泛素水解酶22在肝癌中的表达及siRNA沉默泛素水解酶22基因对肝癌细胞生长的影响

USP22 expression in hepatocellular carcinoma and gene silence of USP22 by siRNA inhibits thegrowth of cancer cells

目的研究泛素水解酶22（ubiquitin carboxyl-terminal hydrolase22，USP22）基因在肝癌中的表达及沉默USP22基因对肝癌细胞的增殖活性、细胞凋亡和细胞周期的影响。方法采用免疫组化检测58例肝细胞癌组织及相应癌旁组织中USP22蛋白的表达情况，采用RT-PCR和Westernblot检测肝癌细胞中USP22的表达水平，并合成针对USP22基因的siRNA转染肝癌细胞；采用RT-PCR检测和Westernblot检测转染效率；MTY法测定细胞生长抑制率；流式细胞仪测定细胞凋亡率和细胞周期；免疫印迹检测CyclinDl、CDK4和CDK6蛋白表达水平。结果发现USP22基因在肝癌组织及细胞中高表达；用针对USP22siRNA干扰后，USP22表达水平明显降低；MTr法检测其细胞生长抑制率为42．24％，明显高于对照组（tsiUSP22组=22．47，P〈0．01）；流式细胞仪检测其细胞凋亡率为33．08％-4-d．53％，明显高于对照组（tsiUSP22组=12．34，P〈0．01）；流式细胞仪检测细胞周期，发现其处于G1期细胞的百分比为68．81％±2．71％，明显高于对照组（tsiUSP22组=33．91，P〈0．01）；Westernblot检测发现细胞周期蛋白CyclinDl、CDK4和CDK6在USP22siRNA组中的表达水平明显低于对照组（tCyclinD1=10．85，tcDK4=10．51，tcDK6=10．59，P〈0．01）。结论UPS22基因在肝癌组织及细胞中存在异常高表达，利用siRNA干扰UPS22表达，可抑制肝癌HepG3B细胞增殖活性，诱导其凋亡，并导致细胞周期被阻滞在G1期以内。

Objective To explore the expression of ubiquitin carboxyl-terminal hydrolase 22 （USP22） in hepatocellular carcinoma （HCC） and the impact of USP22 gene silence by siRNA on the proliferative activity, apoptosis and cell cycle of HCC cell line HepG3B. Methods Immunohistochemistry was used to detect the expression of USP22 in 58 HCC specimens and paraneoplastic liver tissues, RT-PCR and Western blot was used to detect the expression of USP22 in HCC cells, siRNA targeting USP22 mRNA was transfected into cell line HepG3B, RT-PCR and Western blot was used to detect the transfer efficiency, MTF assay was used to detect the cell proliferation inhibiting rate; Flow cytometry was used to analyze the apoptosis rate of cells and the cell cycle; Western blot was used to detect the expression of D1, CDK4 and CDK6 protein. Results USP22 was highly expressed in HCC; after transfected siRNA targeting USP22, the expression of USP22 decreased; The cell proliferation inhibiting rate was 42. 24% , higher than the negative control group （ tsiusP22group = 22.47, P 〈 0. 01 ） ; the apoptosis rate was 33.08 % ＋ 4. 53 % , higher than negative control and blank control group（tsiusP22group = 12. 34,P 〈0. 01 ） ; The rate of cells in the G1 phase was 68.81% ± 2. 71%, higher than negative control and blank control group （ tsiUSP22group= 33.91, P 〈 0. 01 ） ; D1, CDK4 and CDK6 protein expression was higher than negative control and blank control group（tCyclinD1 = 10.85, tCDK4 = 10.51, tCDK6=10.59, P 〈 0.01 ）. Conclusions USP22 gene was highly expressed in HCC, specific siRNA targeting USP22 efficiently inhibits cell proliferation, promotes apoptosis and caused cell cycle arrest in G1 phase in HepG3B cells.