Significant evidence has shown that the miRNA pathway is an important component in the downstream signaling cascades of TGF-β1 pathway. Our previous study has indicated that miR-335-5p expression was significantly down-regulated and acted as a vital player in the metastasis of non-small cell lung cancer (NSCLC), however the underlying mechanism remained unclear. The differential expression level of miR-335-5p and ROCK1 were determined by qRT-PCR and IHC analysis in human tissue samples with or without lymph node metastasis. Transwell assay was conducted to determine cell ability of migration and invasion. SiRNA interference, microRNA transfection and western blot analysis were utilized to clarify the underlying regulatory mechanism. We showed that down-regulated expression of miR-335-5p and up-regulated expression of ROCK1 in NSCLC tissues were associated with lymph node metastasis. Over-expresion of miR-335-5p significantly inhibited TGF-β1-mediated NSCLC migration and invasion. Furthermore, luciferase reporter assays proved that miR-335-5p can bind to 3′-UTR of ROCK1 directly. Moreover, we confirmed that siRNA-mediated silencing of ROCK1 significantly diminished TGF-β1-mediated EMT and migratory and invasive capabilities of A549 and SPC-A1 cells. This is the first time to report that miR-335-5p regulates ROCK1 and impairs its functions, thereby playing a key role in TGF-β1-induced EMT and cell migration and invasion in NSCLC.

中文翻译：

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miR-335-5p通过ROCK1抑制TGF-β1诱导的非小细胞肺癌上皮-间质转化

大量证据表明，miRNA途径是TGF-β1途径下游信号级联反应的重要组成部分。我们以前的研究表明，miR-335-5p表达明显下调，并且在非小细胞肺癌（NSCLC）转移中起着至关重要的作用，但是其潜在机制仍不清楚。通过qRT-PCR和IHC分析测定有或无淋巴结转移的人体组织样品中miR-335-5p和ROCK1的差异表达水平。进行Transwell测定以确定细胞迁移和侵袭的能力。使用SiRNA干扰，microRNA转染和Western blot分析来阐明潜在的调控机制。我们发现在NSCLC组织中miR-335-5p的表达下调和ROCK1的表达上调与淋巴结转移有关。miR-335-5p的过度表达显着抑制TGF-β1介导的NSCLC迁移和侵袭。此外，荧光素酶报告基因检测证明miR-335-5p可以直接与ROCK1的3'-UTR结合。此外，我们证实ROCK1的siRNA介导的沉默显着降低了TGF-β1介导的EMT以及A549和SPC-A1细胞的迁移和侵袭能力。这是首次报道miR-335-5p调节ROCK1并削弱其功能，从而在TGF-β1诱导的EMT以及NSCLC中的细胞迁移和侵袭中发挥关键作用。荧光素酶报告基因检测证明，miR-335-5p可以直接与ROCK1的3'-UTR结合。此外，我们证实了ROCK1的siRNA介导的沉默显着降低了TGF-β1介导的EMT以及A549和SPC-A1细胞的迁移和侵袭能力。这是首次报道miR-335-5p调节ROCK1并削弱其功能，从而在TGF-β1诱导的EMT以及NSCLC中的细胞迁移和侵袭中发挥关键作用。荧光素酶报告基因检测证明，miR-335-5p可以直接与ROCK1的3'-UTR结合。此外，我们证实了ROCK1的siRNA介导的沉默显着降低了TGF-β1介导的EMT以及A549和SPC-A1细胞的迁移和侵袭能力。这是首次报道miR-335-5p调节ROCK1并削弱其功能，从而在TGF-β1诱导的EMT以及NSCLC中的细胞迁移和侵袭中发挥关键作用。