南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (05): 533-.doi: 10.12122/j.issn.1673-4254.2019.05.06

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miR-152 和miR-448 靶向于Rictor并抑制结直肠癌细胞增殖

张杰，韩增胜，董立新，李甄，栗坤，石明，刘志伟，李健   

  • 出版日期:2019-05-20 发布日期:2019-05-20

MicroRNA-152 and microRNA-448 inhibit proliferation of colorectal cancer cells in vitro by targeting Rictor

  • Online:2019-05-20 Published:2019-05-20

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摘要： 目的筛选靶向于Rictor mRNA表达的miRNAs并研究其在结直肠癌中的调控作用。方法采用软件预测筛选可能靶向 于Rictor mRNA表达的miRNAs，用所预测的miRNAs转染KM12SM细胞株，利用qRT-qPCR和Western blotting进一步确定对 Rictor表达具显著调控作用的miRNAs。在此基础上，利用双荧光素酶报告系统进一步确定可结合到Rictor mRNA的3’UTR区 对其转录后进行调节的miRNAs。利用细胞存活检测和克隆形成实验进一步确证所筛选miRNAs对细胞存活和克隆形成的影 响。结果miR-152 和miR-448 对Rictor 表达均具显著抑制作用（P<0.01，P<0.05），且miR-152 和miR-448 可结合到Rictor mRNA的3’UTR区，显著抑制荧光素酶活性（P<0.01，P<0.05），对其进行转录后进行调节，miR-152和miR-448过表达均可显著 抑制结直肠癌细胞株KM12SM的生长和增殖。结论miR-152和miR-448可能通过靶向于Rictor mRNA进而抑制其蛋白表达， 最终负调控结直肠癌的生长和克隆形成。

Abstract: Objective To screen the microRNAs (miRNAs) targeting Rictor and investigate their effects in regulating the biological behaviors of colorectal cancer (CRC). Methods Human colorectal cancer cell line KM12SM was transfected with the miRNAs targeting Rictor identified by prediction software to test inhibitory effects of these miRNAs on Rictor expression using qRT-PCR andWestern blotting. Dual luciferase reporter assay was used to further confirm the binding of these miRNAs to the 3’UTR of Rictor mRNA. Cell survival and colony formation assays were used to investigate the effects of these miRNAs on survival and colony formation in KM12SM cells. Results miR-152 and miR-448 were identified as the Rictor-targeting miRNAs, which significantly inhibited the expression of Rictor in KM12SM cells (P<0.05). The two miRNAs were confirmed to bind to the 3’UTR of Rictor mRNA and significantly inhibited luciferase activity in KM12SM cells (P<0.01, P<0.05); they also showed activities of posttranscriptional modulation of Rictor. Overexpression of miR-152 and miR-448 both significantly inhibited the growth and colony formation of KM12SM cells. Conclusion miR-152 and miR-448 can down-regulate the protein expression of Rictor by targeting Rictor mRNAto negatively regulate the growth and colony formation of colorectal cancer cells.