HIF-1α沉默联合甲基硒酸对宫颈癌细胞增殖与凋亡的影响及其机制探究

Study on effect and mechanism of HIF-1αsilencing combined with methylselenenic acid on proliferation and apoptosis of cervical cancer cells

目的探究缺氧诱导因子1α(HIF-1α)基因沉默联合甲基硒酸(MSA)对宫颈癌细胞增殖、凋亡及迁移的影响及分子机制。方法采用脂质体法将HIF-1α干扰RNA和阴性对照RNA分别转染海拉细胞(HeLa),转染48 h后用MSA处理细胞24 h,CCK-8法和克隆形成实验检测细胞增殖情况;流式细胞法检测细胞凋亡情况;Western blot实验检测HIF-1α、Bcl-2、E-cadherin蛋白表达水平;Transwell实验检测细胞迁移能力;RNA-seq分析差异表达基因和差异信号通路。结果与对照组相比,干扰HIF-1α联合MSA抑制细胞增殖(P<0.01);流式细胞检测结果表明,联合用药组可诱导细胞凋亡;Transwell和Western blot结果表明,干扰HIF-1α联合MSA能够抑制HeLa细胞迁移,并且下调Bcl-2蛋白表达、上调E-cadherin蛋白表达。mRNA测序结合信号通路富集结果显示凋亡信号通路及下游基因的表达受到抑制。结论HIF-1α基因沉默联合MSA药物处理能够协同抑制宫颈癌细胞增殖,诱导癌细胞凋亡,其调控机制可能与Bcl-2家族蛋白的表达和p53信号通路抑制相关。

Objective To investigate the influence and molecular mechanism of hypoxia-inducing factor-1α(HIF-1α)gene silencing combined with methyl selenenic acid(MSA)on cervical cancer cell proliferation,apoptosis and cell migration.Methods HeLa cells were transfected with HIF-1 interference RNA and negative control RNA.After transfection for 48 h,cells were stimulated with MSA for 24 h,and cell proliferation was determined by CCK-8 assay and colony formation.Apoptosis was determined by flow cytometry combined with Annexin V-FITC/PI.The expression levels of HIF-1α,Bcl-2,and E-cadherin were detected by Western blot assay.Cell migration ability was determined by Transwell assay.RNA-seq analysis was used to investigate the differentially expressed genes and differential signaling pathways.Results Compared with the control group,interfering with HIF-1αcombined with MSA significantly inhibited cell proliferation(P<0.01).Flow cytometry results showed that the combined drug group significantly induced apoptosis.Transwell results showed that interfering with HIF-1αcombined with MSA inhibited He La cell migration.Compared with the control group,interfering with HIF-1αcombined with MSA downregulated the expression of Bcl-2 and up-regulated the expression of E-cadherin.RNA-sequencing combined with signal pathway enrichment results showed that the expression of apoptotic signal pathway and downstream genes was inhibited.Conclusion HIF-1αgene silencing combined with MSA can synergically inhibit the proliferation and induce apoptosis of cervical cancer cells,and its regulatory mechanism may be related to the expression of Bcl-2family proteins and the inhibition of p53 signaling pathway.