泛素结合酶E2T对卵巢癌细胞增殖的影响

Effect of UBE2T on proliferation of human ovarian cancer cell line SKOV3

目的 观察泛素结合酶E2T(UBE2T)对人卵巢癌细胞株SKOV3增殖的影响,并探讨其机制。方法 构建UBE2T-shRNA和携带UBE2T基因的慢病毒载体,将其转染至经过培养传代的人卵巢癌细胞株SKOV3,筛选稳定株,分别记为下调组、上调组;另转染NC-shRNA阴性对照慢病毒载体至人卵巢癌细胞株SKOV3,记为阴性对照组;并取未转染的人卵巢癌细胞株SKOV3,记为空白对照组。培养72 h。噻唑蓝(MTT)法、流式细胞仪、反转录-实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质免疫印迹法(WB)分别检测细胞增殖抑制率、细胞周期、UBE2T及其下游相关基因和蛋白表达。结果 下调组细胞增殖抑制率、G1期细胞占比均高于其余3组(P<0.05),S期和G2期细胞占比、UBE2T、蛋白激酶B(AKT)、细胞周期蛋白D1(Cyclin D1)mRNA及蛋白表达、p-AKT水平均低于其余3组(P<0.05),上调组细胞增殖抑制率、G1期细胞占比均低于阴性对照组与空白组(P<0.05),S期和G2期细胞占比、UBE2T、AKT、Cyclin D1 mRNA及蛋白表达、p-AKT水平均高于阴性对照组与空白组(P<0.05)。结论 UBE2T基因下调可抑制人卵巢癌细胞株SKOV3增殖。

Objective To observe the effect of ubiquitin binding enzyme e2t(UBE2T) on the proliferation of human ovarian cancer cell line SKOV3 and explore its mechanism. Methods UBE2T shRNA and lentiviral vector carrying UBE2T gene were constructed and transfected into human ovarian cancer cell line SKOV3 after subculture. In addition, NC shRNA negative control lentiviral vector was transfected into human ovarian cancer cell line SKOV3, which was recorded as negative control group. The untransfected human ovarian cancer cell line SKOV3 was taken as blank control group, which were cultured for 72 hours. MTT assay, flow cytometry, RT-qPCR and Western blot(WB) were used to detected the cell proliferation inhibition rates, cell cycle, m RNA and protein expressions of UBE2T and its downstream. Results The inhibition rate of cell proliferation and the proportion of G1 phase cells in the down regulation group were higher than those in the other three groups(P<0.05), which in the up regulation group were lower than those in the negative control group and blank control group(P<0.05), and the percentage of cells in S phase and G2 phase, the mRNA and protein expressions of UBE2T, AKT,Cyclin D1 and the level of p-Akt in S phase and G2 phase were lower than those in the other three groups(P<0.05), which in the up regulation group were higher than those in the negative control group and blank control group(P<0.05). Conclusion UBE2T gene down regulation can inhibit proliferation of human ovarian cancer cell line SKOV3.