Msi1过表达对宫颈癌细胞增殖及干性维持的影响及其机制

Effect of Msi1 overexpression on proliferation and stemness maintenance of cervical cancer cells and its mechanism

目的探讨Msi1通过Wnt/β-catenin通路促进宫颈癌细胞增殖及干细胞样特性的作用机制。方法取制备成功的Msi1稳定过表达的SiHa、HeLa细胞株及相应的对照细胞株,其中Msi1稳定过表达的SiHa细胞及其对照细胞分别命名为SiHa-Msi1组、SiHa-EGFP组,Msi1稳定过表达的HeLa细胞及其对照细胞分别命名为HeLa-Msi1组、HeLa-EGFP组。采用裸鼠成瘤检测SiHa-Msi1组、HeLa-Msi1组及其相应对照组体内肿瘤形成能力;细胞计数、软琼脂克隆形成实验检测SiHa-Msi1组、HeLa-Msi1组及相应对照组细胞的体外增殖能力;肿瘤细胞成球及连续成球实验检测SiHa-Msi1组、HeLa-Msi1组及相应对照组细胞的肿瘤干细胞样特性;在SiHa-Msi1组、HeLa-Msi1组及相应对照组细胞中,采用报告基因分析(TOP-Flash/FOP-Flash)检测Wnt/β-catenin通路活性的变化,采用real-time PCR检测干细胞相关因子Klf4、Sox2、Oct4、Aldh的表达,采用Western blot检测Wnt信号通路中的β-catenin、SOX2蛋白的表达。结果与相应对照组相比,SiHa-Msi1组和HeLa-Msi1组体内形成肿瘤体积和质量显著增加(P<0.05),体外细胞增殖数量、软琼脂形成的单克隆数量均显著增加(P<0.05),肿瘤细胞连续成球后成球数目及直径均显著增加(P<0.05);SiHa-Msi1组、HeLa-Msi1组Wnt通路活性显著增加(P<0.05),干细胞相关因子Sox2、Oct4、Aldh的RNA表达水平显著上调(P<0.05),Wnt通路中的β-catenin和SOX2蛋白表达显著上调(P<0.05)。结论Msi1过表达促进了宫颈癌细胞增殖并维持其干细胞样特性,可能通过激活Wnt/β-catenin信号通路,上调SOX2在宫颈癌细胞中的表达,从而发挥了促进宫颈癌进展的作用。

Objective To investigate the mechanism of Msi1 promoting cervical cancer cell proliferation and stem cell like characteristics through Wnt/β-catenin pathway.Methods SiHa and HeLa cell lines stably overexpressing Msi1 and their corresponding control cell lines were successfully constructed and named as SiHa-Msi1 group and SiHa-EGFP group,and HeLa-Msi1 group and HeLa-EGFP group.Tumor formation ability in vivo was detected by tumor formation assay in nude mice in SiHa-Msi1 group,HeLa-Msi1 group and their corresponding control groups.Cytometry and soft agar colony formation assay were performed to examine the proliferation ability of cells in vitro in SiHa-Msi1 group,HeLa-Msi1 group and their corresponding control groups.Tumorsphere culture and serial tumorsphere formation assays were performed to detect cancer stem cell-like properties in SiHa-Msi1 group,HeLa-Msi1 group and their corresponding control groups.In SiHa-Msi1 group,HeLa-Msi1 group and their corresponding control groups,the activity of Wnt pathway was detected by reporter assay(TOP-Flash/FOP-Flash),the expression levels of stem cell related factors Klf4,Sox2,Oct4 and Aldh were detected by real-time PCR,and the expression levels of Wnt signaling downstreamβ-catenin and SOX2 proteins were detected by Western blot.Results Compared with the corresponding control groups,the size and the weight of formed tumor in vivo were significantly increased in SiHa-Msi1 group and HeLa-Msi1 group(P<0.05),both the amount of cell proliferation and the monoclones formed in soft agar were significantly increased(P<0.05),and the numbers and diameters of tumor cells were significantly increased after continuous spheroidization(P<0.05).Compared with the corresponding control groups,the RNA levels of stem cell related factors Sox2,Oct4 and Aldh were significantly up-regulated in SiHa-Msi1 group and HeLa-Msi1 group(P<0.05),and the expression levels ofβ-catenin and SOX2 proteins were significantly up-regulated(P<0.05).Conclusion The overexpression of Msi1 promotes the proliferation of cervical cancer cells and maintains their stem cell like characteristics by activating Wnt/β-catenin signaling pathway and up-regulating the expression of SOX2 in cervical cancer cells,which promotes the progression of cervical cancer.