内质网膜蛋白复合物亚单位6调控细胞增殖和凋亡对宫颈癌Hela细胞放射线敏感性的影响

Effect of endoplasmic reticulum membrane protein complex subunit 6 regulating cell proliferation and apoptosis on radiosensitivity of cervical cancer Hela cells

目的探讨内质网膜蛋白复合物亚单位6(EMC6)调控细胞增殖和凋亡对宫颈癌Hela细胞放射线敏感性的影响。方法将宫颈癌Hela细胞分为CON组(正常宫颈癌Hela细胞)、shCtrl组(EMC6基因空白质粒转染宫颈癌Hela细胞)和shEMC6组(EMC6基因短发夹RNA干扰表达质粒转染宫颈癌Hela细胞),给予2、4、6、8 Gy X线外照射处理,照射处理后采用定量聚合酶链反应法检测EMC6 mRNA表达水平,蛋白质印迹法检测EMC6蛋白表达水平,克隆形成实验检测细胞增殖水平,流式细胞术检测细胞凋亡水平和细胞周期。结果shEMC6组EMC6 mRNA表达水平低于shCtrl组[(0.530±0.017)比(1.004±0.108)](P=0.002),EMC6的敲减率为47.24%。shEMC6组EMC6蛋白表达水平低于shCtrl组[(0.506±0.012)比(0.528±0.005)](P=0.023)。2、4 Gy X线照射处理后,shEMC6组细胞增殖水平均低于shCtrl组和CON组(均P<0.05)。2 Gy X线照射后4、8、12 h,shEMC6组细胞凋亡率均高于shCtrl组[(45.71±0.66)%比(34.86±1.16)%、(13.85±0.24)%比(12.85±0.19)%、(22.74±0.14)%比(17.77±0.40)%],差异均有统计学意义(均P<0.05)。2 Gy X线照射前0 h,shEMC6组S期细胞比例低于shCtrl组,G_(2)期细胞比例高于shCtrl组(均P<0.05);照射后4 h,shEMC6组S期细胞比例低于shCtrl组(P<0.05),G_(2)期细胞比例与shCtrl组比较差异无统计学意义(P>0.05);照射后8 h,shEMC6组S期细胞比例高于shCtrl组,G_(2)期细胞比例低于shCtrl组(均P<0.05);照射后12 h,shEMC6组S期细胞比例与shCtrl组比较差异无统计学意义(P>0.05),G_(2)期细胞比例低于shCtrl组(P<0.05)。结论宫颈癌Hela细胞沉默EMC6后,细胞增殖减少,同时细胞凋亡和G_(2)期细胞数量显著增加,对放射线的敏感性显著提高。

Objective To investigate the effect of endoplasmic reticulum membrane protein complex subunit 6(EMC6)regulating cell proliferation and apoptosis on radiosensitivity of cervical cancer Hela cells.Methods Cervical cancer Hela cells were divided into CON group(normal cervical cancer Hela cells),shCtrl group(EMC6 gene blank plasmid was transfected into cervical cancer Hela cells)and shEMC6 group(EMC6 gene short hairpin RNA interference expression plasmid was transfected into cervical cancer Hela cells).They were treated with 2,4,6 and 8 Gy X-ray external irradiation.After irradiation,the expression level of EMC6 mRNA was detected by quantitative polymerase chain reaction and the expression level of EMC6 protein was detected by western blotting.The level of cell proliferation was detected by clone formation assay,and the level of apoptosis and cell cycle were detected by flow cytometry.Results The expression level of EMC6 mRNA in shEMC6 group was lower than that in shCtrl group[(0.530±0.017)vs(1.004±0.108)](P=0.002),and the knockdown rate of EMC6 was 47.24%.The expression level of EMC6 protein in shEMC6 group was lower than that in shCtrl group[(0.506±0.012)vs(0.528±0.005)](P=0.023).After 2,4 Gy X-ray irradiation,the level of cell proliferation in shEMC6 group was lower than that in shCtrl group and CON group(both P<0.05).At 4,8 and 12 h after 2 Gy X-ray irradiation,the apoptosis rates of shEMC6 group were higher than those of shCtrl group[(45.71±0.66)%vs(34.86±1.16)%,(13.85±0.24)%vs(12.85±0.19)%,(22.74±0.14)%vs(17.77±0.40)%](all P<0.05).At 0 h before 2 Gy X-ray irradiation,the proportion of S-phase cells in shEMC6 group was lower than that in shCtrl group,and the proportion of G_(2)-phase cells was higher than that in shCtrl group(both P<0.05);4 h after irradiation,the proportion of S-phase cells in shEMC6 group was lower than that in shCtrl group(P<0.05),and there was no significant difference in the proportion of G_(2)-phase cells between shEMC6 group and shCtrl group(P>0.05);8 h after irradiation,the proportion of S-phase cells in shEMC6 group was higher than that in shCtrl group,and the proportion of G_(2)-phase cells was less than that in shCtrl group(all P<0.05);12 h after irradiation,the proportion of S-phase cells in shEMC6 group was not significantly different from that in shCtrl group(P>0.05),and the proportion of G_(2)-phase cells was lower than that in shCtrl group(P<0.05).Conclusion The silencing of EMC6 in Hela cells resulted in a decrease in cell proliferation,together with a significant increase in apoptosis and the number of G_(2)-phase cells,and a significant increase in sensitivity to radiation.