抑制USP22基因对人结直肠癌细胞增殖的影响

Effects of USP22 inhibition on proliferation of colorectal cancer cells

目的 利用RNA干扰技术探讨泛素特异性肽酶22(USP22)对人结直肠癌细胞增殖的影响。方法免疫荧光检测结直肠癌组织中USP22的表达;Western blotting检测体外培养的HCT-116、SW480、HC-29结直肠癌细胞株中USP22的表达,筛选出表达量最高的细胞作为研究细胞。设计、合成针对USP22特异性siRNA,同时设置阴性对照siRNA,利用脂质体转染结直肠癌细胞后,Western blotting检测siRNA抑制效率;通过MTT法检测细胞的增殖能力;流式细胞术检测细胞的周期及凋亡;Western blotting检测周期相关蛋白CyclinB1、p21、p53、CDK2的表达。结果 结直肠癌组织中USP22表达量与癌旁组织比较,差异有统计学意义(P<0.05),结直肠癌组织中USP22表达量升高。SW480细胞中USP22的表达量最高,因此作为靶细胞进行后续研究。与对照组比较,USP22 siRNA能够降低USP22的表达(P<0.05)。USP22抑制后,与对照组比较,能够抑制SW480细胞的增殖,促使SW480细胞停滞于G0/G1期,抑制细胞周期进程,增加细胞凋亡率(P<0.05),进而抑制细胞增殖(P<0.05);同时,与对照组比较,CyclinB1、CDK2的表达量降低(P<0.05),p53和p21的表达量升高(P<0.05)。结论 USP22抑制后,能够抑制结直肠癌细胞的增殖,其机制可能与抑制细胞周期进程、促进细胞凋亡有关。

Objective To discuss the effect of ubiquitin specifc protease 22(USP22)on the growth of colorectal cancer cells.Methods The expression of USP22 in colorectal cancer tissue was detected by immunofluorescence.The expression of USP22 in the three kinds of colorectal cancer cells(HCT-116,SW480,HT-29)were detected by western blotting analysis to select the highest expressed cell line.The unique siRNA for USP22 gene and the negative control siRNA were designed and produced.After transfection,the efficiency of siRNA was detected by western blotting analysis.The cell proliferation was tested by MTT analysis.The cell cycle and apoptosis were tested by flow cytometry.The altered expressions of cell cycle related genes(CyclinB1,p21,p53,and CDK2)were detected by western blotting.Results The results of immunofluorescence showed that the expression of USP22 in cancer tissue was higher than in para-carcinoma tissue(P<0.05).With the highest USP22 expression of SW480 cells selected to further experiment,the western blotting results showed that the expression of USP22 was significantly reduced by siRNA transfection(P<0.05).Silencing USP22 could significantly suppress the proliferation of SW480 cells,and induce them to arrest during the G0/G1 in order to increasing apoptosis rate(P<0.05)and inhibiting cell proliferation(P<0.05).Meanwhile,compared to negative control group,the expression of CyclinB1 and CDK2 was significantly decreased(P<0.05),and the expression of p21 and p53 was significantly increased(P<0.05).Conclusions Silencing USP22 could suppress the proliferation of colorectal cancer cells,and the mechanism might relate to cell cycle suppression and cell apoptosis promotion.