BACKGROUND Recent evidence indicates that metformin inhibits mammalian cancer growth and metastasis through the regulation of microRNAs. Metformin regulates miR-381 stability, which plays a vital role in tumor progression. Moreover, increased YAP expression and activity induce non-small cell lung cancer (NSCLC) tumor growth and metastasis. However, the molecular mechanism underpinning how metformin-induced upregulation of miR-381 directly targets YAP or its interactions with the epithelial-mesenchymal transition (EMT) marker protein Snail in NSCLC is still unknown. METHODS Levels of RNA and protein were analyzed using qPCR, western blotting and immunofluorescence staining. Cellular proliferation was detected using a CCK8 assay. Cell migration and invasion were analyzed using wound healing and transwell assays. Promoter activity and transcription were investigated using the luciferase reporter assay. Chromatin immunoprecipitation was used to detect the binding of YAP to the promoter of Snail. The interaction between miR-381 and the 3'UTR of YAP mRNA was analyzed using the MS2 expression system and co-immunoprecipitation with biotin. RESULTS We observed that miR-381 expression is negatively correlated with YAP expression and plays an opposite role to YAP in the regulation of cellular proliferation, invasion, migration, and EMT of NSCLC cells. The miR-381 function as a tumor suppressor was significantly downregulated in lung cancer tissue specimens and cell lines, which decreased the expression of its direct target YAP. In addition, metformin decreased cell growth, migration, invasion, and EMT via up-regulation of miR-381. Moreover, YAP, which functions as a co-transcription factor, enhanced NSCLC progression and metastasis by upregulation of Snail. Snail knockdown downregulated the mesenchymal marker vimentin and upregulated the epithelial marker E-cadherin in lung cancer cells. Furthermore, miR-381, YAP, and Snail constitute the miR-381-YAP-Snail signal axis, which is repressed by metformin, and enhances cancer cell invasiveness by directly regulating EMT. CONCLUSIONS Metformin-induced repression of miR-381-YAP-Snail axis activity disrupts NSCLC growth and metastasis. Thus, we believe that the miR-381-YAP-Snail signal axis may be a suitable diagnostic marker and a potential therapeutic target for lung cancer.

中文翻译：

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二甲双胍抑制的 miR-381-YAP-snail 轴活性会破坏 NSCLC 的生长和转移。

背景最近的证据表明二甲双胍通过调节微小RNA抑制哺乳动物癌症的生长和转移。二甲双胍调节 miR-381 的稳定性，这在肿瘤进展中起着至关重要的作用。此外，增加的 YAP 表达和活性会诱导非小细胞肺癌 (NSCLC) 肿瘤的生长和转移。然而，支持二甲双胍诱导的 miR-381 上调如何直接靶向 YAP 或其与 NSCLC 中上皮-间质转化 (EMT) 标记蛋白 Snail 相互作用的分子机制仍然未知。方法 使用 qPCR、蛋白质印迹和免疫荧光染色分析 RNA 和蛋白质水平。使用CCK8测定法检测细胞增殖。使用伤口愈合和 transwell 测定分析细胞迁移和侵袭。使用荧光素酶报告基因测定研究启动子活性和转录。染色质免疫沉淀用于检测 YAP 与 Snail 启动子的结合。使用 MS2 表达系统和与生物素的免疫共沉淀分析 miR-381 和 YAP mRNA 的 3'UTR 之间的相互作用。结果我们观察到miR-381表达与YAP表达呈负相关，在NSCLC细胞的细胞增殖、侵袭、迁移和EMT的调节中与YAP起着相反的作用。miR-381 作为肿瘤抑制因子的功能在肺癌组织标本和细胞系中显着下调，从而降低其直接靶标 YAP 的表达。此外，二甲双胍通过上调 miR-381 降低细胞生长、迁移、侵袭和 EMT。此外，YAP，其作为共转录因子发挥作用，通过上调 Snail 增强 NSCLC 的进展和转移。Snail 敲除下调间充质标志物波形蛋白并上调肺癌细胞中的上皮标志物 E-钙粘蛋白。此外，miR-381、YAP和Snail构成miR-381-YAP-Snail信号轴，被二甲双胍抑制，通过直接调控EMT增强癌细胞侵袭力。结论 二甲双胍诱导的 miR-381-YAP-Snail 轴活性抑制会破坏 NSCLC 的生长和转移。因此，我们认为 miR-381-YAP-Snail 信号轴可能是肺癌的合适诊断标志物和潜在治疗靶点。Snail 敲除下调间充质标志物波形蛋白并上调肺癌细胞中的上皮标志物 E-钙粘蛋白。此外，miR-381、YAP和Snail构成miR-381-YAP-Snail信号轴，被二甲双胍抑制，通过直接调控EMT增强癌细胞侵袭力。结论 二甲双胍诱导的 miR-381-YAP-Snail 轴活性抑制会破坏 NSCLC 的生长和转移。因此，我们认为 miR-381-YAP-Snail 信号轴可能是肺癌的合适诊断标志物和潜在治疗靶点。Snail 敲除下调间充质标志物波形蛋白并上调肺癌细胞中的上皮标志物 E-钙粘蛋白。此外，miR-381、YAP和Snail构成miR-381-YAP-Snail信号轴，被二甲双胍抑制，通过直接调控EMT增强癌细胞侵袭力。结论 二甲双胍诱导的 miR-381-YAP-Snail 轴活性抑制会破坏 NSCLC 的生长和转移。因此，我们认为 miR-381-YAP-Snail 信号轴可能是肺癌的合适诊断标志物和潜在治疗靶点。结论 二甲双胍诱导的 miR-381-YAP-Snail 轴活性抑制会破坏 NSCLC 的生长和转移。因此，我们认为 miR-381-YAP-Snail 信号轴可能是肺癌的合适诊断标志物和潜在治疗靶点。结论 二甲双胍诱导的 miR-381-YAP-Snail 轴活性抑制会破坏 NSCLC 的生长和转移。因此，我们认为 miR-381-YAP-Snail 信号轴可能是肺癌的合适诊断标志物和潜在治疗靶点。