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Mechanism of epithelial?mesenchymal transition inhibited by miR?203 in non?small cell lung cancer.

ABSTRACT: The aim of the present study was to investigate whether miR?203 can inhibit transforming growth factor?? (TGF??)?induced epithelial?mesenchymal transition (EMT), and the migration and invasion ability of non?small cell lung cancer (NSCLC) cells by targeting SMAD3. In the present study, the expression levels of miR?203, SMAD3 mRNA and protein in NSCLC tissues were examined, as well as their corresponding paracancerous samples. The miR?203 mimics and miR?203 inhibitor were transfected into the H226 cell line. RT?qPCR was used to assess the expression levels of E?cadherin, Snail, N?cadherin and vimentin mRNA, and western blotting was performed to detect the expression levels of p?SMAD2, SMAD2, p?SMAD3, SMAD3 and SMAD4. The cell migration and invasion abilities were detected by Transwell assays. The target site of SMAD3 was predicted by the combined action between miR?203 and dual luciferase. The results revealed that the RNA levels of miR?203, compared with paracancerous tissues, were decreased in NSCLC tissues, while SMAD3 mRNA and protein levels were upregulated, and miR?203 inhibited SMAD3 expression. Induction of TGF?? led to decreased E?cadherin mRNA levels, upregulation of Snail, N?cadherin and vimentin mRNA levels (P<0.05), and significant increase in cell migration and invasion, whereas transfection of miR?203 mimics reversed the aforementioned results (P<0.05). Conversely, miR?203 inhibitor could further aggravate the aforementioned results (P<0.05). Western blot results revealed that transfection of miR?203 mimics significantly reduced the protein expression of SMAD3 and p?SMAD3 (P<0.05). Furthermore, the results of the Dual?Luciferase assay revealed that miR?203 inhibited SMAD3 expression by interacting with specific regions of its 3'?UTR. Overall, a novel mechanism is revealed, in which, miR?203 can inhibit SMAD3 by interacting with specific regions of the 3'?UTR of SMAD3, thereby restraining TGF???induced EMT progression and migration and invasion of NSCLC cells.

SUBMITTER: Huang W

PROVIDER: S-EPMC6967097 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Mechanism of epithelial‑mesenchymal transition inhibited by miR‑203 in non‑small cell lung cancer.

Huang Weicong W Wu Yuanbo Y Cheng Dezhi D He Zhifeng Z

Oncology reports 20191213 2

The aim of the present study was to investigate whether miR‑203 can inhibit transforming growth factor‑β (TGF‑β)‑induced epithelial‑mesenchymal transition (EMT), and the migration and invasion ability of non‑small cell lung cancer (NSCLC) cells by targeting SMAD3. In the present study, the expression levels of miR‑203, SMAD3 mRNA and protein in NSCLC tissues were examined, as well as their corresponding paracancerous samples. The miR‑203 mimics and miR‑203 inhibitor were transfected into the H22 ...[more]

PMID: 31894278

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Project description:BACKGROUND: Although tumor invasion and metastasis are both classical hallmarks of cancer malignancy and the major causes of poor clinical outcomes among cancer patients, the underlying master regulators of invasion and metastasis remain largely unknown. In this study, we observed that an overexpression of microspherule protein 1 (MCRS1) promotes the invasion and metastasis of non-small cell lung cancer (NSCLC) cells. Furthermore, we sought to systematically investigate the pathophysiological functions and related mechanisms of MCRS1. METHODS: Retrovirus-mediated RNA interference was employed to knockdown MCRS1 expression in NSCLC cell lines. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot respectively were used to measure levels of mRNA and protein. Further cell permeability assessment, invasion and proliferation assays were conducted to evaluate MCRS1 functions in vitro while nude mice experiments were performed to examine metastatic capability in vivo. Microarray analysis and microRNA (miRNA) sequencing were respectively carried out for mRNA and miRNA expression profiling, while chromatin immunoprecipitation (ChIP), luciferase reporter assay, and miRNA transfection were used to investigate the interaction between MCRS1 and miRNAs. RESULTS: MCRS1 knockdown induced morphological alterations, increased monolayer integrity, decreased cellular invasion and metastasis, and attenuated stemness and drug resistance among tested NSCLC cells. The levels of MCRS1 expression were likewise correlated with tumor metastasis among NSCLC patients. We identified differentially expressed genes after MCRS1 silencing, which included cell junction molecules, such as ZO-1, Occludin, E-cadherin, and DSG2. However, these differentially expressed genes were not directly recognized by a transcriptional complex containing MCRS1. Furthermore, we found that MCRS1 binds to the miR-155 promoter and regulates its expression, as well as MCRS1 promotes epithelial-mesenchymal transition (EMT), invasion, and metastasis through the up-regulation of miR-155. Systematic investigations ultimately showed that MCRS1 was directly and negatively regulated by the binding of miR-129* to its 3'-UTR, with miR-129* overexpression suppressing the growth and invasion of NSCLC cells. CONCLUSIONS: MiR-129* down-regulation induced MCRS1 overexpression, which promotes EMT and invasion/metastasis of NSCLC cells through both the up-regulation of miR-155 and down-regulation of cell junction molecules. This miR-129*/MCRS1/miR-155 axis provides a new angle in understanding the basis for the invasion and metastasis of lung cancer.

Project description:Background: Dysregulation of microRNAs (miRNAs) is associated with the pathogenesis of non-small cell lung cancer (NSCLC). However, the mechanisms through which miR-182-5p regulate NSCLC progression have not been established. This study aimed at evaluating the expression levels of miR-182-5p in human NSCLC and its function in lung cancer cells. Endothelial PAS Domain-containing protein 1 (EPAS1; also referred to as hypoxia-inducing factor 2A, HIF-2α) is a transcription factor that is responsible for induction of genes related to cell survival under hypoxia conditions. Hypoxia, an inherent feature of solid tumors, is associated with aggressive phenotypes, as well as resistance to radiotherapy and chemotherapy, which predict metastasis and poor prognosis. Methods: The Cancer Genome Atlas (TCGA) dataset was used to investigate the association between miR-182-5p expression and clinicopathological characteristics as well as prognosis of NSCLC patients. Target genes of miR-182-5p were identified using the PITA, miRmap, microT, miRanda, PicTar, and TargetScan prediction tools. Transwell assays were performed to determine the potential functions of miR-182-5p in lung cancer cells. Luciferase reporter assays were performed to analyze regulation of the putative target of miR-182-5p while western blot assays were used to validate the luciferase results. Results: miR-182-5p was found to be upregulated in NSCLC tissues and acted as an independent prognostic factor for tumor recurrence in NSCLC patients. Functionally, overexpression of miR-182-5p promoted lung cancer cell migration and invasion. Genome-wide gene expression analysis and luciferase report assays revealed that EPAS1 is a direct target of miR-182-5p. EPAS1 was negatively correlated with miR-182-5p expression in NSCLC tissues. Univariate and multivariate survival analyses identified EPAS1 as an independent prognostic factor for overall survival (OS) in NSCLC. Conclusions: These findings imply that miR-182-5p promotes NSCLC progression by targeting EPAS1 and is, therefore, a potential indicator of tumor recurrence in NSCLC patients.

Dataset Information

Mechanism of epithelial?mesenchymal transition inhibited by miR?203 in non?small cell lung cancer.

Publications

Mechanism of epithelial‑mesenchymal transition inhibited by miR‑203 in non‑small cell lung cancer.

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