MiR-191靶向调控TIMP3促进肺癌细胞恶性转化进程的研究

MiR-191 Promotes Malignant Transformation of Lung Cancer Cells by Directly Targeting TIMP3

目的探索miR-191靶向调控基质金属蛋白酶抑制剂3(TIMP3)对肺癌细胞增殖、迁移、侵袭影响及相关机制,为深入了解肺癌发生发展机制及寻找新的诊断和治疗靶标提供新思路。方法 Target Scan软件预测miR-191的潜在靶基因;双荧光素酶报告实验检测miR-191对TIMP3调控作用; Western blot和实时定量聚合酶链式反应(qPCR)检测正常肺细胞CCD-19Lu和肺癌细胞A549中TIMP3、miR-191表达水平;肺癌细胞A549中转染anti-miR-191后检测TIMP3蛋白表达变化;噻唑蓝(MTT)检测细胞增殖能力; Transwell检测细胞迁移、侵袭能力; Western blot检测细胞中MMP2、JNK、p-JNK、β-actin蛋白表达水平。结果 Target Scan预测软件发现TIMP3是miR-191的潜在靶基因;双荧光素酶报告实验证实TIMP3是miR-191的靶标;正常肺细胞CCD-19Lu中miR-191表达较低,TIMP3 mRNA表达较高;肺癌细胞A549中miR-191表达较高,TIMP3 mRNA表达较低;肺癌细胞A549中转染anti-miR-191可显著增加TIMP3蛋白表达;MTT结果显示肺癌细胞增殖未受明显影响; Transwell实验结果表明anti-miR-191可抑制细胞迁移和侵袭; Western blot结果显示TIMP3下游癌蛋白MMP2、p-JNK显著下调。结论 TIMP3是miR-191的一个靶标,肺癌中高表达的miR-191可靶向抑制TIMP3激活MMP2、JNK等癌基因促使肺癌细胞发生恶性转化; miR-191/TIMP3信号轴在肺癌恶性病变过程中可能起重要作用,可成为肺癌早期诊断与靶向治疗的分子靶标。

Objective To explore the effect and mechanism of miR-191 on malignant transformation of lung cancer cells by directly targeting TIMP3,to gain insights into the mechanism of lung cancer development and to find new diagnostic and therapeutic targets.Methods Target Scan software was used to predict the promising miRNA targeting TIMP3. The effect of miR-191 on TIMP3 was detected by dual luciferase reporter assay. Western blot and qPCR were used to detect the expressions of TIMP3 and miR-191 in normal lung cell CCD-19 Lu and lung cancer cell A549.The expression of TIMP3 protein was detected in lung cancer cell A549 transfected with anti-miR191. The ability of cell proliferation was detected by MTT assay while the ability of cell invasion and migration was detected by transwell chamber. The expressions of MMP2、JNK、p-JNK、β-actin were detected by Western blot.Results Target Scan software demonstrated that TIMP3 was a possible target of miR-191. Dual luciferase reporter assay confirmed that TIMP3 was a promissing target of miR-191. The expression of miR-191 was higher in normal lung cell line CCD-19 Lu and lower in lung cancer cell line A549,whereas TIMP3 showed a reverse trend to miR-191. Its expression was higher in lung cancer cell line A549,but dramatically declined in normal lung cell CCD-19 Lu. Transfection of anti-miR-191 in lung cancer cell A549 significantly increased TIMP3 protein levels. MTT assay showed that the proliferation of lung cancer cells was not significantly affected. Transwell assays verified that anti-miR-191 could inhibit cell migration and invasion. Western blot found that MMP2 and p-JNK were strikingly down-regulated. Conclusion TIMP3 is a directly target of miR-191 which,usually overexpressed in lung cancer,can promote the malignant transformation of lung cancer cells by targeting TIMP3 and activating MMP2,JNK simultaneously. The miR-191/TIMP3 axis might play an important role in malignant transformation of lung cancer cells and can be a potential molecular target for early diagnosis and targeted therapy of lung cancer.