Differential Gene Expression Profiling In HCC And The Effect Of EIF3B On The Proliferation Of HCC

Background and Objective:Hepatocellular carcinoma(HCC) is the secondcause of cancer-related death in the world，and half occurred in our country.multi-step processes including genetic and epigenetic alterations are thought to beaccumulated during progression of HCC.most of the abnormal expressed genes areplay a key role in the process of malignant transformation of liver cells, such asregulation of the cell cycle, cell growth, apoptosis, cell migration and diffusion.screening and identification of molecular targets involved in hepatic cell malignanttransformation is very important, and maybe a potential clinical therapeutic targetin HCC patients. In recent years, with the emergence and development of cDNAmicroarray (gene chip) technology, it has become a powerful means to look inside theabnormal expressed genes involved in tumorigenesis and progress in the wholegenome. In this study, we use the Affymetrix Human Geome U133plus2.0geneexpression profile chip, by comparing the gene expression of hcc tissues with it ofnormal liver tissues, we found the differently expressed genes. Combining withbioinformatics analysis, we can learn more about the molecular mechanism of hcctransformation and select the sensitive genes as a potential molecular targets for thegene therapy of hcc. The purpose of this study is to screen out the hepatocellularcarcinoma related genes in the whole genome.Then, made a preliminary research tostudy the influence of objective gene on the cell proliferation and apoptosis ofhepatocellular carcinoma, and develop a potential therapeutic target in HCC.PartⅠScreening differentially expressed gene of HCC usingmicroarray technologyAim：To investigate and establish the differential mRNA expression profiling betweenHepatocellular carcinoma and adjacent normal liver tissue using Affymetrix microarray chip．Then,screening of the pathogenic gene for further study.Methods：the hepatocellular carcinoma tissues of patients with HCC in the SecondAffiliated Hospital of Nanchang University were confirmed by pathologicalexamination and collected from May toDecember2013.And,the hepatocellularcarcinoma specimen bank was setup.When collectting,processing and storingspecimen,we follow the standard operating procedures. All of the patients gaveinformed consent to take part in this study. And this study was approved by the EthicsCommittee of the Second Affiliated Hospital of Nanchang University.A total of ten pairs of hepatocellular carcinoma and adjacent normal tissue in theSecond Affiliated Hospital of Nanchang University hepatocellular carcinomaspecimen bank were randomly selected and detected using the Affymetrix microarraychip. After extraction of total RNA and quality control，the RNA was processed forfluorescent labeling，hybridization，washing，scanning and signal digitizing．Then,the scanned image was input to GeneChip Operating software (GCOS1.4) softwarefor the hybridization intensity ratio. The average ratio of gray scale equal or morethan2times was considered significant．Results：The result of total RNA quality identification showed that all samples werehigh-quality and they were all suitable for microarray experiments.Significantlydifferentially expressed genes between hcc and adjacent tissues were obtained usingmicroarray and SAM software.Compared with those of adjacent normal tissue，93mRNAs were significantly up-regulated and68mRNAs were down-regulated in hcctissues．Conclusion:1. There are significantly differentially expressed mRNAs between hcc andadjacent normal liver tissues．These differentially expressed mRNAs may play animportant role in the occurrence and development of hcc and become the newmolecular markers or potential therapeutic targets of hcc in the future．2. The overexpression of EIF3B maybe play an important role in the occurrenceand development of hcc. ParⅡThe study of EIF3B on the proliferation of HCC cell lineAim：We studied the effect of EIF3B on the proliferation of HCC cell line in order toacquire a better understanding of the biological function of EIF3B in HCC．Methods：A lentiviral vector for RNA interference of the EIF3B gene were builed by jikaigene company and transfected into hcc SMMC-7721cell lines. The experiment wasdivided into two groups：negative control group(NC) and EIF3B-siRNA group．Cellcounting and colony formation experiments were performed to observe the effect ofEIF3B on proliferation of SMMC-7721cell. Flow cytometry assay were performed toevaluate the influence of down-regulation of EIF3B on the cell cycle of HCC cellline.Results：1．The result of Cell counting showed that：the number of living cells wassignificantly reduced in EIF3B-siRNA group compared with the negative controlgroup(P<0．05)，and the ability of proliferation was significantly inhibited．2．Colony formation assay revealed that：colony formation number in EIF3B-siRNA group was significantly lower than the NC group(P<0.01)．SMMC-7721cellproliferation was significantly promoted by the overexpression of EIF3B．3． Flow cytometry showed that inhibition of EIF3B could decrease theproportion of G0/G1phase hcc cells，increase that of S phase and G2/M phase cells，indicating that G2/M cell cycle arrest and the cell proliferation was inhibited．Conclusion:1. Down-reguation of EIF3B may inhibit the proliferation of hcc cells.2. EIF3B may promote the proliferation of hcc cells through cell cycle relatedpathways.