miR-32-5p靶向TOB1基因调控结直肠癌细胞放射增敏性及迁移侵袭能力机制研究

Mechanism of miR-32-5p targeting TOB1 gene in regulating radiosensitization,migration and invasion of colorectal cancer cells

目的探讨miR-32-5p对结直肠癌细胞放射敏感性及迁移侵袭能力的影响及其潜在作用机制。方法培养人结直肠癌SW480细胞和正常结肠上皮NCM460细胞,将结直肠癌细胞分为未转染组和转染组(分别转染anti-miR-NC、anti-miR-32-5p、pcDNA、pcDNA-TOB1、anti-miR-32-5p+si-NC、nti-miR-32-5p+si-TOB1)。RT-qPCR和Western blot分别检测细胞中miR-32-5p、TOB1 mRNA和蛋白表达,克隆形成实验检测转染各组细胞放射敏感性,Transwell实验检测转染各组细胞迁移、侵袭能力,双荧光素酶报告基因实验和Western blot验证miR-32-5p是否靶向TOB1。结果与人正常结肠上皮细胞相比,结肠癌细胞中miR-32-5p表达明显上调(P<0.05),TOB1 mRNA和蛋白表达明显下调(P<0.05)。与anti-miR-NC比较,anti-miR-32-5p组细胞放射增敏比1.801,细胞迁移和侵袭数目均明显减少(P<0.05);与pcDNA组比较pcDNA-TOB1组细胞放射增敏比1.764,细胞迁移数目和侵袭数目均明显减少(P<0.05)。双荧光素酶报告基因实验和Western blot结果证实miR-32-5p靶向负调控TOB1蛋白表达。与anti-miR-32-5p+si-NC组比较,anti-miR-32-5p+si-TOB1组细胞放射增敏比为0.591,细胞迁移数目和侵袭数目均明显增多(P<0.05)。结论抑制miR-32-5p表达能够明显增强结直肠癌细胞放射敏感性,抑制细胞迁移和侵袭,其作用机制可能与靶向促进TOB1表达有关。

Objection To investigate the effect of miR-32-5p on the radiosensitivity,migration and invasion of colorectal cancer cells and the underlying mechanism.Methods Human colorectal cancer SW480 cells and normal colonic epithelial NCM460 cells were cultured.The colorectal cancer cells were divided into the non-transfected and transfected groups(transfected with anti-miR-NC,anti-miR-32-5p,pcDNA,pcDNA-TOB1,anti-miR-32-5p+si-NC and anti-miR-32-5p+si-TOB1,respectively).The expression of miR-32-5p and TOB1 at the mRNA and protein levels was detected by RT-qPCR and Western blot.The radiosensitivity of the transfected cells was determined by colony formation assay.The migration and invasion ability of the transfected cells were detected by Transwell assay.Whether miR-32-5p targeted TOB1 was validated by dual luciferase reporter gene assay and Western blot.Results Compared with human colonic epithelial cells,the expression of miR-32-5p was significantly up-regulated,whereas the expression of TOB1 mRNA and protein was remarkably down-regulated in the colon cancer cells(all P<0.05).Compared with the anti-miR-NC,the quantity of cell migration and invasion was significantly decreased(both P<0.05)and the radiosensitivity ratio was 1.801 in the anti-miR-32-5p group.Compared with the pcDNA group,the quantity of cell migration and invasion was significantly decreased(both P<0.05)and the radiosensitivity ratio was 1.764 in the pcDNA-TOB1 group.Dual luciferase reporter gene assay and Western blot confirmed that miR-32-5p negatively regulated the expression of TOB1 protein.Compared with the anti-miR-32-5p+si-NC group,the quantity of cell migration and invasion was significantly increased(both P<0.05)and the radiosensitivity ratio was 0.591 in the anti-miR-32-5p+si-TOB1 group.Conclusions Inhibition of miR-32-5p expression can significantly enhance the radiosensitivity of colorectal cancer cells and suppress cell migration and invasion.The underlying mechanism might be related to the targeted up-regulation of TOB1 expression.