miRNA-21靶向调控Wnt/β-catenin对非小细胞肺癌细胞增殖与侵袭的影响

Effect of microRNA-21 on the proliferation and invasion of non-small cell lung cancer through targeting Wnt/β-catenin pathway

目的研究microRNA-21(miR-21)是否通过调控wnt/β-catenin信号通路从而影响人非小细胞肺癌(non-small cell lung cancer, NSCLC)细胞株NCI-H1975细胞体外增殖与侵袭能力。方法预测和构建含有miR-21候选靶基因和β-连环蛋白基因的双荧光素酶报告质粒。将miR-21模拟物通过Lipofactamine 2000脂质体转染法转染NCI-H1975细胞,应用qRTPCR检测细胞中miR-21和β-catenin mRNA表达情况,应用Western blot检测β-catenin蛋白水平,CCK-8法检测miR-21mimic对NCI-H1975细胞增殖能力的影响,Transwell侵袭实验检测miR-21 mimic对NCI-H1975细胞侵袭能力的影响。结果qRT-PCR肺癌组织中的miR-21与邻近正常组织相比表达明显升高;双荧光素酶报告基因结果显示miR-21能和β-catenin的3’UTR端结合且显著促进荧光素酶活性;上调miR-21可使β-catenin的mRNA和蛋白表达水平显著升高,同时上调miR-21可显著增强NCI-H1975细胞的增殖与侵袭能力。结论 miR-21在转录后水平调控β-catenin表达来促进NCI-H1975细胞的增殖与侵袭能力,可能是NSCLC潜在的治疗靶点。

Objective To investigate whether miR-21 affects the proliferation and invasion of non-small-cell lung cancer (NSCLC) cell line NCI-H1975 by regulating Wnt/β-catenin signaling pathway in vitro. Methods The dual luciferase reporter plasmids containing the candidate target gene of miR-21 and the β-catenin gene were predicted and constructed. miR-21 mimics were transfected into NCI-H1975 cells by lipofection with lipofactamine 2000. The expression level of miR-21 and β-catenin mRNA in non-small lung cancer tissues and adjacent normal lung tissues was detected by quantitative real-time PCR (qRT-PCR). The expression level of β-catenin protein was detected by Western blot. The effect of miR-21 mimics on the proliferation of NCI-H1975 cells was detected by CCK-8 method and on the invasion ability was detected by transwell invasion assay. Results Compared with adjacent normal lung tissues, miR-21 detected by qRT-PCR was highly expressed in non-small cell lung cancer tissues. Dual luciferase reporter gene assay showed that miR-21 could directly bind to the 3′-untranslated region (3’ UTR) of the β-catenin gene and significantly enhance luciferase activity. qRT-PCR and western blot results indicated that the upregulation of miR-21 expression could significantly increase the mRNA and protein expression levels of β-catenin in NCI-H1975 cell line. Moreover, CCK-8 and Transwell invasion assay showed that the upregulation of miR-21 expression significantly promoted the proliferation and invasion of NCI-H1975 cell line. Conclusion miR-21 can promote the proliferation and invasion of NSCLC cell line NCI-H1975 by regulating the expression of β-catenin at the post-transcriptional level, which may be a potential treatment target of NSCLC.