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MiR-203 down-regulates Rap1A and suppresses cell proliferation, adhesion and invasion in prostate cancer.

Xiang J, Bian C, Wang H, Huang S, Wu D - J. Exp. Clin. Cancer Res. (2015)

Bottom Line: Here, we confirmed that the expression of miR-203 was significantly downregulated in prostate cancer specimens compared with matched adjacent normal prostate specimens.In addition, we identified Rap1A as a direct target suppressed by miR-203, and there was an inverse relationship between the expression of miR-203 and Rap1A in PCa.These findings provide further evidence that a crucial role for miR-203 in inhibiting metastasis of PCa through the suppression of Rap1A expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Tongji Hospital, Tongji University School of Medicine, NO 389 Xinchun road, Shanghai, 200065, China. zhuxufifa2007@163.com.

ABSTRACT

Objective: Evidence supports an important role for miR-203 in the regulation of the proliferation, migration and invasion of prostate cancer (PCa) cells. However, the exact mechanisms of miR-203 in PCa are not entirely clear.

Methods: We examined the expression of miR-203 in prostate cancer tissues, adjacent normal tissues, PCa cell lines and normal prostate epithelial cells by qRT-PCR. Then, the effects of miR-203 or Rap1A on proliferation, adhesion and invasion of PCa cells were assayed using CKK-8, adhesion analysis, and transwell invasion assays. Luciferase reporter assay was performed to assess miR-203 binding to Rap1A mRNA. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice.

Results: Here, we confirmed that the expression of miR-203 was significantly downregulated in prostate cancer specimens compared with matched adjacent normal prostate specimens. Mechanistic dissection revealed that miR-203 mediated cell proliferation, adhesion and invasion in vitro, and tumor growth in vivo, as evidenced by reduced RAC1, p-PAK1, and p-MEK1 expression. In addition, we identified Rap1A as a direct target suppressed by miR-203, and there was an inverse relationship between the expression of miR-203 and Rap1A in PCa. Knockdown of Rap1A phenocopied the effects of miR-203 on PCa cell growth and invasion. Furthermore, Rap1A over-expression in PCa cells partially reversed the effects of miR-203-expression on cell adhesion and invasion.

Conclusions: These findings provide further evidence that a crucial role for miR-203 in inhibiting metastasis of PCa through the suppression of Rap1A expression.

No MeSH data available.


Related in: MedlinePlus

Rap1A is a new target of miR-203. (A) The putative miR-203 binding sequence was in the 3′-UTR of Rap1A mRNA. (B) A dual luciferase report assay was performed in HEK-293 T cells cotransfected with psi-CHECK2- Rap1A and miR-203. The dual luciferase report assay of HEK-293 T cells cotransfected with RAP1A-3′-UTR-WT or Rap1A-3′-UTR-MUT and empty vector control or pCDH-miR-203. The Renilla luciferase activity was used as control. (C) Western blot analysis of the endogenous expressions of Rap1A upon forced expression of miR-203. (D) The protein expression of Rap1A in PC-3 and DU145 cells transfected with miR-203 inhibitor or NC was analyzed by western blotting. (E) Quantitative real-time PCR analysis showing the mRNA levels of Rap1A. (F) The relative expression of Rap1A in PCa tissues and matched adjacent normal tissues was determined by qRT-PCR. (G) Plots showing a negative correlation between the relative expression levels of miR-203 and Rap1A in the above samples. * p < 0.05, ** p < 0.01.
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Fig3: Rap1A is a new target of miR-203. (A) The putative miR-203 binding sequence was in the 3′-UTR of Rap1A mRNA. (B) A dual luciferase report assay was performed in HEK-293 T cells cotransfected with psi-CHECK2- Rap1A and miR-203. The dual luciferase report assay of HEK-293 T cells cotransfected with RAP1A-3′-UTR-WT or Rap1A-3′-UTR-MUT and empty vector control or pCDH-miR-203. The Renilla luciferase activity was used as control. (C) Western blot analysis of the endogenous expressions of Rap1A upon forced expression of miR-203. (D) The protein expression of Rap1A in PC-3 and DU145 cells transfected with miR-203 inhibitor or NC was analyzed by western blotting. (E) Quantitative real-time PCR analysis showing the mRNA levels of Rap1A. (F) The relative expression of Rap1A in PCa tissues and matched adjacent normal tissues was determined by qRT-PCR. (G) Plots showing a negative correlation between the relative expression levels of miR-203 and Rap1A in the above samples. * p < 0.05, ** p < 0.01.

