PSMB8在人绒毛膜滋养层细胞中对细胞凋亡和自噬的影响

Effect of PSMB8 on Apoptosis and Autophagy in Human Chorionic Trophoblast Cells

目的初步探讨蛋白酶体亚基PSMB8(proteasome subunit beta 8)对人绒毛膜滋养层细胞(HTR-8/Svneo)凋亡及自噬的影响。方法将HTR-8细胞分为转染组和对照组,采用瞬时转染的方法分别将PSMB8 siRNA-1、PSMB8 siRNA-2转入转染组中,将NC siRNA转入对照组中。转染48h后用qRT-PCR进行干涉效率验证,CCK-8检测细胞增殖情况,TUNEL和流式细胞术分析细胞凋亡情况,免疫荧光染色检测细胞内caspase-3(cleaved)和LC3蛋白的表达,qRT-PCR检测细胞中自噬相关基因mRNA表达水平。结果qRT-PCR结果显示,PSMB8 siRNA-1组干扰效果较好。与对照组比较,PSMB8 siRNA-1组细胞增殖能力减弱,HTR-8细胞中TUNEL(15.05±2.62 vs 33.40±2.44,P=0.000)和caspase-3(cleaved)(21.70±5.87 vs 53.29±10.29,P<0.01)阳性率增加。流式结果显示,与对照组比较,PSMB8 siRNA-1组存活细胞比例减少(89.10±0.78 vs 77.00±1.68,P=0.000),早期凋亡和晚期凋亡细胞比例增加(9.09±0.65 vs 10.71±0.61,P<0.05),坏死细胞比例也增加(2.49±0.73 vs 12.49±2.02,P<0.01)。与对照组比较,PSMB8 siRNA-1组LC3蛋白阳性细胞率增加(19.02±3.78 vs 31.90±4.87,P<0.05),各自噬相关基因mRNA相对表达水平均上调。结论在HTR-8细胞中沉默PSMB8基因表达可以诱导细胞凋亡并且激活自噬。

Objective The effects of down-regulation of proteasome subunit beta 8 on apoptosis and autophagy of human chorionic trophoblast cells(HTR-8/Svneo)were investigated.The effects of PSMB8 on apoptosis and autophagy of human chorionic trophoblast cells(HTR-8/Svneo)were investigated in this study.Methods HTR-8 cells were divided into transfection group and control group.PSMB8 siRNA-1 and PSMB8 siRNA-2 were transfected into the transfection group respectively,and NC siRNA was transferred into the control group.After 48 hours of transfection,qRT-PCR was used to verify the interference efficiency,CCK-8 was used to detect cell proliferation,TUNEL and flow cytometry were used to analyze apoptosis,and immunofluorescence staining was used to detect the expression of caspase-3(cleaved)and LC3 protein in cells.The expression level of autophagy-related gene mRNA in the cells was examined by qRT-PCR.Results qRT-PCR results showed that PSMB8 siRNA-1 group had better interference effect.Compared with the control group,the cell proliferation ability of PSMB8 siRNA-1 group was weakened,TUNEL(15.05±2.62 vs 33.40±2.44,P=0.000)and caspase-3(cleaved)(21.70±5.87 vs 53.29±10.29,P<0.01)positive cell rates were increased.Flow cytometry results showed that the proportion of viable cells in the PSMB8 siRNA-1 group was reduced compared with the control group(89.10±0.78 vs 77.00±1.68,P=0.000),and the proportion of early apoptosis and late apoptotic cells increased(9.09±0.65 vs 10.71±0.61,P<0.05),the proportion of necrotic cells also increased(2.49±0.73 vs 12.49±2.02,P<0.01).Compared with the control group,the rate of LC3 protein positive cells in PSMB8 siRNA-1 group was increased(19.02±3.78 vs 31.90±4.87,P<0.05),and the relative mRNA expression levels of autophagy related genes were up-regulated.Conclusion Down-regulation of PSMB8 can induce apoptosis and activate autophagy in HTR-8 cells.