Study On The Function And Mechanism Of NCOA5 In Hepatocellular Carcinoma

Background and objectives:In recent years,the incidence and mortality of tumors have continued to rise.Cancer has become the leading cause of death among Chinese patients and an important public health issue.As one of the countries with the largest number of hepatitis B virus carriers in the world,more than 100,000 people died from primary liver cancer each year in China.Hepatocellular carcinoma(HCC)is the most common type of primary liver cancer,which is the sixth most common tumor in the world,ranking third in tumor-related causes of death,second only to lung cancer and gastric cancer.The main treatment of liver cancer is surgery,the efficacy of radiotherapy or chemotherapy is poor.And like most tumcors,the proportion of early diagnosis and sturgery of hepatocellular carcinoma is only 10%-20%,the majority of patients are diagnosed at the advanced stage,with limited treatment and poor prognosis.The pathology of liver cancer is complicated,with multistep and multifactorial carcinogenesis remain to be explored.NCOA5(Nuclear receptor coactivator 5),also known as CIA(coactivator independent of AF-2 function),is an AF2(activation function 2)-free co-stimulatory factor that regulates ERa transcription.Previous studies have shown that NCOA5 plays an important role in the development and progression of primary liver cancer and diabetes.The defects of NCOA5 are not only the causative factors of T2D and HCC,but also the important factors in linking T2D and HCC.NCOA5 also involves in the progression of esophageal cancer,gastric cancer,colorectal cancer and other tumors,but the specific pathologic mechanism is unclear yet.Studies have shown that NCOA5 plays an important role in maintaining self-renewal of pluripotent stem cells.Therefore,the purpose of this study is to clarify the important function and mechanism of NCOA5 in hepatocellular carcinoma,and provide a molecular mechanism for the development of hepatocellular carcinoma,and to find potential targets for the treatment of hepatocellular carcinoma.Methods:1.The expression of NCOA5 in human hepatocellular carcinoma cell lines HuH-7,HepG2,Bel-7402,Bel-7404,HCCLM3,SK-Hep-1 and immortalized human liver epithelial cells L02 were studied by immunoblotting.2.The NCOA5 in HCCLM3 cells were knocked out by CRISPR/Cas9 technique,and identified by protein immunoblotting and genomic cloning and sequencing.The results showed that the NCOA5 knockout cell lines were successfully generated.3.To evaluate the sgRNA sequences used in the experiment and predict the potential off targets(POTs)based on the CRISPR design online tool.4.NCOA5-KO-1,NCOA5-KO-2 cells and wild type cells(WT)were divided into three groups to explore the effects of NCOA5 knockout on the proliferation,migration and formation of tumor microspheres.5.The cells of NCOA5-KO-1 and WT were sent for whole-transcriptome analysis(RNA-sequencing).The differentially expressed genes were searched and clustered.6.NCOA5-KO-1 cells,NCOA5-KO-2 cells and WT cells were divided into three groups.The total protein was extracted and identified by immunoblotting to analyze the expression of epithelial mesenchymal transition and cancer stem cell related proteins.7.Statistical analysis:Metrological data were expressed as(x±s)and analyzed with SPSS 13.0.Comparisons of two groups or three groups mean values were analyzed by independent sample t test or One-way ANOVA followed by LSD or Dunnett multiple comparison test.P-Values were considered to be significant at<0.05.Results:The NCOA5 gene was knocked out successfully by CRISPR/Cas9 technique,which was identified by genomic sequencing and western blot.All POTs were PCR amplified and then sequenced.Sequencing results showed that no mutation was detected in these POTs.Cell function experiments showed that,knockout of NCOA5 downregulates the viability,migration and formation of tumor spheres of HCCLM3 cell.Transcriptome sequencing(RNA-seq)showed that 1335 genes were altered after NCOA5 knockout,in which 602 genes were up-regulated and 733 genes were down-regulated.The results of cluster analysis showed that differentially expressed genes were associated with cell adhesion,cell matrix synthesis,cell metabolism and so on.At the same time,it was found that knockout of NCOA5 in HCCLM3 cells inhibited the expression of epithelial mesenchymal transition(EMT)and cancer stem cell related genes.Further studies showed that knockout of NCOA5 inhibited the expression of EMT and cancer stem cell related proteins in cells.Conclusion:The knockout of NCOA5 gene in HCCLM3 cells by CRISPR/Cas9 technique provides an important cellular model for exploring the role of NCOA5 in hepatocellular carcinoma.The knockout of NCOA5 in hepatocarcinomas cells may inhibit the formation of hepatocellular stem cells by inhibiting EMT,thus inhibiting the proliferation and metastasis of hepatocellular carcinoma cells and the formation of tumor stem cells.