 gene on the proliferation,migration and invasion of HCC 3.To explore the possible molecular mechanism of HOXA9 gene functions in HCC.Methods 1.The expression of HOXA9 in HCC paraffin embedded tissues was detected by immunohistochemistry(IHC),and the relationship between the expression of HOXA9 and clinicopathological parameters was explored.2.The expression of HOXA9 gene in HCC cells were detected by real time quantitative polymerase chain reaction(qRT-PCR)and western-blot(WB).3.HOXA9 gene eukaryotic expression plasmid and siRNA were transfected into HCC cells by liposome 2000 or transfection reagent.qRT-PCR and WB were used to detect gene expression and interference efficiency.MTT and Colony formation assay were used to estimate the proliferation ability of HCC,and Wound healing and Transwell assay were used to estimate migration and invasion abilities.4.Different doses of TGF-?1 were used to stimulate HCC cells.qRT-PCR was used to detect expression of HOXA9 gene and EMT related molecules(E-cadherin and N-cadherin).Wound healing and Transwell experiments were used to detect the effect of TGF-?1 on migration and invasion abilities of HCC.5.The effect of HOXA9 gene on EMT was detected by qRT-PCR and WB.Relevant cell functional experiments were used to compare the migration and invasion abilities of the TGF-?1 stimulation group,TGF-?1 stimulation with HOXA9 overexpression group,and control group.Results 1.The positive rate of HOXA9 expression in 81 cases of HCC was significantly lower than that in adjacent tissues,and correlated with the classification of HCC.2.The mRNA and protein expression of HOXA9 were low in HCC cells compared with the normal human hepatocyte HL-7702.3.qRT-PCR and WB results found that HOXA9 eukaryotic expression promotes the expression of HCC,while siRNA inhibits its expression.MTT and Colony formation assay found that HOXA9 could inhibit the proliferation ability of HCC.Wound healing and Transwell assay found that HOXA9 could significantly inhibit migration and invasion abilities.4.TGF-?1 could significantly attenuate HOXA9 mRNA expression level,and this effect was most significant at the dose of 5ng/ml.TGF-?1 could inhibit E-cadherin expression while increase N-cadherin expression in HepG2 cell.Besides,TGF-?1 could promote the migration and invasion abilities of HCC.5.Contrast to cells transfected with mock vector,cells transfected with HOXA9 expression vector had a high E-cadherin mRNA and protein level while a low N-cadherin expression level.Wound healing and transwell assays found that TGF-?1 could significantly enhance the migration and invasion ability of HCC cells,while after transfecting HOXA9 gene,this effect was attenuated.Conclusion 1.The expression of HOXA9 gene in hepatocellular carcinoma tissues and cells was decreased and correlated with the classification of HCC.2.HOXA9 inhibits proliferation,migration and invasion abilities of HCC cells.3.TGF-?1 induces down-regulation of HOXA9 and promotes EMT and migration and invasion abilities of HCC.4.HOXA9 inhibits migration and invasion of HCC through TGF-?1-mediated EMT pathway.