葡萄糖调节蛋白78对宫颈癌SiHa细胞生物学行为的影响

Effects of glucose-regulated protein 78 on biological behavior of cervical cancer SiHa cells

目的:探究GRP78对宫颈癌SiHa细胞生物学行为的影响以及作用机制。方法:采用RNAi技术沉默SiHa细胞中GRP78基因的表达。将针对不同位点设计的siRNA通过脂质体介导分别转染入SiHa细胞,通过免疫印迹法和qRT-PCR分别检测转染前后SiHa细胞GRP78蛋白和GRP78 mRNA的表达。划痕实验检测SiHa细胞的迁移能力;Transwell实验检测SiHa细胞的侵袭能力;免疫印迹法检测上皮间质转化相关蛋白及增殖相关蛋白的表达;流式细胞术检测SiHa细胞的凋亡率。结果:转染后,GRP78 mRNA和GRP78蛋白表达水平均明显降低且GRP78-siRNA1501的沉默效果最显著,基因水平和蛋白水平的检测总体趋势相符合;此外,与空白组和NC-siRNA组相比,GRP78-siRNA1501组SiHa细胞中E-cadherin蛋白表达水平明显增高,N-cadherin、Vimentin蛋白表达水平明显降低,且增殖相关蛋白P-AKT^(ser473)、P-GSK3β^(ser9)、Cyclin D1、CDK4表达水平也明显降低;Annexin V-FITC/PI双染结果显示,GRP78-siRNA1501组SiHa细胞的凋亡率明显高于NC-siRNA组。结论:沉默SiHa细胞中GRP78基因,会导致SiHa细胞增殖、侵袭和迁移能力减弱,并促进其凋亡。增殖能力减弱可能与抑制AKT/GSK3β/Cy-clin D1信号通路有关,侵袭和迁移能力减弱可能与抑制上皮间质转化的进程有关。

Objective:To investigate the effect of GRP78 on the biological behavior of cervical cancer SiHa cells and its mech-anism.Methods:The expression of GRP78 gene in SiHa cells was silenced by RNAi technology.siRNA designed for different sites was transfected into SiHa cells through liposome mediated transfection.Western blot and qRT-PCR were used to detect the expression of GRP78 protein and GRP78 mRNA in SiHa cells before and after transfection.The migration and invasion ability of SiHa cells were detected by scratch test and Transwell assay respectively.Western blot was used to detect the expression of the proteins associated with epithelial-mesenchymal transition and proliferation.Flow cytometry was used to detect the apoptosis rate of SiHa cells.Results:GRP78 mRNA and GRP78 protein expression levels were significantly decreased after transfection,and GRP78-siRNA1501 demon-strated the most significant silencing effect.The gene level was consistent with the overall trend of the protein level.In addition,com-pared with the blank group and the NC-siRNA group,the expression level of E-cadherin protein in SiHa cells was significantly in-creased in the GRP78-siRNA1501 group,but the expression levels of N-cadherin and Vimentin protein were significantly decreased,and the expression levels of proliferation-related proteins P-AKT^(ser473),P-GSK3β^(ser9),Cyclin D1 and CDK4 were also significantly de-creased.Annexin V-FITC/PI double staining results showed that the apoptosis rate of SiHa cells in GRP78-siRNA1501 group was sig-nificantly higher than that in NC-siRNA group.Conclusion:Silencing GRP78 gene in SiHa cells leads to decreased proliferation,inva-sion and migration ability of SiHa cells,and promotes their apoptosis.The decreased proliferation ability may be related to the inhibi-tion of AKT/GSK3β/Cyclin D1 signaling pathway,and the decreased invasion and migration ability may be related to the inhibition of the process of epithelial-mesenchymal transition.