Mentions: To identify the potential target genes of miR-203 that might contribute to its prometastatic function, we used TargetScan (http://www.targetscan.org/) to predict the targets of miR-203 and found that Rap1A was a putative target (Figure 3A). To determine whether Rap1A was a direct target of miR-203, reporter constructs containing full-length Rap1A 3′-UTR were made, along with their corresponding mutant counterpart at the miR-203 target site. Cotransfection of the reporters with miR-203 caused a 50% decrease in luciferase activity compared with the control vector (Figure 3B). Mutating the miRNA binding sites on Rap1A abrogated the miRNA-mediated degradation and rescued the luciferase activity (Figure 3B). These results suggested that Rap1A was a direct target of miR-203. Rap1A protein expression was also significantly decreased in miR-203 over-expressing cells (Figure 3C) and Rap1A protein levels in PC-3 and DU 145 cells with overexpression of miR-203 were increased after transfection with miR-203 inhibitor, compared with a control miRNA inhibitor (Figure 3D). To demonstrate further that Rap1A was negatively regulated by miR-203, we assessed the expression of Rap1A at the mRNA level in the same panel of specimens. The expression levels of Rap1A mRNA were higher in cancer tissues compared with matched adjacent non-tumor tissues (Figure 3E). An inverse correlation between the expression of miR-203 and Rap1A was observed by linear regression analysis. Together, these data demonstrated that that Rap1A is a direct target of miR-203. As Rap1A is reported to function as an oncogene, this may indicate that miR-203 suppresses tumor metastasis through inhibiting Rap1A.Figure 3


MiR-203 down-regulates Rap1A and suppresses cell proliferation, adhesion and invasion in prostate cancer.

Xiang J, Bian C, Wang H, Huang S, Wu D - J. Exp. Clin. Cancer Res. (2015)

Rap1A is a new target of miR-203. (A) The putative miR-203 binding sequence was in the 3′-UTR of Rap1A mRNA. (B) A dual luciferase report assay was performed in HEK-293 T cells cotransfected with psi-CHECK2- Rap1A and miR-203. The dual luciferase report assay of HEK-293 T cells cotransfected with RAP1A-3′-UTR-WT or Rap1A-3′-UTR-MUT and empty vector control or pCDH-miR-203. The Renilla luciferase activity was used as control. (C) Western blot analysis of the endogenous expressions of Rap1A upon forced expression of miR-203. (D) The protein expression of Rap1A in PC-3 and DU145 cells transfected with miR-203 inhibitor or NC was analyzed by western blotting. (E) Quantitative real-time PCR analysis showing the mRNA levels of Rap1A. (F) The relative expression of Rap1A in PCa tissues and matched adjacent normal tissues was determined by qRT-PCR. (G) Plots showing a negative correlation between the relative expression levels of miR-203 and Rap1A in the above samples. * p < 0.05, ** p < 0.01.
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Related In: Results  -  Collection

