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MicroRNA-4723 inhibits prostate cancer growth through inactivation of the Abelson family of nonreceptor protein tyrosine kinases.

Arora S, Saini S, Fukuhara S, Majid S, Shahryari V, Yamamura S, Chiyomaru T, Deng G, Tanaka Y, Dahiya R - PLoS ONE (2013)

Bottom Line: However, the mechanism of regulation of c-Abl is not known.Analysis of putative miR-4723 targets showed that miR-4723 targets integrin alpha 3 and Methyl CpG binding protein in addition to Abl1 and Abl2 kinases.In conclusion, we have identified a novel microRNA that mediates regulation of Abl kinases in prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Veterans Affairs Medical Center, San Francisco and University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
The Abelson (c-Abl) proto-oncogene encodes a highly conserved nonreceptor protein tyrosine kinase that plays a role in cell proliferation, differentiation, apoptosis and cell adhesion. c-Abl represents a specific anti-cancer target in prostate cancer as aberrant activity of this kinase has been implicated in the stimulation of prostate cancer growth and progression. However, the mechanism of regulation of c-Abl is not known. Here we report that Abl kinases are regulated by a novel microRNA, miR-4723, in prostate cancer. Expression profiling of miR-4723 expression in a cohort of prostate cancer clinical specimens showed that miR-4723 expression is widely attenuated in prostate cancer. Low miR-4723 expression was significantly correlated with poor survival outcome and our analyses suggest that miR-4723 has significant potential as a disease biomarker for diagnosis and prognosis in prostate cancer. To evaluate the functional significance of decreased miR-4723 expression in prostate cancer, miR-4723 was overexpressed in prostate cancer cell lines followed by functional assays. miR-4723 overexpression led to significant decreases in cell growth, clonability, invasion and migration. Importantly, miR-4723 expression led to dramatic induction of apoptosis in prostate cancer cell lines suggesting that miR-4723 is a pro-apoptotic miRNA regulating prostate carcinogenesis. Analysis of putative miR-4723 targets showed that miR-4723 targets integrin alpha 3 and Methyl CpG binding protein in addition to Abl1 and Abl2 kinases. Further, we found that the expression of Abl kinase is inversely correlated with miR-4723 expression in prostate cancer clinical specimens. Also, Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells suggesting that Abl is a functionally relevant target of miR-4723 in prostate cancer. In conclusion, we have identified a novel microRNA that mediates regulation of Abl kinases in prostate cancer. This study suggests that miR-4723 may be an attractive target for therapeutic intervention in prostate cancer.

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Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells.PC3 cells were transfected with Abl1 siRNA/nonspecific (NS) control siRNA for 72 h followed by various assays. Two sets of siRNA against Abl1- Abl1 siRNA1 and siRNA2- were used for functional assays. (A) Relative Abl1 protein expression after siRNA transfections as assessed by immunoblotting. GAPDH was used as a loading control. (B) Cell viability assay, (C) Colony formation assay, (D) Cell cycle analysis, (E) Apoptosis assay in PC3 cells after NS siRNA (left panel)/Abl1 siRNA1 (middle panel)/Abl1 siRNA2 (right panel) treatments.
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pone-0078023-g007: Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells.PC3 cells were transfected with Abl1 siRNA/nonspecific (NS) control siRNA for 72 h followed by various assays. Two sets of siRNA against Abl1- Abl1 siRNA1 and siRNA2- were used for functional assays. (A) Relative Abl1 protein expression after siRNA transfections as assessed by immunoblotting. GAPDH was used as a loading control. (B) Cell viability assay, (C) Colony formation assay, (D) Cell cycle analysis, (E) Apoptosis assay in PC3 cells after NS siRNA (left panel)/Abl1 siRNA1 (middle panel)/Abl1 siRNA2 (right panel) treatments.

Mentions: To determine if Abl is a functionally relevant target of miR-4723 in prostate cancer, we inhibited Abl1 expression using siRNA to see if Abl1 knockdown functionally mimics the effects of miR-4723 overexpression. We treated PC3 cells with Abl1 siRNA followed by in vitro functional assays (Fig. 7). Two sets of siRNA against Abl1- Abl1 siRNA1 and siRNA2- were used to achieve efficient knockdown as assessed by immunoblot analysis (Fig. 7A). Non-specific siRNA (NS) was used as a control. A significant decrease in cell viability was observed in Abl1 siRNA treated PC3 cells as compared to cells treated with control siRNA (Fig. 7B). Abl1 knockdown also led to decreased clonogenicity of PC3 cells compared to control (Fig. 7C). Cell cycle analysis showed that Abl1 knockdown led to G2-M phase arrest similar to miR-4723 overexpression (Fig. 7D). Apoptosis assay showed that apoptotic cell fractions were significantly increased upon Abl1 knockdown compared to control siRNA-treated cells, an effect similar to that observed upon miR-4723 reintroduction in PC3 cells (Fig. 7E). These results suggest that Abl1 is an important determinant of the tumorigenic properties of prostate cancer cells and that Abl1 inhibition partially phenocopies the anti-tumorigenic effects of miR-4723 in prostate cancer.


