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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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miR-129* directly regulated MCRS1 expression in NSCLC cells. (a) Expression of MCRS1 and miR-129* in NSCLC cell lines. (b) The level of miR-129* in clinical samples (Student’s t-test, *P <0.05). (c) Correlation analysis of miR-129* and MCRS1 mRNA levels in NSCLC tissues (Pearson’s method, R = -0.513, *P <0.05). (d) Top: MCRS1 was predicted as a target of miR-129*. Bottom: Validation of the direct targeting of MCRS1 by miR-129* using a luciferase reporter assay (Student’s t-test, *P <0.05). (e and f) Relative mRNA and protein expressions of MCRS1, E-cadherin, and Vimentin in cells transfected with the miR-129* mimic or mimic NC (Student’s t-test, *P <0.05). MTT assays (g) and matrigel invasion assays (h) of EPLC-32 M1 and NCI-H292 cells transfected with the miR-129* mimic or mimic NC (Student’s t-test, *P <0.05). (i) The relationship between the level of miR-129* expression and the status of tumor metastasis (*P <0.05; one-way ANOVA and Student’s t-test). NC: negative control; Noncancerous: noncancerous lung tissues; No metastasis: NSCLCs without metastasis; Metastasis: NSCLCs with metastasis.
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Fig6: miR-129* directly regulated MCRS1 expression in NSCLC cells. (a) Expression of MCRS1 and miR-129* in NSCLC cell lines. (b) The level of miR-129* in clinical samples (Student’s t-test, *P <0.05). (c) Correlation analysis of miR-129* and MCRS1 mRNA levels in NSCLC tissues (Pearson’s method, R = -0.513, *P <0.05). (d) Top: MCRS1 was predicted as a target of miR-129*. Bottom: Validation of the direct targeting of MCRS1 by miR-129* using a luciferase reporter assay (Student’s t-test, *P <0.05). (e and f) Relative mRNA and protein expressions of MCRS1, E-cadherin, and Vimentin in cells transfected with the miR-129* mimic or mimic NC (Student’s t-test, *P <0.05). MTT assays (g) and matrigel invasion assays (h) of EPLC-32 M1 and NCI-H292 cells transfected with the miR-129* mimic or mimic NC (Student’s t-test, *P <0.05). (i) The relationship between the level of miR-129* expression and the status of tumor metastasis (*P <0.05; one-way ANOVA and Student’s t-test). NC: negative control; Noncancerous: noncancerous lung tissues; No metastasis: NSCLCs without metastasis; Metastasis: NSCLCs with metastasis.

Mentions: Many oncogenes and tumor suppressor genes are regulated by miRNAs [18]. Thus, to identify miRNAs that may regulate MCRS1 expression, miRNA profiles were determined in the NSCLC cell lines (EPLC-32 M1, A549, and 801D) and 16HBE. Compared with 16HBE, in total, 87 consensus miRNAs were differentially expressed in NSCLC cells (Additional file 8). We then used the miRWalk web tool to predict seven miRNAs that target MCRS1 (Additional file 9). The research strategy is described in Additional file 10, and miR-129* and miR-1299 were identified as candidates. We examined the expression of these miRNAs in a series of lung cancer cell lines and found that the expression of miR-129* correlated inversely with MCRS1 expression (Figure 6a). Moreover, we confirmed the data from the cultured cells in the 15 NSCLC tissues and 6 noncancerous lung tissues. As shown in Figure 6b and 6c, miR-129* was remarkably down-regulated in the NSCLC tissues and appeared to be inversely correlated with MCRS1 mRNA expression.


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)

miR-129* directly regulated MCRS1 expression in NSCLC cells. (a) Expression of MCRS1 and miR-129* in NSCLC cell lines. (b) The level of miR-129* in clinical samples (Student’s t-test, *P <0.05). (c) Correlation analysis of miR-129* and MCRS1 mRNA levels in NSCLC tissues (Pearson’s method, R = -0.513, *P <0.05). (d) Top: MCRS1 was predicted as a target of miR-129*. Bottom: Validation of the direct targeting of MCRS1 by miR-129* using a luciferase reporter assay (Student’s t-test, *P <0.05). (e and f) Relative mRNA and protein expressions of MCRS1, E-cadherin, and Vimentin in cells transfected with the miR-129* mimic or mimic NC (Student’s t-test, *P <0.05). MTT assays (g) and matrigel invasion assays (h) of EPLC-32 M1 and NCI-H292 cells transfected with the miR-129* mimic or mimic NC (Student’s t-test, *P <0.05). (i) The relationship between the level of miR-129* expression and the status of tumor metastasis (*P <0.05; one-way ANOVA and Student’s t-test). NC: negative control; Noncancerous: noncancerous lung tissues; No metastasis: NSCLCs without metastasis; Metastasis: NSCLCs with metastasis.
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Fig6: miR-129* directly regulated MCRS1 expression in NSCLC cells. (a) Expression of MCRS1 and miR-129* in NSCLC cell lines. (b) The level of miR-129* in clinical samples (Student’s t-test, *P <0.05). (c) Correlation analysis of miR-129* and MCRS1 mRNA levels in NSCLC tissues (Pearson’s method, R = -0.513, *P <0.05). (d) Top: MCRS1 was predicted as a target of miR-129*. Bottom: Validation of the direct targeting of MCRS1 by miR-129* using a luciferase reporter assay (Student’s t-test, *P <0.05). (e and f) Relative mRNA and protein expressions of MCRS1, E-cadherin, and Vimentin in cells transfected with the miR-129* mimic or mimic NC (Student’s t-test, *P <0.05). MTT assays (g) and matrigel invasion assays (h) of EPLC-32 M1 and NCI-H292 cells transfected with the miR-129* mimic or mimic NC (Student’s t-test, *P <0.05). (i) The relationship between the level of miR-129* expression and the status of tumor metastasis (*P <0.05; one-way ANOVA and Student’s t-test). NC: negative control; Noncancerous: noncancerous lung tissues; No metastasis: NSCLCs without metastasis; Metastasis: NSCLCs with metastasis.
Mentions: Many oncogenes and tumor suppressor genes are regulated by miRNAs [18]. Thus, to identify miRNAs that may regulate MCRS1 expression, miRNA profiles were determined in the NSCLC cell lines (EPLC-32 M1, A549, and 801D) and 16HBE. Compared with 16HBE, in total, 87 consensus miRNAs were differentially expressed in NSCLC cells (Additional file 8). We then used the miRWalk web tool to predict seven miRNAs that target MCRS1 (Additional file 9). The research strategy is described in Additional file 10, and miR-129* and miR-1299 were identified as candidates. We examined the expression of these miRNAs in a series of lung cancer cell lines and found that the expression of miR-129* correlated inversely with MCRS1 expression (Figure 6a). Moreover, we confirmed the data from the cultured cells in the 15 NSCLC tissues and 6 noncancerous lung tissues. As shown in Figure 6b and 6c, miR-129* was remarkably down-regulated in the NSCLC tissues and appeared to be inversely correlated with MCRS1 mRNA expression.

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