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miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 by directly targeting E2F3 in gastric cancer cells.

Chang S, Gao L, Yang Y, Tong D, Guo B, Liu L, Li Z, Song T, Huang C - Oncotarget (2015)

Bottom Line: Inhibition of miR-145 reverses the antiproliferative effect of 1,25(OH)2D3.Furthermore, miR-145 expression was lower in tumors compared with matched normal samples and correlated with increased the E2F3 transcription factor protein staining.Altogether, our results indicate that miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 in GC cells and might hold promise for prognosis and therapeutic strategies for GC treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Molecular Biology/Key Laboratory of Environment and Genes Related to Diseases, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi, P. R. China.

ABSTRACT
VitaminD3 signaling is involved in inhibiting the development and progression of gastric cancer (GC), while the active vitamin D metabolite 1-alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3)-mediated gene regulatory mechanisms in GC remain unclear. We found that miR-145 is induced by 1,25(OH)2D3 in a dose- and vitamin D receptor (VDR)-dependent manner in GC cells. Inhibition of miR-145 reverses the antiproliferative effect of 1,25(OH)2D3. Furthermore, miR-145 expression was lower in tumors compared with matched normal samples and correlated with increased the E2F3 transcription factor protein staining. Overexpression of miR-145 inhibited colony formation, cell viability and induced cell arrest in S-phase in GC cells by targeting E2F3 and CDK6. miR-145 inhibition consistently abrogates the 1,25(OH)2D3-mediated suppression of E2F3, CDK6, CDK2 and CCNA2 genes. Altogether, our results indicate that miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 in GC cells and might hold promise for prognosis and therapeutic strategies for GC treatment.

No MeSH data available.


Related in: MedlinePlus

E2F3 and CDK6 are both direct targets of miR-145(A) Scheme of the potential binding sites of miR-145 in the 3′ UTR of E2F3 and CDK6. (B) Luciferase assay in SGC-7901 cells. Pre-miR-145 was cotransfected with target gene reporter construct (WT or MUT version of pGLO constructs) or NS-control. Luciferase activity in pGLO-E2F3 and pGLO-CDK6 group displayed a significant decrease following ectopic expression of miR-145. (*p < 0.05; **p < 0.01; ***p < 0.001, Student's t-test) (C) E2F3 protein expression level measured by IHC in gastric cancer tissue (G1, poorly differentiated;G2 moderately differentiated;G3 well differentiated) (D) E2F3 protein level measured by western blotting in GC and adjust normal tissue, 2 paired sample presented. (E) Inverse correlation between miR-145 and E2F3 expression in GC tissues. Statistical analysis was performed using Pearson ’ s correlation coefficient (r = − 0.47, *P < 0.05).
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Figure 5: E2F3 and CDK6 are both direct targets of miR-145(A) Scheme of the potential binding sites of miR-145 in the 3′ UTR of E2F3 and CDK6. (B) Luciferase assay in SGC-7901 cells. Pre-miR-145 was cotransfected with target gene reporter construct (WT or MUT version of pGLO constructs) or NS-control. Luciferase activity in pGLO-E2F3 and pGLO-CDK6 group displayed a significant decrease following ectopic expression of miR-145. (*p < 0.05; **p < 0.01; ***p < 0.001, Student's t-test) (C) E2F3 protein expression level measured by IHC in gastric cancer tissue (G1, poorly differentiated;G2 moderately differentiated;G3 well differentiated) (D) E2F3 protein level measured by western blotting in GC and adjust normal tissue, 2 paired sample presented. (E) Inverse correlation between miR-145 and E2F3 expression in GC tissues. Statistical analysis was performed using Pearson ’ s correlation coefficient (r = − 0.47, *P < 0.05).

Mentions: To understand the mechanism of miR-145-induced inhibition of cell proliferation in GC, miR-145 targets were identified using computer-aided miRNA target prediction programs, such as TargetScan (http://www.targetscan.org/) and other miRBase linked websites (http://www.mirbase.org/). Four putative miR-145 target genes that might play a role in cell proliferation were identified, including CDK6, E2F3, CCND2, and ERBB4, as shown in Figure 5A, the binding sites at E2F3 and CDK6 3′-untranslated region (UTR) were displayed. Luciferase reporter assays showed that two of them, CDK6 and E2F3, induced 70% and 60% reduction in luciferase activity compared with vector control, respectively (Figure 5B). We focused on E2F3 because CDK6 has been reported as a target of miR-145 in colon cancer [1]. The miR-145 target sequence in the 3′ - UTR of E2F3 is highly conserved in human and other species. E2F3 is an important cell cycle regulation gene that is highly expressed in GC tissues (Figure 5C and 5D). Next, we determined whether ectopic expression of miR-145 suppressed endogenous E2F3 at the protein level by western blot. As shown in Figure 6D, miR-145 suppressed both E2F3 and CDK6. miR-145 also induced downregulation of CDK2 and CCNA2, which are vital cell cycle regulators (Figure 6D). Interestingly, miR-145 expression level in vivo was inversely-correlated with E2F3 mRNA expression level, which was verified by Pearson's correlation coefficient test (Figure 5E). Taken together, our data demonstrated that miR-145 target E2F3 and CDK6 directly and suppress their expression at translation level in SGC-7901 cells.


miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 by directly targeting E2F3 in gastric cancer cells.