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Fig3: Rap1A is a new target of miR-203. (A) The putative miR-203 binding sequence was in the 3′-UTR of Rap1A mRNA. (B) A dual luciferase report assay was performed in HEK-293 T cells cotransfected with psi-CHECK2- Rap1A and miR-203. The dual luciferase report assay of HEK-293 T cells cotransfected with RAP1A-3′-UTR-WT or Rap1A-3′-UTR-MUT and empty vector control or pCDH-miR-203. The Renilla luciferase activity was used as control. (C) Western blot analysis of the endogenous expressions of Rap1A upon forced expression of miR-203. (D) The protein expression of Rap1A in PC-3 and DU145 cells transfected with miR-203 inhibitor or NC was analyzed by western blotting. (E) Quantitative real-time PCR analysis showing the mRNA levels of Rap1A. (F) The relative expression of Rap1A in PCa tissues and matched adjacent normal tissues was determined by qRT-PCR. (G) Plots showing a negative correlation between the relative expression levels of miR-203 and Rap1A in the above samples. * p < 0.05, ** p < 0.01.
Mentions: To identify the potential target genes of miR-203 that might contribute to its prometastatic function, we used TargetScan (http://www.targetscan.org/) to predict the targets of miR-203 and found that Rap1A was a putative target (Figure 3A). To determine whether Rap1A was a direct target of miR-203, reporter constructs containing full-length Rap1A 3′-UTR were made, along with their corresponding mutant counterpart at the miR-203 target site. Cotransfection of the reporters with miR-203 caused a 50% decrease in luciferase activity compared with the control vector (Figure 3B). Mutating the miRNA binding sites on Rap1A abrogated the miRNA-mediated degradation and rescued the luciferase activity (Figure 3B). These results suggested that Rap1A was a direct target of miR-203. Rap1A protein expression was also significantly decreased in miR-203 over-expressing cells (Figure 3C) and Rap1A protein levels in PC-3 and DU 145 cells with overexpression of miR-203 were increased after transfection with miR-203 inhibitor, compared with a control miRNA inhibitor (Figure 3D). To demonstrate further that Rap1A was negatively regulated by miR-203, we assessed the expression of Rap1A at the mRNA level in the same panel of specimens. The expression levels of Rap1A mRNA were higher in cancer tissues compared with matched adjacent non-tumor tissues (Figure 3E). An inverse correlation between the expression of miR-203 and Rap1A was observed by linear regression analysis. Together, these data demonstrated that that Rap1A is a direct target of miR-203. As Rap1A is reported to function as an oncogene, this may indicate that miR-203 suppresses tumor metastasis through inhibiting Rap1A.Figure 3

Bottom Line: Here, we confirmed that the expression of miR-203 was significantly downregulated in prostate cancer specimens compared with matched adjacent normal prostate specimens.In addition, we identified Rap1A as a direct target suppressed by miR-203, and there was an inverse relationship between the expression of miR-203 and Rap1A in PCa.These findings provide further evidence that a crucial role for miR-203 in inhibiting metastasis of PCa through the suppression of Rap1A expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Tongji Hospital, Tongji University School of Medicine, NO 389 Xinchun road, Shanghai, 200065, China. zhuxufifa2007@163.com.

ABSTRACT

Objective: Evidence supports an important role for miR-203 in the regulation of the proliferation, migration and invasion of prostate cancer (PCa) cells. However, the exact mechanisms of miR-203 in PCa are not entirely clear.

Methods: We examined the expression of miR-203 in prostate cancer tissues, adjacent normal tissues, PCa cell lines and normal prostate epithelial cells by qRT-PCR. Then, the effects of miR-203 or Rap1A on proliferation, adhesion and invasion of PCa cells were assayed using CKK-8, adhesion analysis, and transwell invasion assays. Luciferase reporter assay was performed to assess miR-203 binding to Rap1A mRNA. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice.

Results: Here, we confirmed that the expression of miR-203 was significantly downregulated in prostate cancer specimens compared with matched adjacent normal prostate specimens. Mechanistic dissection revealed that miR-203 mediated cell proliferation, adhesion and invasion in vitro, and tumor growth in vivo, as evidenced by reduced RAC1, p-PAK1, and p-MEK1 expression. In addition, we identified Rap1A as a direct target suppressed by miR-203, and there was an inverse relationship between the expression of miR-203 and Rap1A in PCa. Knockdown of Rap1A phenocopied the effects of miR-203 on PCa cell growth and invasion. Furthermore, Rap1A over-expression in PCa cells partially reversed the effects of miR-203-expression on cell adhesion and invasion.

Conclusions: These findings provide further evidence that a crucial role for miR-203 in inhibiting metastasis of PCa through the suppression of Rap1A expression.

No MeSH data available.


Related in: MedlinePlus