MiRNA-424对人结肠癌细胞系Lovo细胞增殖及Rspo3基因表达的影响

Effects of MicoRNA-424 on Proliferation and Rspo3 Gene Expression in Human Colon Cancer Lovo Cell Line

[目的]探讨miR-424对人结肠癌Lovo细胞增殖及Rspo3基因表达的影响。[方法]运用实时荧光定量PCR检测Lovo细胞和正常结肠上皮组织NCM460细胞中miR-424与Rspo3 mRNA的表达水平。miR-424抑制剂（inhibitor）转染Lovo细胞后,观察细胞增殖能力,及miR-424、Rspo3 mRNA、miR-15/16/103表达水平,Western blot检测Rspo3蛋白表达水平。通过在线软件预测靶向Rspo3基因的miRNA,并用双荧光素酶报告基因实验验证。[结果]Mi R-424与Rspo3 mRNA在结肠癌Lovo细胞中过表达（P〈0.05）。miR-424 inhibitor组Lovo细胞增殖受到明显抑制（P〈0.05）,且Rspo3基因表达量显著下降（P〈0.05）,miR-15/16/103均上调,其中miR-103上调最显著（P〈0.05）。双荧光素酶报告基因实验验证miR-103可靶向调控Rspo3基因的表达。[结论]miR-424抑制剂可阻滞Lovo细胞增殖且通过反馈性上调miR-103靶向抑制Rspo3基因的表达。

[Purpose] To investigate the effect of miR-424 on proliferation and RSPO3 gene expression in human colon cancer Lovo cell line. [Methods] Real-time quantitative PCR was used to detect the expressions of miR-424 and Rspo3 mRNA in colon cancer Lovo cells and normal colorectal epithelial NCM460 cells. After transfected with miR-424 inhibitor,negative control and liposome respectively,the proliferation of Lovo cells was detected by CCK-8 method. The expressions of Rspo3 mRNA and miR-15/16/103 were detected by real-time q RCR. The miRNAs targeting Rspo3 gene were predicted by prediction software and verified by dual luciferase reporter assay.[Results] Mi R-424 and Rspo3 mRNA were overexpressed in Lovo cells（P〈0.05）. The proliferation of Lovo cells transfected with miR-424 inhibitor was significantly inhibited（P〈0.05）,and the expression of Rspo3 gene was significantly decreased（P〈0.05）,while miR-15/16/103 was upregulated（P〈0.05）. Dual luciferase reporter gene experiments confirmed that miR-103 targeted Rspo3 gene expression. [Conclusion] Inhibition of miR-424 can reduce the proliferation of Lovo cells and down-regulate the Rspo3 expression through the up-regulation of miR-103.