内质膜蛋白复合体亚基6对海拉细胞自噬的调控作用

Regulation of endoplasmic reticulum membrane protein complex subunit 6 on autophagy of HeLa cells

目的 探讨内质膜蛋白复合体亚基6（EMC6）基因在海拉（HeLa）细胞中的自噬调解活动。 方法 （1）构建3种质粒：EMC6真核表达质粒、特异性干扰EMC6质粒、空白质粒。分别将3种质粒转染HeLa细胞，筛选3组中的稳定表达细胞株。（2）用实时定量聚合酶链反应（Real-time PCR）、Western blot检测3组质粒转染后Hela细胞中EMC6 mRNA表达结果。（3）电镜下观察HeLa细胞内自噬体；用自噬抑制剂Bafilomycin A1和3-甲基腺嘌呤（3-MA）分别作用于转染EMC6质粒的处理组、空白质粒组和HeLa组，在共焦显微镜下观察比较细胞株中绿色荧光蛋白（GFP）-微管相关蛋白1轻链3（LC3）的变化。 结果 （1）Real-time PCR法检测转染不同质粒进入HeLa细胞株后，处理组、干扰组及空白质粒组各5例，2－ΔΔCt的平均值分别为3.29±0.62、0.38±0.04、1.11±0.20，EMC6 mRNA拷贝数量在3组之间差异有统计学意义（P＝0.020）。（2）在转染了EMC6质粒的HeLa细胞组中可观察到自噬体形成，并观察到Bafilomycin A1作用于3组细胞后，可见GFP-LC3斑点聚集，而EMC6过表达组的LC3斑点明显较空白质粒组及HeLa组丰富。 结论 过表达EMC6蛋白可使HeLa细胞内自噬体数目增加，可能使宫颈癌细胞内自噬活动增强；EMC6过表达细胞株中，磷酸肌醇3激酶（PI3K）抑制剂抑制了EMC6所诱发的自噬过程。

Objective To study the regulatory effects of endoplasmic reticulum membrane protein complex subunit 6 （EMC6） gene on autophagy of HeLa cells. Methods （1） Three kinds of plasmids were constructed： EMC6 eukaryotic expression plasmid, specific interference EMC6 plasmid and empty plasmid. Three plasmids were transfected into HeLa cells. The stable expression cell lines were screened in three groups. （2） The expression of EMC6 mRNA in HeLa cells transfected with three groups of plasmids was detected by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting. （3） The autophagy of HeLa cells was observed by electron microscopy. Bafilomycin A1 and 3-methyladenine （3-MA）, respectively, were used to treat the transfected EMC6 plasmid in the treatment group, and the blank plasmid group and the HeLa group. The changes of green fluorescent protein （GFP）-microtubule-associated protein 1 light chain 3 （LC3） in cell lines were observed under confocal microscope. Results （1） Acorrding to the the transfection with different plasmids into HeLa cells detected by Real-time PCR, the cells were divided into three groups： the treatment group, the interference group and the empty plasmid group with 5 cases in each group. 2-ΔΔCt, on average, was 3.29±0.62, 0.38±0.04, and 1.11±0.20 respectively. EMC6 mRNA copies showed statistically significant differences among the three groups （P=0.020）. （2） In the HeLa cells transfected with EMC6 plasmid, autophagosome formation could be observed. After treatment with A1, on the three groups of cells, the spot aggregation of GFP-LC3 was seen, while in EMC6 overexpression group LC3 spots were increased as compared with those in empty plasmid group and HeLa group.Conclusion Overexpression of EMC6 protein can increase the number of autophagy in HeLa cells, which may enhance the activity of autophagy in cervical cancer cells. In EMC6 overexpressing cell lines, phosphatidylinositol 3 kinase （PI3K） inhibitors inhibit autophagy induced by EMC6.