内质网应激-自噬反应在顺铂诱导宫颈癌HeLa细胞死亡中的作用

Role of endoplasmic reticulum stress-autophagic response in cisplatin-induced cervical cancer HeLa cells death

目的研究内质网应激-自噬反应在顺铂诱导宫颈癌HeLa细胞死亡中的作用,以及自噬基因Beclin 1对HeLa细胞顺铂敏感性的影响。方法不同浓度顺铂作用HeLa细胞后,丹磺酰戊二胺染色法观察细胞中自噬体的形成,Western blot检测自噬相关蛋白Beclin 1和LC3的表达及内质网应激相关蛋白IRE1、PERK和ATF6的表达。将自噬基因Beclin 1的真核表达载体pcDNA3.1(+)-Beclin1转染HeLa细胞,Western blot检测Beclin 1在HeLa细胞中的蛋白表达,噻唑蓝比色(MTT)法检测顺铂对HeLa细胞半数抑制浓度(IC50)的变化,流式细胞仪检测凋亡细胞的百分率。结果顺铂作用HeLa细胞后,细胞内自噬体形成增加,自噬蛋白Beclin 1和LC3表达增加,且均呈现时间依赖性(P<0.05),内质网应激相关蛋白IRE1、PERK和ATF6的表达增强。pcDNA3.1(+)-Beclin1转染HeLa后,Beclin 1蛋白表达增加(P<0.05);MTT检测IC50由转染前的1.0μg/ml下降到0.5μg/ml;凋亡细胞百分率升高,与对照组相比差异有统计学意义(P<0.05)。结论内质网应激-自噬反应参与了顺铂对宫颈癌HeLa细胞的细胞毒性作用,自噬基因Beclin 1过表达能够增加HeLa细胞对顺铂的敏感性。

Objective To investigate the role of endoplasmic reticulum（ER） stress - autophagic response in cisplatin-induced cervical cancer HeLa cell death, and to explore the effect of Beclin 1 on sensitivity of HeLa cells to cisplatin. Methods Hela cells were treat ed with different concentrations of cisplatin, respectively. Monodansylcadaverin （MDC） method was explored to measure the formation of autophagic vacuoles, and Western blot was used to detect the protein expression of Beclin 1, LC3, IRE1, PERK and ATF6. The eu-karyotic expression vector of Beclin 1 was transfected via lipofectamine into HeLa cells, MTY assay was employed to observe the effect of cisplatin on the IC50 of HeLa cells, and flow cytometry was used to observe the percentage of autophagic cells and apoptotic cells. Re- sults The autophagosomes were increased after treated with cisplatin, and the expression of Beclin 1 and LC3 was significantly in-creased in a dose-dependent manner in HeLa ceils （ P 〈 0.05）, while the expression of IRE 1, PERK and ATF6 was increased too. The the expression of Beclinl significantly increased after pcDNA3. 1 （ ＋ ）-Beclinl vector being transfected into HeLa cells （ P 〈 0.05 ）. MTY assay showed the ICs0 value decreased from 1.0 μg/ml to 0.5 μg/ml, and more apoptotic cells were identified after pcD-NA3.1 （ ＋ ） - Beclinl being transfected （ P 〈 0.05 ）. Conclusion ER stress-autophagic response plays an important role in the process of cisplatin-induced HeLa cells death, and Beclin 1 overexpression can increase the sensitivity of HeLa cells to cisplatin.