miR-129靶向调节Twist1抑制乳腺癌细胞的增殖、迁移和侵袭

miR-129 inhibits proliferation,migration and invasion of breast cancer cells by targeting Twist1

目的研究miR-129对乳腺癌细胞增殖、迁移及侵袭的影响及其作用机制。方法选取乳腺癌SKBR3、AU565细胞系,建立过表达与敲低miR-129细胞系,将细胞分为Control-mimic组、miR-129-mimic组和miR-129-mimic+miR-mimic-shRNA组。采用RT-PCR检测SKBR3、AU565细胞系中miR-129的表达水平,CCK-8法检测SKBR3、AU565细胞系的增殖能力,通过划痕实验及Transwell实验测定SKBR3、AU565细胞系的迁移及侵袭能力。采用生物信息学和双荧光素酶报告系统检测miR-129与Twist1的互补结合位点,采用Western blot对miR-129是否是Twist1的转录靶点进行分析验证。结果miR-129-mimic组SKBR3和AU565细胞中miR-129的表达水平显著高于Control-mimic组(P<0.05)。miR-129-mimic组SKBR3和AU565细胞的增殖能力明显低于Control-mimic组(P<0.01),而miR-129-mimic+miR-mimic-shRNA组逆转了miR-129过表达导致的细胞增殖能力降低。与Control-mimic组比较,miR-129-mimic组中SKBR3和AU565细胞的迁移及侵袭能力明显下降(P<0.01)。生物信息学软件分析显示,miR-129与Twist1的3’-UTR端有互补结合位点,且双荧光素酶试验结果显示,miR-129-mimic组中WT-3’Twist-UTR的荧光素酶活性明显低于Control-mimic组(P<0.01);而miR-129-mimic组中Mut-3’Twist-UTR的荧光素酶活性与Control-mimic组比较,差异无统计学意义(P>0.05);Western blot结果显示,与Control-mimic组比较,miR-129-mimic组SKBR3和AU565细胞中Twist1蛋白水平明显下降(P<0.01)。结论miR-129可通过靶向调节Twist1抑制乳腺癌细胞增殖、迁移和侵袭,是乳腺癌诊断及治疗的潜在靶点。

Objective To study the effect of miR-129 on proliferation,migration and invasion of breast cancer cells and its mechanism.Methods Breast cancer SKBR3 and AU565 cell lines were selected to establish overexpression and knockdown miR-129 cell lines.The cells were divided into the Control-mimic group,the miR-129-mimic group and the miR-129-mimic+miR-mimic-shRNA group.The expression level of miR-129 in SKBR3 and AU565 cell lines was detected by RT-PCR.The proliferation of SKBR3 and AU565 cell lines was detected by CCK-8 assay.The migration and invasion of SKBR3 and AU565 cell lines were detected by scratch assay and Transwell assay.The complementary binding sites of miR-129 and Twist1 were detected by bioinformatics and dual-luciferase reporter system.Western blot was used to analyze and verify whether miR-129 was a transcription target of Twist1.Results The expression level of miR-129 in SKBR3 and AU565 cells in the miR-129-mimic group was significantly higher than that in the Control-mimic group(P<0.05).The proliferation of SKBR3 and AU565 cells in the miR-129-mimic group was significantly lower than that in the Control-mimic group(P<0.01),while the miR-129-mimic+miR-mimic-shRNA group could reverse the decrease of cell proliferation caused by overexpression of miR-129.Compared with the Control-mimic group,the migration and invasion of SKBR3 and AU565 cells in the miR-129-mimic group were significantly decreased(P<0.01).Bioinformatics software analysis showed that miR-129 had complementary binding sites with the 3’-UTR end of Twist1.The results of dual-luciferase assay showed that the luciferase assay of WT-3’Twist-UTR in the miR-129-mimic group was significantly lower than that in the Control-mimic group(P<0.01);but there was no significant difference in luciferase activity of Mut-3’Twist-UTR between the miR-129-mimic group and the Control-mimic group(P>0.05).Western blot results showed that compared with the Control-mimic group,the Twist1 protein level in SKBR3 and AU565 cells of the miR-129-mimic group was significantly decreased(P<0.01).Conclusion miR-129 can inhibit the proliferation,migration and invasion of breast cancer cells by targeting Twist1,which is a potential target for diagnosis and treatment of breast cancer.