沉默泛素特异性肽酶22对人胃癌细胞增殖的影响及其机制

Potential mechanism of specific small interfering RNA against ubiquitin - specific protease 22 gene on proliferation of human gastric carcinoma cells

目的探讨泛素特异性肽酶22（USP22）对人胃癌细胞增殖的影响及其作用机制。方法设计、合成针对USP22特异性小干扰RNA（siRNA）片段，通过实时定量反转录聚合酶链反应（RT-qPCR）、Western blot筛选抑制效率最高的siRNA片段以备后续实验；抑制USP22后，噻唑蓝（MTT）法检测胃癌细胞SGC-7901细胞增殖，流式细胞术检测细胞周期及凋亡变化；抑制USP22后，Western blot检测周期、凋亡相关蛋白的表达变化。结果成功设计、合成针对USP22 siRNA片段，其抑制效率高达80%；USP22抑制后，胃癌细胞SGC-7901增殖明显受到抑制，细胞停滞于G0/G1期[（52.87±1.41）%比（73.73±1.78）%，t＝15.491，P＝0.000]。同时凋亡率显著增加[（19.63±0.82）%比（4.54±0.46）%，t＝58.051，P＝0.000]；Western blot结果显示bcl-2相关X蛋白（bax）表达水平显著上升（t＝9.580，P＝0.001）、B细胞淋巴瘤/白血病-2（bcl-2）及pro-半胱氨酰天冬氨酸特异性蛋白酶-3（Caspase-3）的表达量显著降低（t＝6.674、7.269；P＝0.000、0.000）；磷酸化蛋白激酶B（p-Akt）及下游磷酸化糖原合成酶激酶3β（p-GSK3β）表达水平显著降低（t＝13.512、6.846；P＝0.003）。结论抑制USP22后，可能通过影响Akt活化，抑制胃癌细胞增殖及诱导细胞凋亡。

Objective To explore the effects of ubiquitin-specific protease 22 （USP22） on the proliferation of human gastric carcinoma cells and the potential mechanism.MethodsThe specific small interfering RNA （siRNA） fragments of USP22 were designed, and the highest efficient siRNA was selected by real-time quantitative reverse transcriptase-polymerase chain reaction （RT-qPCR） and Western blotting. The proliferation of SGC-7901 cells was tested using methyl thiazol tetrazolium （MTT） assay after USP22 silencing, and the cell cycle and apoptosis were tested by flow cytometry. The altered expression of cell cycle and apoptosis related proteins was detected by Western blotting.ResultsThe efficiency of USP22 siRNA was more than 80%. The proliferation of SGC-7901 cells was significantly suppressed after USP22 silencing, and the cell cycle was arrested in G0/G1 phase [（52.87±1.41）% vs. （73.73±1.78）%, t=15.491, P=0.000]. Meanwhile, the apoptosis rate was significantly increased by USP22 silencing [（19.63±0.82）% vs. （4.54±0.46）%, t=58.051, P=0.000]. The results of Western blotting revealed that the expression of B cell lymphoma/leukemia-2 associated X protein （bax） was significantly increased （t=10.630, P=0.000）, whereas that of B-cell lymphoma/leukemia-2 （bcl-2） and pro-cysteinyl aspartate-specific protease （Caspase）-3 was significantly reduced （t=6.674, 7.269; P=0.000）. Furthermore, the phosphorylation of protein kinase B （Akt） and glycogen synthase kinase 3β （GSK3β） was significantly reduced （t=13.512, 6.846; P=0.003）.ConclusionCell growth of SGC-7901 cells was inhibited by USP22 silencing via reducing the activity of Akt.