MicroRNA-4723 inhibits prostate cancer growth through inactivation of the Abelson family of nonreceptor protein tyrosine kinases.

Arora S, Saini S, Fukuhara S, Majid S, Shahryari V, Yamamura S, Chiyomaru T, Deng G, Tanaka Y, Dahiya R - PLoS ONE (2013)

Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells.PC3 cells were transfected with Abl1 siRNA/nonspecific (NS) control siRNA for 72 h followed by various assays. Two sets of siRNA against Abl1- Abl1 siRNA1 and siRNA2- were used for functional assays. (A) Relative Abl1 protein expression after siRNA transfections as assessed by immunoblotting. GAPDH was used as a loading control. (B) Cell viability assay, (C) Colony formation assay, (D) Cell cycle analysis, (E) Apoptosis assay in PC3 cells after NS siRNA (left panel)/Abl1 siRNA1 (middle panel)/Abl1 siRNA2 (right panel) treatments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815229&req=5

pone-0078023-g007: Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells.PC3 cells were transfected with Abl1 siRNA/nonspecific (NS) control siRNA for 72 h followed by various assays. Two sets of siRNA against Abl1- Abl1 siRNA1 and siRNA2- were used for functional assays. (A) Relative Abl1 protein expression after siRNA transfections as assessed by immunoblotting. GAPDH was used as a loading control. (B) Cell viability assay, (C) Colony formation assay, (D) Cell cycle analysis, (E) Apoptosis assay in PC3 cells after NS siRNA (left panel)/Abl1 siRNA1 (middle panel)/Abl1 siRNA2 (right panel) treatments.
Mentions: To determine if Abl is a functionally relevant target of miR-4723 in prostate cancer, we inhibited Abl1 expression using siRNA to see if Abl1 knockdown functionally mimics the effects of miR-4723 overexpression. We treated PC3 cells with Abl1 siRNA followed by in vitro functional assays (Fig. 7). Two sets of siRNA against Abl1- Abl1 siRNA1 and siRNA2- were used to achieve efficient knockdown as assessed by immunoblot analysis (Fig. 7A). Non-specific siRNA (NS) was used as a control. A significant decrease in cell viability was observed in Abl1 siRNA treated PC3 cells as compared to cells treated with control siRNA (Fig. 7B). Abl1 knockdown also led to decreased clonogenicity of PC3 cells compared to control (Fig. 7C). Cell cycle analysis showed that Abl1 knockdown led to G2-M phase arrest similar to miR-4723 overexpression (Fig. 7D). Apoptosis assay showed that apoptotic cell fractions were significantly increased upon Abl1 knockdown compared to control siRNA-treated cells, an effect similar to that observed upon miR-4723 reintroduction in PC3 cells (Fig. 7E). These results suggest that Abl1 is an important determinant of the tumorigenic properties of prostate cancer cells and that Abl1 inhibition partially phenocopies the anti-tumorigenic effects of miR-4723 in prostate cancer.

Bottom Line: However, the mechanism of regulation of c-Abl is not known.Analysis of putative miR-4723 targets showed that miR-4723 targets integrin alpha 3 and Methyl CpG binding protein in addition to Abl1 and Abl2 kinases.In conclusion, we have identified a novel microRNA that mediates regulation of Abl kinases in prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Veterans Affairs Medical Center, San Francisco and University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
The Abelson (c-Abl) proto-oncogene encodes a highly conserved nonreceptor protein tyrosine kinase that plays a role in cell proliferation, differentiation, apoptosis and cell adhesion. c-Abl represents a specific anti-cancer target in prostate cancer as aberrant activity of this kinase has been implicated in the stimulation of prostate cancer growth and progression. However, the mechanism of regulation of c-Abl is not known. Here we report that Abl kinases are regulated by a novel microRNA, miR-4723, in prostate cancer. Expression profiling of miR-4723 expression in a cohort of prostate cancer clinical specimens showed that miR-4723 expression is widely attenuated in prostate cancer. Low miR-4723 expression was significantly correlated with poor survival outcome and our analyses suggest that miR-4723 has significant potential as a disease biomarker for diagnosis and prognosis in prostate cancer. To evaluate the functional significance of decreased miR-4723 expression in prostate cancer, miR-4723 was overexpressed in prostate cancer cell lines followed by functional assays. miR-4723 overexpression led to significant decreases in cell growth, clonability, invasion and migration. Importantly, miR-4723 expression led to dramatic induction of apoptosis in prostate cancer cell lines suggesting that miR-4723 is a pro-apoptotic miRNA regulating prostate carcinogenesis. Analysis of putative miR-4723 targets showed that miR-4723 targets integrin alpha 3 and Methyl CpG binding protein in addition to Abl1 and Abl2 kinases. Further, we found that the expression of Abl kinase is inversely correlated with miR-4723 expression in prostate cancer clinical specimens. Also, Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells suggesting that Abl is a functionally relevant target of miR-4723 in prostate cancer. In conclusion, we have identified a novel microRNA that mediates regulation of Abl kinases in prostate cancer. This study suggests that miR-4723 may be an attractive target for therapeutic intervention in prostate cancer.

Show MeSH
Related in: MedlinePlus