miR-148a靶向Wnt1通路对结直肠癌细胞侵袭、迁移能力的影响

Role of miR-148a targeting Wnt1 pathway on invasion and migration of colorectal cancer cells

目的观察微小RNA-148a(miR-148a)对结直肠癌(CRC)细胞迁移、侵袭能力的影响,并探讨其作用机制与Wnt1信号通路的关系。方法取人正常结直肠黏膜细胞FHC及人CRC细胞HCT116、SW620,采用qRT-PCR法检测Wnt1 mRNA、miR-148a表达。取HCT116细胞,分为Control组、miR-148a mimic组、miR-148a NC组。转染后48 h,用Transwell法检测HCT116细胞侵袭、迁移情况;通过荧光实时定量PCR(qRT-PCR)法检测各组细胞miR-148a、Wnt1 mRNA表达;Western blotting法检测各组细胞Wnt1蛋白及通路蛋白相关蛋白β-连环蛋白(β-catenin)、侵袭和迁移相关蛋白N-钙黏蛋白(N-cadherin)和基质金属蛋白酶2(MMP-2)表达;采用双荧光素酶报告实验验证miR-148a与Wnt1的靶向关系。结果与FHC细胞相比,HCT116、SW620细胞中Wnt1 mRNA表达升高,miR-148a表达降低(P均<0.05);HCT116与SW620细胞相比差异无统计学意义(P>0.05)。与Control组和miR-148a NC组相比,miR-148a mimic组Wnt1 mRNA表达水平、细胞侵袭、迁移能力及Wnt1、β-catenin、MMP-2、N-cadherin蛋白均表达降低,miR-148a表达水平升高(P均<0.05);Control组与miR-148a NC组相比,上述指标差异均无统计学意义(P均>0.05)。双荧光素酶报告基因检测显示,与miR-148a NC+Wnt1-3′UTR-WT组比较,miR-148a mimic+Wnt1-3′UTR-WT组荧光素酶活性降低(P<0.05)。结论miR-148a能够靶向抑制Wnt1/β-catenin通路激活,降低N-cadherin、MMP-2蛋白表达,从而抑制CRC细胞的侵袭、迁移能力。

Objective To observe the effects of microRNA-148a(miR-148a)on the migration and invasion of colorectal cancer(CRC)cells and to explore the relationship between its mechanism of action and Wnt1 signaling pathway.Methods Human colorectal mucosa cell line FHC and human CRC cell lines HCT116 and SW620 were cultured for 48 h,and the expression of miR-148a was detected by real-time quantitative PCR(qRT-PCR).HCT116 cells were divided into the control group,miR-148a mimic group,and miR-148a NC group;at 48 h after transfection,the invasion and migration of HCT116 cells were detected by Transwell method;the expression levels of miR-148a and Wnt1 mRNA were detected by real-time quantitative PCR;Western blotting was used to detect the expression levels of Wnt1 protein,pathway protein-related proteinβ-catenin,invasion and migration-related proteins N-cadherin and matrix metalloproteinase-2(MMP-2);and the targeting relationship between miR-148a and Wnt1 was verified by Doub-luciferase report.Results Compared with the FHC cells,the relative expression level of Wnt1 mRNA in HCT116 and SW620 cells was significantly higher(P<0.05),and the relative expression level of miR-148a was significantly lower(P<0.05);there was no significant difference between the HCT116 and SW620 cells(P>0.05).Compared with the control group and miR-148a NC group,the relative expression level of Wnt1 mRNA,the abilities of cell invasion and migration,and protein expression levels of Wnt1,β-catenin,MMP-2 and N-cadherin in the miR-148a mimic group were significantly lower(all P<0.05),and the relative expression level of miR-148a was significantly higher(P<0.05);while there was no significant difference in the above indexes between the control group and miR-148a NC group(all P>0.05).Detection of Doub-luciferase reporter gene showed that,compared with the miR-148a NC+Wnt1-3′UTR-WT group,the activity of luciferase in the miR-148a mimic+Wnt1-3′UTR-WT group was lower(P<0.05).Conclusion MiR-148a can inhibit Wnt1/β-catenin pathway activation,reduce the expression of N-cadherin and MMP-2 protein,and thus inhibit the invasion and migration of CRC cells.