USP22in Hepatoma And The Gene Silence Of USP22by SiRNA Influence The Growth Of Hepatoma Cells

Objective: Hepatocellular carcinoma (HCC) is the fifth most commonmalignant tumor worldwide and causes approximately600,000deaths globallyeach year. Despite improvements in clinical treatments, such as surgicalresection, liver transplantation and interventional therapy, HCC prognosis is stillvery poor. Because tumorigenesis and tumor progression in hepatic cells are theresults of multiple genetic alterations, a single molecule targeting therapy hasyet to be discovered. Thus, identification of target molecules that control thebiological characteristics of HCC is of great importance. Ubiquitin carboxylterminal-22hydrolase（USP22) is a newly discovered the ubiquitin proteasomefamily, can be applied to ubiquitin-proteasome system of ubiquitin proteinsubstrates, by cutting off and the connection between the substrate proteinubiquitin chain, and play a wide range of biological functions, it has proved witha wide variety of tumor malignant degree, proliferation, metastasis andprognosis related, its expression in HCC is unknown. This study examined theUSP22mRNA and protein expression level in liver cancer tissue and cells, thedesign and synthesis of USP22gene specific siRNA, and transfection HepG3Bhepatocellular carcinoma cell line, silent USP22gene expression in HepG3B cells, preliminarily USP22gene for liver cancer HepG3B cell proliferation,apoptosis and cell cycle. To explore the expression of ubiquitincarboxyl-terminal hydrolase22(USP22) in hepatoma and the gene silence ofUSP22by siRNA influence the proliferative activity, apoptosis and cell cycle ofthe hepatocellular carcinoma cell line HepG3B. Methods: A total of104primary HCC patients from the archives of the Department of Pathology in theAffiliated Hospital of Guilin Medical University (Guilin, China) were initiallyrecruited in our study. Immunohistochemistry was used to detect the expressionof USP22in58specimens of hepatocellular carcinoma(HCC) andparaneoplastic liver tissue,RT-PCR and Western blot was used to detect theexpression of USP22in hepatocellular carcinoma cells, receive operatingcharacteristic (ROC) curve analysis was employed to determine a cutoff scorefor USP22expression in a training set (n=59). For validation, the ROC-derivedcutoff score was subjected to analysis of the association of USP22expressionwith patient outcome and clinical characteristics in a testing set (n=59) andoverall patients (n=104).siRNA targeting USP22mRNA was transfected intohepatocellular carcinoma cell line HepG3B, RT-PCR and Western blot was usedto detect the transfer efficiency, MTT assay was used to detect the cellproliferation inhibiting rate; Flow cytometry was used to analyze the apoptosisrate of cells and the cell cycle; Western blot was used to detect the expression ofD1、CDK4and CDK6protein. Results: Immunohistochemical results showthat the ubiquitin carboxyl terminal-22hydrolase(USP22) in liver tissue of thepositive expression rate significantly higher than the corresponding tissueadjacent to carcinoma and normal liver tissues (p <0.01);Rt-pcr results showedthat the ubiquitin carboxyl terminal-22(USP22) RNA in liver cancer tissue of expression is significantly higher than the corresponding adjacent tissues andnormal liver tissue;Western blot results showed that the protein ubiquitincarboxyl terminal-22(USP22) in the tissue of liver cancer is significantlyhigher than the corresponding tissues and normal liver tissue adjacent tocarcinoma;Rt-pcr and Western blot test showed that human liver cell linesHepG2, HepG3B, Bel-7402USP22relative expression level was significantlyhigher than in human normal liver cell line Chang live; after transfected with thesiRNA which targeting USP22mRNA, the expression of USP22were greatlydecreased; The cell proliferation inhibiting rate by MTT were42.24%,significantly higher than the negative control group（tsiUSP22group=22.47，p＜0.01）; the apoptosis rate by flow cytometry were33.08%±4.53%, significantlyhigher than the negative control group and the blank control group（tsiUSP22group=12.34，p＜0.01）; the rate of cells in the G1phase by flow cytometry were68.81%±2.71%, significantly higher than the negative control group and theblank control group（tsiUSP22group=33.91，p＜0.01）; Western blot analysisshowed that the expression of D1、CDK4and CDK6protein higher than thenegative control group and the blank control group（tCyclinD1=10.85，tCDK4=10.51，tCDK6=10.59，p＜0.01）. Conclusions: The ubiquitin carboxylterminal-22hydrolase (USP22) in liver cancer tissue of expression issignificantly higher than the corresponding adjacent tissues and normal livertissue; In human liver cancer cell HepG2, HepG3B, Bel-7402expression ofUSP22was obviously higher than that of normal liver cells in Chang liver;Usedfor the ubiquitin carboxyl terminal-22hydrolase (USP22) gene transfectionspecific siRNA sequences HepG3B cell, HepG3B inhibits hepatocellularcarcinoma cell proliferation activity, can induce the apoptosis rate increases, and leads to cell cycle protein CyclinD1and CDK4and CDK6expression levelsdrop, which in turn caused cell G1cell cycle arrest.