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目的 :探讨微RNA-200c(micro RNA-200c,mi R-200c)对耐受甲氨蝶呤(methotrexate,MTX)的人非小细胞肺癌A549细胞迁移和侵袭能力的影响,及其可能的分子作用机制。方法 :通过浓度递增结合低剂量持续诱导,获得耐受MTX的人肺癌A549/MTX细胞系后,观察诱导前后细胞的形态学改变。将mi R-200c模拟物(mimic)转染A549/MTX细胞株后,分别采用细胞划痕愈合实验和Transwell细胞迁移和侵袭实验检测细胞的迁移和侵袭能力;再用实时荧光定量PCR法检测mi R-200c过表达的A549/MTX细胞中人Zeste基因增强子同源物2(human enhancer of Zeste homolog 2,EZH2)和E-钙黏蛋白(E-cadherin,E-cad)的m RNA表达水平。结果 :肺癌A549/MTX耐药细胞构建成功。miR-200c minic转染后A549/MTX耐药细胞表达mi R-200c水平比转染阴性片段组高6.41倍(P<0.05),表明转染成功。mi R-200c mimic转染后A549/MTX细胞的迁移和侵袭能力显著降低(P值均<0.05);而且A549/MTX细胞中EZH2 m RNA表达水平明显降低,而E-cad m RNA水平明显升高(P值均<0.05)。结论 :mi R-200c高表达可以抑制A549/MTX耐药细胞的迁移和侵袭能力,其机制可能与其下调EZH2表达和上调E-cad水平有关。
OBJECTIVE: To investigate the effect of microRNA-200c (mi R-200c) on the migration and invasion of human non-small cell lung cancer A549 cells resistant to methotrexate (MTX) and its possible molecular Mechanism. Methods: MTX human lung cancer A549 / MTX cell line was obtained by increasing the concentration and combining with low dose. The morphological changes of the cells were observed before and after induction. After transfection of mi R-200c mimic into A549 / MTX cell line, the cell migration and invasion ability were detected by cell scratch healing assay and Transwell cell migration and invasion assay respectively. Real-time quantitative PCR The m RNA expression level of human enhancer of Zeste homolog 2 (EZH2) and E-cadherin (E-cad) in A549 / MTX cells overexpressing R-200c . Results: The lung cancer A549 / MTX resistant cells were successfully constructed. The level of mi R-200c in A549 / MTX-resistant cells transfected with miR-200c minic was 6.41-fold higher than that in the transfected negative group (P <0.05), indicating that the transfection was successful. The migration and invasion ability of A549 / MTX cells was significantly decreased after transfected with mi R-200c mimic (all P <0.05), and the expression of EZH2 m RNA in A549 / MTX cells was significantly decreased High (all P <0.05). Conclusion: The overexpression of mi R-200c can inhibit the migration and invasion of A549 / MTX-resistant cells, which may be related to its down-regulation of EZH2 expression and up-regulation of E-cad.