肝细胞生长因子激活剂的抑制剂-1基因对宫颈癌细胞自噬及凋亡调控的影响

Effect of hepatocyte growth factor activator inhibitor-1 gene on autophagy and apoptosis of cervical cancer cells

目的探讨肝细胞生长因子激活剂的抑制剂-1(hepatocyte growth factor activator inhibitor-1,HAI-1)基因对宫颈癌细胞自噬及凋亡调控的机制。方法对数生长期HeLa细胞分为转染组和对照组,转染组转染HAI-1质粒,对照组细胞正常培养。采用实时荧光定量PCR法检测2组细胞HAI-1 mRNA表达情况,采用AO染色和流式细胞术检测2组细胞自噬情况,采用ELISA检测2组细胞自噬相关因子LC3-Ⅱ、LC3-Ⅰ和Beclin-1蛋白水平,应用流式细胞仪检测2组细胞凋亡率,采用Western blot法检测细胞Bax和Bcl-2蛋白相对表达量。结果转染组细胞HAI-1 mRNA相对表达量(1.57±0.26)、AO细胞荧光强度(52.14±3.88)、LC3-Ⅱ蛋白[(3.42±0.85)μg/L]、Beclin-1蛋白[(14.69±1.96)μg/L]水平、细胞凋亡率[(86.75±6.42)%]、Bax蛋白相对表达量(1.24±0.13)和Bax/Bcl-2(7.75±0.83)高于对照组[0.48±0.09、5.47±0.32、(1.10±0.22)μg/L、(10.12±0.53)μg/L、(12.64±1.33)%、0.25±0.06、0.17±0.05](P<0.05),LC3-Ⅰ蛋白水平[(0.59±0.11)μg/L]、Bcl-2蛋白相对表达量(0.16±0.04)低于对照组[(3.26±0.82)μg/L、1.47±0.15](P<0.05)。结论HAI-1转染通过增加HeLa细胞中LC3-Ⅱ和Beclin-1蛋白表达,降低LC3-Ⅰ蛋白表达,提高Bax/Bcl-2来调节宫颈癌细胞的自噬和凋亡。

Objective To investigate the mechanism of hepatocyte growth factor activator inhibitor-1(HAI-1)gene on regulating the autophagy and apoptosis of cervical cancer cells.Methods HeLa cells in logarithmic growth phase were divided into transfection group transfected with HAI-1 plasmid and control group cultured normally.HAI-1 mRNA expression was detected by real-time fluorescence quantitative PCR,the autophagy was observed by AO reagent staining and flow cytometry test,the relative expressions of LC3-Ⅱ,LC3-Ⅰand Beclin-1 protein were detected by ELISA,the apoptotic rate was detected by flow cytometer,and the relative expressions of Bax and Bcl-2 protein were detected by Western blot.Results The relative expression of HAI-1 mRNA(1.57±0.26),the fluorescence intensity(52.14±3.88),the levels of LC3-Ⅱprotein((3.42±0.85)g/L)and Beclin-1 protein((14.69±1.96)μg/L),the apoptotic rate((86.75±6.42)%),and the relative expressions of Bax protein and Bax/Bcl-2(1.24±0.13,7.75±0.83)in transfection group were higher than those in control group(0.48±0.09,5.47±0.32,(1.10±0.22)μg/L,(10.12±0.53)μg/L,(12.64±1.33)%,0.25±0.06,0.17±0.05)(P<0.05),while the LC3-Ⅰlevel and the relative expression of Bcl-2 protein were lower in transfection group((0.59±0.11)μg/L,0.16±0.04)than those in control group((3.26±0.82)μg/L,1.47±0.15)(P<0.05).Conclusion HAI-1 transfection can regulate the autophagy and apoptosis of cervical cancer cells by increasing the expressions of LC3-Ⅱand Beclin-1 in HeLa cells,reducing LC3-Ⅰand increasing the expression of Bax/Bcl-2 protein.