中国全科医学Issue(33)：4077-4083,7.DOI:10.3969/j.issn.1007-9572.2015.33.013
泛素特异性蛋白酶18在肝癌细胞中的表达及其对生物学活性的影响研究
Expression of USP18 in Hepatoma Carcinoma Cells and Its Influence on Biological Activity
摘要
Abstract
Objective To detect the expression of USP18 in hepatoma carcinoma cells and its influence on biological activity(cell proliferation,cell clone,cell cycle and cell apoptosis). Methods Selected 5 kinds of hepatoma cell lines from July 2013 to July 2014( Hep G2,Hep G2. 2. 15,Hep 3B,Huh - 7 and SMMC - 7721). RT - PCR and Western blotting method were used to detect their expression levels of USP18 mRNA and USP18,by which we found out the hepatoma cell line with the highest expression levels and conducted siRNA transfection on it. LipofectamineTM 2000 was used to divide USP18 siRNA transfected hepatoma carcinoma cells into normal group,negative control group,siRNA1 group,siRNA2 group and siRNA3 group. RT - PCR and Western blotting were employed to measure interference efficiency,and MTT method was used to make cell growth curves. Plate clone formation assay was employed to measure clone formation rate,flow cytometry was undertaken to examine the cycle distribution of cells,and flow cytometry of Annexin V - FITC/ PI staining was used to detect cell apoptosis rate. Results The five kinds of hepatoma carcinoma cell lines were significantly different(P < 0. 05)in the expression levels of USP18 mRNA and USP18,among which Hep 3B was found higher(P < 0. 05)than other four kinds of hepatoma cell line in the expression levels of USP18 mRNA and USP18,so we chose Hep 3B to conduct the following tests. At 48 hours after USP18 siRNA transfection of Hep 3B,the transfection rate reached( 91. 00 ± 5. 67 )% . The five groups were significantly different in interference efficiency(P < 0. 05),among which siRNA1 group,siRNA2 group and siRNA3 group were higher than normal group and negative control group in interference efficiency(P < 0. 05). On day 1,day 2 and day 3 after transfection,siRNA1 group,siRNA2 group and siRNA3 group were lower than normal group and negative control group in absorbancy(P < 0. 05). siRNA1 group,siRNA2 group and siRNA3 group were lower(P < 0. 05)in clone formation rate and higher(P < 0. 05)in cell apoptosis rate than normal group and negative control group. The five groups were significantly different in cell cycle distribution (P < 0. 05). Normal group and negative control group were not significantly different in absorbance value,clone formation rate, cell cycle distribution and cell apoptosis rate( P > 0. 05). Conclusion Hep 3B has high expression level of USP18,and USP18 gene knockout can inhibit the proliferation of hepatoma carcinoma cells,reduce cell clone,retard cell cycle progress and promote cell apoptosis.关键词
肝肿瘤/泛素特异性蛋白酶/细胞增殖/细胞凋亡Key words
Liver neoplasms/Ubiquitin specific peptidase/Cell proliferation/Apoptosis分类
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颜伟,刘安文,蔡婧,汪弋汀,曾小莉,陈雅洁,熊乐,国畅阔..泛素特异性蛋白酶18在肝癌细胞中的表达及其对生物学活性的影响研究[J].中国全科医学,2015,(33):4077-4083,7.基金项目
江西省自然科学基金资助项目 （）
