miR-145通过靶向抑制SMAD3的表达抑制非小细胞肺癌细胞侵袭能力

目的:探讨miR-145靶向调控SMAD3的表达对非小细胞肺癌细胞侵袭能力的影响。方法:采用qRT-PCR检测miR-145和SMAD3在30例非小细胞肺癌组织和癌旁组织中的表达水平并分析其相关性。利用靶基因预测网站预测miR-145的潜在靶基因SMAD3，利用双荧光素酶报告基因实验验证。利用细胞转染实验对非小细胞肺癌A549细胞进行了miR-145 mimics、miR-145 inhibitor、siRNA SMAD3及其相关对照的细胞转染，采用Transwell实验检测转染后A549细胞侵袭能力的变化。使用Western blot检测上调或下调miR-145表达对A549细胞中SMAD3蛋白表达的影响。结果:qRT-PCR结果显示，非小细胞肺癌组织中miR-145低表达，SMAD3高表达，且两者表达水平呈负相关。靶基因预测及验证实验结果显示miR-145可以与SMAD3 的 3'-UTR特异性结合，SMAD3是miR-145的靶基因。Western blot 结果显示，转染miR-145 mimics可以显著降低SMAD3蛋白的表达水平（P<0.001），转染miR-145 inhibitor则显著提高SMAD3蛋白的表达水平（P<0.01）。Transwell体外侵袭实验结果显示，与对照组相比，转染miR-145 mimic显著降低了A549细胞的侵袭能力（P<0.001），转染miR-145 inhibitor显著提高了A549细胞的侵袭能力（P<0.05）；与对照组相比，敲低SMAD3的表达A549细胞的侵袭能力减弱（P<0.001）；与单独敲低SMAD3相比，共同转染siRNA SMAD3和miR-145 mimics A549细胞的侵袭能力减弱（P<0.05），共同转染siRNA SMAD3和miR-145 inhibitor A549细胞的侵袭能力无显著差异（P>0.05）。结论:miR-145在非小细胞肺癌组织中低表达，miR-145通过靶向抑制SMAD3的表达发挥对非小细胞肺癌细胞侵袭能力的抑制作用，为临床综合治疗提供了新的作用靶点。

miR-145 inhibits the invasion of non-small cell lung cancer cells by targeting inhibition of SMAD3 expression

Objective:To investigate the effect of miR-145 targeting the regulation of SMAD3 expression on the invasive ability of non-small cell lung cancer cells.Methods:The expression levels of miR-145 and SMAD3 in 30 non-small cell lung cancer tissues and adjacent tissues were detected by qRT-PCR and their correlations were analyzed.The target gene prediction site was used to predict the potential target gene SMAD3 of miR-145,which was verified by the dual luciferase reporter gene assay.The transfected cells of miR-145 mimics,miR-145 inhibitor,siRNA SMAD3 and related controls were transfected into non-small cell lung cancer A549 cells by cell transfection experiments.Transwell assay was used to detect the invasive ability of A549 cells after transfection.The effect of miR-145 on the expression of SMAD3 protein was up-regulated or down-regulated in A549 cells by Western blot.Results:The results of qRT-PCR showed that miR-145 was down-regulated and SMAD3 was highly expressed in non-small cell lung cancer tissues,and the expression levels of both were negatively correlated.Target gene prediction and validation experiments showed that miR-145 can specifically bind to the 3'-UTR of SMAD3,which was a target gene of miR-145.Western blot analysis showed that transfection of miR-145 mimics significantly decreased the expression of SMAD3 protein(P<0.001),and transfection of miR-145 inhibitor significantly increased the expression of SMAD3 protein(P<0.01).Transwell in vitro invasion assay showed that the transfected miR-145 mimic group significantly reduced the invasive ability of A549 cells compared with the control group(P<0.001),and the transfected miR-145 inhibitor group significantly increased the invasive ability of A549 cells(P<0.05).Compared with the control group,the invasive ability of knockdown of SMAD3 expressing A549 cells was weakened(P<0.001).The invasive ability of co-transfected siRNA SMAD3 and miR-145 mimics A549 cells was weaker than that of knockdown of SMAD3 alone(P<0.05).There was no significant difference in the invasive ability of co-transfected siRNA SMAD3 and miR-145 inhibitor A549 cells(P>0.05).Conclusion:miR-145 is lowly expressed in non-small cell lung cancer tissues,and miR-145 exerts an inhibitory effect on the invasive ability of non-small cell lung cancer cells by targeting inhibition of SMAD3 expression,providing a new target for clinical comprehensive treatment.