miR-496过表达抑制结肠癌细胞生长和转移

miR-496 over-expression inhibits growth and metastasis in colon cancer cells

目的:研究miR-496过表达对结肠癌细胞生长和转移的影响及其分子机制。方法:运用生物信息学软件筛选miR-496靶向相互作用蛋白;real-time PCR和Western blot法测定结肠癌细胞系HT29、HCT116、SW480以及正常结肠上皮细胞NCM460中miR-496、CTNNB1 mRNA和β-catenin蛋白的表达;运用Lipofectamine 2000将miR-496 mimics转染HT29、HCT116和SW480细胞,分别命名为HT29-miR-496 mimics、HCT116-miR-496 mimics和SW480-miR-496 mimics细胞,转染scramble为阴性对照;运用MTT法、乳酸脱氢酶(LDH)试剂盒法、克隆形成实验和Transwell分别测定细胞活力、LDH漏出率、克隆形成能力和转移能力;萤光素酶报告基因实验测定miR-496启动子活性;Western blot法测定β-catenin、真核细胞翻译起始因子4E结合蛋白1(4E-BP1)、p-4E-BP1、低密度脂蛋白受体相关蛋白6(LRP6)、p-LRP6、MMP-7、MMP-9、MMP-13以及TIMP-2的蛋白水平。结果:miR-496与β-catenin内源性相互作用;miR-496在HT29、HCT116和SW480细胞中低表达,而在NCM460高表达;β-catenin在HT29、HCT116和SW480细胞中高表达,而在NCM460低表达;培养24 h、48 h、72 h、96 h的HT29-miR-496 mimics、HCT116-miR-496 mimics和SW480-miR-496 mimics细胞活力、LDH漏出率、克隆形成率和转移的细胞数均显著低于对照组(P<0.05);萤光素酶报告基因实验结果显示转染miR-496 mimics细胞中的miR-496启动子活性明显增加(P<0.05),分别是对照组的1.75倍、2.04倍和1.61倍。Western blot实验结果显示miR-496过表达抑制β-catenin蛋白表达,p-4E-BP1和p-LRP6的蛋白水平降低;siRNA或miR-496过表达介导的β-catenin表达下调能显著抑制MMP-7和MMP-9的表达,促进TIMP-2的表达。结论:miR-496在结肠癌细胞中低表达,在正常结肠上皮细胞中高表达;miR-496过表达抑制结肠癌细胞的生长和转移,其机制是通过抑制Wnt/β-catenin通路进一步抑制MMP-7和MMP-9表达,促进TIMP-2表达,从而抑制结肠癌细胞的恶性表型。

AIM： To investigate the effect of miR-496 overexpression on the growth and metastasis of colon cancer cells and its molecular mechanism .METHODS：The proteins interacting with miR-496 were screened by bioinformatic method.The levels of miR-496, CTNNB1 mRNA and β-catenin protein in colon cancer cell lines , HT29, HCT116 and SW480, and normal colonic epithelial cell line NCM 460 were detected by real-time PCR and Western blot .HT29, HCT116 and SW480 cells were transfected with miR-496 mimics using Lipofectamine 2000 and named as HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimics cells, respectively, and the cells transfected with the scramble served as negative control .The cell viability, lactate dehydrogenase （LDH） leakage, and colony formation and metastatic abilities were determined by MTT assay , LDH assay, colony formation assay and Transwell method , respectively.The promoter activity of miR-496 was measured using luciferase reporter gene assay .The protein levels of β-catenin, eukaryotic translation initiation factor 4E-binding protein 1 （4E-BP1）, p-4E-BP1, low-density lipoprotein receptor-related protein 6（LRP6）, p-LRP6, MMP-7, MMP-9, MMP-13 and TIMP-2 were monitored by Western blot.RESULTS：Endogenous miR-406 interacted with β-catenin was found in the colon cancer cells .Low miR-496 expression in the HT29, HCT116 and SW480 cells and high miR-496 expression in NCM460 cells were detected.In contrast, high β-catenin ex-pression was found in the HT29, HCT116 and SW480 cells and low β-catenin expression was observed in the NCM460 cells.Compared with control group , the cell viability, colony formation rate and the number of metastatic cells remarkably decreased in the HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimic cells （P〈0.05）.The promoter activity of miR-496 was significantly increased in colon cancer cells transfected with miR-496 mimics, and was 1.75, 2.04 and 1.61 times as high as control group .miR-496 over-expression inhibited β-catenin levels, and p-4E-BP1 and p-LRP6 protein levels were also reduced .siRNA-or over-expressed miR-496-mediated β-catenin down-regulation in-hibited MMP-7 and MMP-9 expression , but promoted TIMP-2 expression .CONCLUSION：The expression level of miR-496 in the colon cancer cells is low , but in the normal colonic epithelial cells is high .miR-496 over-expression inhibits the protein levels of MMP-7 and MMP-9, and promotes the protein expression of TIMP-2 via inhibiting Wnt/β-catenin pathway, thus suppressing malignant phenotype in the colon cancer cells .