Chang S, Gao L, Yang Y, Tong D, Guo B, Liu L, Li Z, Song T, Huang C - Oncotarget (2015)

E2F3 and CDK6 are both direct targets of miR-145(A) Scheme of the potential binding sites of miR-145 in the 3′ UTR of E2F3 and CDK6. (B) Luciferase assay in SGC-7901 cells. Pre-miR-145 was cotransfected with target gene reporter construct (WT or MUT version of pGLO constructs) or NS-control. Luciferase activity in pGLO-E2F3 and pGLO-CDK6 group displayed a significant decrease following ectopic expression of miR-145. (*p < 0.05; **p < 0.01; ***p < 0.001, Student's t-test) (C) E2F3 protein expression level measured by IHC in gastric cancer tissue (G1, poorly differentiated;G2 moderately differentiated;G3 well differentiated) (D) E2F3 protein level measured by western blotting in GC and adjust normal tissue, 2 paired sample presented. (E) Inverse correlation between miR-145 and E2F3 expression in GC tissues. Statistical analysis was performed using Pearson ’ s correlation coefficient (r = − 0.47, *P < 0.05).
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Figure 5: E2F3 and CDK6 are both direct targets of miR-145(A) Scheme of the potential binding sites of miR-145 in the 3′ UTR of E2F3 and CDK6. (B) Luciferase assay in SGC-7901 cells. Pre-miR-145 was cotransfected with target gene reporter construct (WT or MUT version of pGLO constructs) or NS-control. Luciferase activity in pGLO-E2F3 and pGLO-CDK6 group displayed a significant decrease following ectopic expression of miR-145. (*p < 0.05; **p < 0.01; ***p < 0.001, Student's t-test) (C) E2F3 protein expression level measured by IHC in gastric cancer tissue (G1, poorly differentiated;G2 moderately differentiated;G3 well differentiated) (D) E2F3 protein level measured by western blotting in GC and adjust normal tissue, 2 paired sample presented. (E) Inverse correlation between miR-145 and E2F3 expression in GC tissues. Statistical analysis was performed using Pearson ’ s correlation coefficient (r = − 0.47, *P < 0.05).
Mentions: To understand the mechanism of miR-145-induced inhibition of cell proliferation in GC, miR-145 targets were identified using computer-aided miRNA target prediction programs, such as TargetScan (http://www.targetscan.org/) and other miRBase linked websites (http://www.mirbase.org/). Four putative miR-145 target genes that might play a role in cell proliferation were identified, including CDK6, E2F3, CCND2, and ERBB4, as shown in Figure 5A, the binding sites at E2F3 and CDK6 3′-untranslated region (UTR) were displayed. Luciferase reporter assays showed that two of them, CDK6 and E2F3, induced 70% and 60% reduction in luciferase activity compared with vector control, respectively (Figure 5B). We focused on E2F3 because CDK6 has been reported as a target of miR-145 in colon cancer [1]. The miR-145 target sequence in the 3′ - UTR of E2F3 is highly conserved in human and other species. E2F3 is an important cell cycle regulation gene that is highly expressed in GC tissues (Figure 5C and 5D). Next, we determined whether ectopic expression of miR-145 suppressed endogenous E2F3 at the protein level by western blot. As shown in Figure 6D, miR-145 suppressed both E2F3 and CDK6. miR-145 also induced downregulation of CDK2 and CCNA2, which are vital cell cycle regulators (Figure 6D). Interestingly, miR-145 expression level in vivo was inversely-correlated with E2F3 mRNA expression level, which was verified by Pearson's correlation coefficient test (Figure 5E). Taken together, our data demonstrated that miR-145 target E2F3 and CDK6 directly and suppress their expression at translation level in SGC-7901 cells.

Bottom Line: Inhibition of miR-145 reverses the antiproliferative effect of 1,25(OH)2D3.Furthermore, miR-145 expression was lower in tumors compared with matched normal samples and correlated with increased the E2F3 transcription factor protein staining.Altogether, our results indicate that miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 in GC cells and might hold promise for prognosis and therapeutic strategies for GC treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Molecular Biology/Key Laboratory of Environment and Genes Related to Diseases, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi, P. R. China.

ABSTRACT
VitaminD3 signaling is involved in inhibiting the development and progression of gastric cancer (GC), while the active vitamin D metabolite 1-alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3)-mediated gene regulatory mechanisms in GC remain unclear. We found that miR-145 is induced by 1,25(OH)2D3 in a dose- and vitamin D receptor (VDR)-dependent manner in GC cells. Inhibition of miR-145 reverses the antiproliferative effect of 1,25(OH)2D3. Furthermore, miR-145 expression was lower in tumors compared with matched normal samples and correlated with increased the E2F3 transcription factor protein staining. Overexpression of miR-145 inhibited colony formation, cell viability and induced cell arrest in S-phase in GC cells by targeting E2F3 and CDK6. miR-145 inhibition consistently abrogates the 1,25(OH)2D3-mediated suppression of E2F3, CDK6, CDK2 and CCNA2 genes. Altogether, our results indicate that miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 in GC cells and might hold promise for prognosis and therapeutic strategies for GC treatment.

No MeSH data available.


Related in: MedlinePlus