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MicroRNA-495 induces breast cancer cell migration by targeting JAM-A.

Cao M, Nie W, Li J, Zhang Y, Yan X, Guan X, Chen X, Zen K, Zhang CY, Jiang X, Hou D - Protein Cell (2014)

Bottom Line: MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression.In the present study, we found that miR-495 was markedly up-regulated in clinical breast cancer samples by quantitative real time-PCR (qRT-PCR).Taken together, our findings suggest that miR-495 could facilitate breast cancer progression through the repression of JAM-A, making this miRNA a potential therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, China.

ABSTRACT
MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression. The deregulated expression of miRNAs is associated with a variety of diseases, including breast cancer. In the present study, we found that miR-495 was markedly up-regulated in clinical breast cancer samples by quantitative real time-PCR (qRT-PCR). Junctional adhesion molecule A (JAM-A) was predicted to be a potential target of miR-495 by bioinformatics analysis and was subsequently verified by luciferase assay and Western blotting. JAM-A was found to be negatively correlated with the migration of breast cancer cells through loss-of-function and gain-of-function assays, and the inhibition of JAM-A by miR-495 promoted the migration of MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of JAM-A could restore miR-495-induced breast cancer cell migration. Taken together, our findings suggest that miR-495 could facilitate breast cancer progression through the repression of JAM-A, making this miRNA a potential therapeutic target.

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MiR-495 induces breast cancer cell migration by targeting JAM-A. (A) Western blotting analysis of the protein levels of JAM-A in MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. (B) Left panel: Representative image of wound healing assay of MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. Right panel: Quantitative analysis of the wound closure rates. (C) Top panel: Representative image of Transwell assay of MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. Bottom panel: Quantitative analysis of the migration rates. (D) Western blotting analysis of the protein levels of JAM-A in MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. (E) Left panel: Representative image of wound healing assay of MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. Right panel: Quantitative analysis of the wound closure rates. (F) Left panel: Representative image of Transwell assay of MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. Right panel: Quantitative analysis of the migration rates. *P < 0.05; **P < 0.01
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Fig5: MiR-495 induces breast cancer cell migration by targeting JAM-A. (A) Western blotting analysis of the protein levels of JAM-A in MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. (B) Left panel: Representative image of wound healing assay of MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. Right panel: Quantitative analysis of the wound closure rates. (C) Top panel: Representative image of Transwell assay of MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. Bottom panel: Quantitative analysis of the migration rates. (D) Western blotting analysis of the protein levels of JAM-A in MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. (E) Left panel: Representative image of wound healing assay of MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. Right panel: Quantitative analysis of the wound closure rates. (F) Left panel: Representative image of Transwell assay of MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. Right panel: Quantitative analysis of the migration rates. *P < 0.05; **P < 0.01

Mentions: To further confirm that the effects of miR-495 are mediated by repression of JAM-A in breast cancer cells, knockdown of JAM-A in MCF-7 cells by miR-495 mimic was restored by transfecting JAM-A cDNA vector, the expression of which is not regulated by miR-495 due to its lack of the 3′-UTR (Fig. 5A). Compared with the cells transfected with miR-495 mimic and control vector, MCF-7 cells transfected with miR-495 mimic and JAM-A vector exhibited impaired migration ability (Fig. 5B and 5C). Moreover, JAM-A siRNA was transfected into MDA-MB-231 cells to counteract the increase of JAM-A protein caused by miR-495 inhibitor (Fig. 5D). Fig. 5E and 5F show that the inhibition of MDA-MB-231 cell migration by miR-495 inhibitor can be reversed by JAM-A siRNA. Taken together, these results demonstrated that miR-495 promotes breast cancer cell migration by inhibiting JAM-A.Figure 5


MicroRNA-495 induces breast cancer cell migration by targeting JAM-A.

