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MCRS1 overexpression, which is specifically inhibited by miR-129*, promotes the epithelial-mesenchymal transition and metastasis in non-small cell lung cancer.

Liu MX, Zhou KC, Cao Y - Mol. Cancer (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Experimental Pathology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China. caoy@mail.kiz.ac.cn.

ABSTRACT

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.

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Differentially expressed genes related to EMT process after MCRS1 silencing. (a) A mRNA microarray analysis of the expression changes of genes related to EMT in EPLC-32 M1 cells following stable MCRS1 knockdown. +, up-regulation; -, down-regulation. (b, c) qRT-PCR validation of differentially expressed genes with and without MCRS1 knockdown in EPLC-32 M1 and NCI-H292 cells. Luc: cells without MCRS1 silencing; Msh3: cells with stable MCRS1 silencing. The results were analyzed by student’s t-test (P <0.05).
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Fig4: Differentially expressed genes related to EMT process after MCRS1 silencing. (a) A mRNA microarray analysis of the expression changes of genes related to EMT in EPLC-32 M1 cells following stable MCRS1 knockdown. +, up-regulation; -, down-regulation. (b, c) qRT-PCR validation of differentially expressed genes with and without MCRS1 knockdown in EPLC-32 M1 and NCI-H292 cells. Luc: cells without MCRS1 silencing; Msh3: cells with stable MCRS1 silencing. The results were analyzed by student’s t-test (P <0.05).

Mentions: To explore the molecular effectors associated with MCRS1 activity, we performed a microarray analysis to determine the mRNA profiles of EPLC-32 M1 following MCRS1 knockdown. The results revealed that 237 and 132 genes were up-regulated and down-regulated, respectively, after MCRS1 silencing. Through a bioinformatics analysis using Gene Ontology (GO) terms, the genes regulated by MCRS1 were grouped into several categories (Additional file 2). Additionally, using a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis we were particularly interested in how cell junctions and Notch signaling were affected by MCRS1 due to their potential roles in EMT and tumor metastasis (Figure 4a, Additional file 3). The expression levels of EMT-related molecules, such as ZO-1 (TJP1), Occludin (OCLN), desmoglein 2 (DSG2), E-cadherin (CDH1), ITGA6, Notch 3 (NOTCH3) and Vimentin (VIM), were verified by qRT-PCR (Figure 4b, 4c). Subsequently, we investigated the relationship between MCRS1 and its downstream molecules (ZO-1, Occludin, E-cadherin, DSG-2). Through bioinformatics analysis and prediction, MCRS1 could not directly bind the promoters of these downstream molecules. Using chromatin immunoprecipitation (ChIP) assay, we also did not find that MCRS1 bound directly the promoters of these genes (Additional file 4). Based on these data, MCRS1 may regulate the expressions of the genes related to cell junctions and the Notch pathway, however, those genes were not directly recognized by a transcriptional complex containing MCRS1.Figure 4


MCRS1 overexpression, which is specifically inhibited by miR-129*, promotes the epithelial-mesenchymal transition and metastasis in non-small cell lung cancer.

Liu MX, Zhou KC, Cao Y - Mol. Cancer (2014)

Differentially expressed genes related to EMT process after MCRS1 silencing. (a) A mRNA microarray analysis of the expression changes of genes related to EMT in EPLC-32 M1 cells following stable MCRS1 knockdown. +, up-regulation; -, down-regulation. (b, c) qRT-PCR validation of differentially expressed genes with and without MCRS1 knockdown in EPLC-32 M1 and NCI-H292 cells. Luc: cells without MCRS1 silencing; Msh3: cells with stable MCRS1 silencing. The results were analyzed by student’s t-test (P <0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4233086&req=5

Fig4: Differentially expressed genes related to EMT process after MCRS1 silencing. (a) A mRNA microarray analysis of the expression changes of genes related to EMT in EPLC-32 M1 cells following stable MCRS1 knockdown. +, up-regulation; -, down-regulation. (b, c) qRT-PCR validation of differentially expressed genes with and without MCRS1 knockdown in EPLC-32 M1 and NCI-H292 cells. Luc: cells without MCRS1 silencing; Msh3: cells with stable MCRS1 silencing. The results were analyzed by student’s t-test (P <0.05).
Mentions: To explore the molecular effectors associated with MCRS1 activity, we performed a microarray analysis to determine the mRNA profiles of EPLC-32 M1 following MCRS1 knockdown. The results revealed that 237 and 132 genes were up-regulated and down-regulated, respectively, after MCRS1 silencing. Through a bioinformatics analysis using Gene Ontology (GO) terms, the genes regulated by MCRS1 were grouped into several categories (Additional file 2). Additionally, using a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis we were particularly interested in how cell junctions and Notch signaling were affected by MCRS1 due to their potential roles in EMT and tumor metastasis (Figure 4a, Additional file 3). The expression levels of EMT-related molecules, such as ZO-1 (TJP1), Occludin (OCLN), desmoglein 2 (DSG2), E-cadherin (CDH1), ITGA6, Notch 3 (NOTCH3) and Vimentin (VIM), were verified by qRT-PCR (Figure 4b, 4c). Subsequently, we investigated the relationship between MCRS1 and its downstream molecules (ZO-1, Occludin, E-cadherin, DSG-2). Through bioinformatics analysis and prediction, MCRS1 could not directly bind the promoters of these downstream molecules. Using chromatin immunoprecipitation (ChIP) assay, we also did not find that MCRS1 bound directly the promoters of these genes (Additional file 4). Based on these data, MCRS1 may regulate the expressions of the genes related to cell junctions and the Notch pathway, however, those genes were not directly recognized by a transcriptional complex containing MCRS1.Figure 4

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Experimental Pathology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China. caoy@mail.kiz.ac.cn.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus