miR-372-5p促进结直肠癌细胞的转移:通过靶向PTEN调控PI3K/AKT/CXCL12信号通路

MiR-372-5p regulates PI3K/AKT/CXCL12 signaling pathway by targeting PTEN to promote colorectal cancer cell metastasis

目的探讨miR-372-5p通过靶向PTEN调控PI3K/AKT/CXCL12信号通路对结直肠癌细胞迁移能力的影响及机制。方法qRT-RCR检测miR-372-5p在结直肠癌和癌旁组织及结直肠癌细胞和正常肠上皮细胞中的表达;生物信息学和双荧光素酶实验验证miR-372-5p与PTEN的靶向关系;Western blotting检测转染inhibitor-NC/mimics-NC(miR-372-5p抑制剂/类似物的阴性对照)、miR-inhibitor(miR-372-5p抑制剂)、miR-mimics(miR-372-5p类似物)以及共转染miR-inhibitor+si-PTEN(PTEN干扰)、miR-mimics+PI3K抑制剂对PTEN、CXCL12表达和PI3K/AKT信号通路激活水平的影响;Transwell实验、划痕实验检测以上各组、共转染miR-mimics+si-CXCL12(CXCL12干扰)以及转染mimics-NC、miR-mimics后的HCT116条件培养基对细胞迁移能力的影响。结果结直肠癌组织中miR-372-5p较癌旁组织表达升高,相对于NCM460,HCT116较SW620中miR-372-5p表达上调更显著(P<0.01);双荧光素酶实验证实PTEN是miR-372-5p的潜在靶基因(P<0.05)。相较于NC组,miR-mimics可降低PTEN蛋白表达水平,增加CXCL12蛋白表达水平和AKT磷酸化水平,提高HCT116迁移能力;而miR-inhibitor则导致相反的结果(P<0.05),si-PTEN可中和miR-inhibitor的作用(P<0.01);PI3K抑制剂可降低CXCL12的蛋白表达水平,并抑制HCT116迁移能力(P<0.05),而miR-mimics可中和这一作用(P<0.01);miR-mimics转染HCT116的条件培养基可提高NCM460的迁移能力(P<0.01),联合si-CXCL12可逆转miR-mimics对HCT116转移的促进作用(P<0.01)。结论miR-372-5p通过靶向PTEN激活PI3K/AKT信号通路,上调CXCL12的表达,从而促进结直肠癌细胞的迁移能力。

Objective To investigate whether mi R-372-5p regulates PI3K/AKT/CXCL12 signaling pathway by targeting PTEN to promote metastasis of colorectal cancer cells.Methods We detected the differential expression of miR-372-5p using RT-qRCR in colorectal cancer and adjacent tissues,colorectal cancer cells and normal intestinal epithelial cells.Bioinformatic analysis and double luciferase assay were performed for verification of the targeting relationship between miR-372-5p and PTEN.Western blotting was used to assess the effects of transfection with miR-372-5p inhibitor and miR-372-5p mimics alone,co-transfection with miR-372-5p inhibitor and si-PTEN,and co-transfection with miR-372-5p mimics and PI3K inhibitor on the expressions of PTEN and CXCL12 and the activation of PI3K/AKT signal pathway;Transwell assay and scratch assay were used to examine the changes in the migration ability of the transfected cells,the cells co-transfected with miR-372-5p mimics and si-CXCL12,and the cells treated with conditioned medium from HCT116 cells transfected with miR-372-5p mimics.Results The expression of miR-372-5p was significantly higher in colorectal cancer tissues than in adjacent tissues,and higher in HCT116 and SW620 cells than in NCM460 cells(P<0.01).Double luciferase assay confirmed that PTEN was a potential target gene of miR-372-5p(P<0.05).Transfection of HCT116 cells with miR-372-5p mimics obviously decreased PTEN protein expression,increase CXCL12 expression and the phosphorylation level of AKT,and lowered the cell migration ability,while transfection with miR-372-5p inhibitor produced the opposite effects(P<0.05);si-PTEN obviously neutralized the effect of miR-372-5p inhibitor(P<0.01).PI3K inhibitor significantly decreased CXCL12 expression and inhibited the cell migration(P<0.05),and this effect was mitigated by miR-372-5p mimics(P<0.01).Treatment with the conditioned medium from HCT116cells transfected with miR-372-5p mimics significantly enhanced the migration ability of NCM460 cells,and this effect was suppressed by transfection with si-CXCL12(P<0.01).Conclusion MiR-372-5p activates PI3K/AKT signaling pathway by targeting PTEN and up-regulates CXCL12 expression to promoting metastasis of colorectal cancer cells.