Cao M, Nie W, Li J, Zhang Y, Yan X, Guan X, Chen X, Zen K, Zhang CY, Jiang X, Hou D - Protein Cell (2014)

MiR-495 induces breast cancer cell migration by targeting JAM-A. (A) Western blotting analysis of the protein levels of JAM-A in MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. (B) Left panel: Representative image of wound healing assay of MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. Right panel: Quantitative analysis of the wound closure rates. (C) Top panel: Representative image of Transwell assay of MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. Bottom panel: Quantitative analysis of the migration rates. (D) Western blotting analysis of the protein levels of JAM-A in MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. (E) Left panel: Representative image of wound healing assay of MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. Right panel: Quantitative analysis of the wound closure rates. (F) Left panel: Representative image of Transwell assay of MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. Right panel: Quantitative analysis of the migration rates. *P < 0.05; **P < 0.01
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Fig5: MiR-495 induces breast cancer cell migration by targeting JAM-A. (A) Western blotting analysis of the protein levels of JAM-A in MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. (B) Left panel: Representative image of wound healing assay of MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. Right panel: Quantitative analysis of the wound closure rates. (C) Top panel: Representative image of Transwell assay of MCF-7 cells transfected with control mimic plus control plasmid, miR-495 mimic plus control plasmid or miR-495 mimic plus JAM-A overexpression plasmid. Bottom panel: Quantitative analysis of the migration rates. (D) Western blotting analysis of the protein levels of JAM-A in MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. (E) Left panel: Representative image of wound healing assay of MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. Right panel: Quantitative analysis of the wound closure rates. (F) Left panel: Representative image of Transwell assay of MDA-MB-231 cells transfected with control inhibitor plus control siRNA, miR-495 inhibitor plus control siRNA or miR-495 inhibitor plus JAM-A siRNA. Right panel: Quantitative analysis of the migration rates. *P < 0.05; **P < 0.01
Mentions: To further confirm that the effects of miR-495 are mediated by repression of JAM-A in breast cancer cells, knockdown of JAM-A in MCF-7 cells by miR-495 mimic was restored by transfecting JAM-A cDNA vector, the expression of which is not regulated by miR-495 due to its lack of the 3′-UTR (Fig. 5A). Compared with the cells transfected with miR-495 mimic and control vector, MCF-7 cells transfected with miR-495 mimic and JAM-A vector exhibited impaired migration ability (Fig. 5B and 5C). Moreover, JAM-A siRNA was transfected into MDA-MB-231 cells to counteract the increase of JAM-A protein caused by miR-495 inhibitor (Fig. 5D). Fig. 5E and 5F show that the inhibition of MDA-MB-231 cell migration by miR-495 inhibitor can be reversed by JAM-A siRNA. Taken together, these results demonstrated that miR-495 promotes breast cancer cell migration by inhibiting JAM-A.Figure 5

Bottom Line: MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression.In the present study, we found that miR-495 was markedly up-regulated in clinical breast cancer samples by quantitative real time-PCR (qRT-PCR).Taken together, our findings suggest that miR-495 could facilitate breast cancer progression through the repression of JAM-A, making this miRNA a potential therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, China.

ABSTRACT
MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression. The deregulated expression of miRNAs is associated with a variety of diseases, including breast cancer. In the present study, we found that miR-495 was markedly up-regulated in clinical breast cancer samples by quantitative real time-PCR (qRT-PCR). Junctional adhesion molecule A (JAM-A) was predicted to be a potential target of miR-495 by bioinformatics analysis and was subsequently verified by luciferase assay and Western blotting. JAM-A was found to be negatively correlated with the migration of breast cancer cells through loss-of-function and gain-of-function assays, and the inhibition of JAM-A by miR-495 promoted the migration of MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of JAM-A could restore miR-495-induced breast cancer cell migration. Taken together, our findings suggest that miR-495 could facilitate breast cancer progression through the repression of JAM-A, making this miRNA a potential therapeutic target.

Show MeSH
Related in: MedlinePlus