Limits...
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

miR-145 inhibits GC SGC-7901 cell growth in vitro(A) qRT-PCR analysis of miR-145 in SGC-7901 cells transfected with miR-145 over-expression construct or miR-145 inhibitor. All qRT-PCR results are expressed as mean ± SEM from at least three independent experiments. (B) The effects of miR-145 on SGC-7901 cell viability were determined by MTT assay at 24, 48 and 72 h after transfection with miR-145 over-expression construct or miR-145 inhibitor, with empty vector or ASO-NC, respectively. (C) Representative micrographs of crystal violet-stained cell colonies were analyzed by colony formation assay at day 12 after transfection. (D) Histogram indicated the percentage of cells in G1, S and G2 phases after transfection for 48 h based on the flow-cytometric analysis. Data were presented as mean ± SEM. (*p < 0.05; **p < 0.01; ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480708&req=5

Figure 4: miR-145 inhibits GC SGC-7901 cell growth in vitro(A) qRT-PCR analysis of miR-145 in SGC-7901 cells transfected with miR-145 over-expression construct or miR-145 inhibitor. All qRT-PCR results are expressed as mean ± SEM from at least three independent experiments. (B) The effects of miR-145 on SGC-7901 cell viability were determined by MTT assay at 24, 48 and 72 h after transfection with miR-145 over-expression construct or miR-145 inhibitor, with empty vector or ASO-NC, respectively. (C) Representative micrographs of crystal violet-stained cell colonies were analyzed by colony formation assay at day 12 after transfection. (D) Histogram indicated the percentage of cells in G1, S and G2 phases after transfection for 48 h based on the flow-cytometric analysis. Data were presented as mean ± SEM. (*p < 0.05; **p < 0.01; ***p < 0.001).

Mentions: To investigate the functional role of miR-145, we performed gain-of-function and loss-of function studies by transfecting miR-145 expression vector, empty vector, miR-145 inhibitor, and control oligos into the SGC-7901 and AGS cell lines. qRT-PCR was then performed to validate miR-145 expression after transfection. miR-145 expression level was increased 200-fold 48 h after transfection of the miR-145 expression vector and almostly no fold change after miR-145 inhibitor tranefection (Figure 4A). The MTT assay and colony formation assay dispalyed a significant inhibition of cell growth and colony formation after miR-145 transfection when compared with cells transfected with the empty vector (control) in both cell lines, but increased by transfecting miR-145 inhibitor (Figure 4B and 4C; Supplementary Figure 2A–2D). We then study the impact of miR-145 on cell cycle progression. Both cell lines showed overexpression of miR-145 caused the accumulation of cells in the S phase and a corresponding reduction of cells in the G2/M phase, it's consistent there was a decrease percentage of cells in S phase when transfected miR-145 inhibitor (Figure 4D; Supplementary Figure 2E and 2F). These results suggested that miR-145 blocked the S/G2 transition in GC cells in vitro.


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)

miR-145 inhibits GC SGC-7901 cell growth in vitro(A) qRT-PCR analysis of miR-145 in SGC-7901 cells transfected with miR-145 over-expression construct or miR-145 inhibitor. All qRT-PCR results are expressed as mean ± SEM from at least three independent experiments. (B) The effects of miR-145 on SGC-7901 cell viability were determined by MTT assay at 24, 48 and 72 h after transfection with miR-145 over-expression construct or miR-145 inhibitor, with empty vector or ASO-NC, respectively. (C) Representative micrographs of crystal violet-stained cell colonies were analyzed by colony formation assay at day 12 after transfection. (D) Histogram indicated the percentage of cells in G1, S and G2 phases after transfection for 48 h based on the flow-cytometric analysis. Data were presented as mean ± SEM. (*p < 0.05; **p < 0.01; ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480708&req=5

Figure 4: miR-145 inhibits GC SGC-7901 cell growth in vitro(A) qRT-PCR analysis of miR-145 in SGC-7901 cells transfected with miR-145 over-expression construct or miR-145 inhibitor. All qRT-PCR results are expressed as mean ± SEM from at least three independent experiments. (B) The effects of miR-145 on SGC-7901 cell viability were determined by MTT assay at 24, 48 and 72 h after transfection with miR-145 over-expression construct or miR-145 inhibitor, with empty vector or ASO-NC, respectively. (C) Representative micrographs of crystal violet-stained cell colonies were analyzed by colony formation assay at day 12 after transfection. (D) Histogram indicated the percentage of cells in G1, S and G2 phases after transfection for 48 h based on the flow-cytometric analysis. Data were presented as mean ± SEM. (*p < 0.05; **p < 0.01; ***p < 0.001).
Mentions: To investigate the functional role of miR-145, we performed gain-of-function and loss-of function studies by transfecting miR-145 expression vector, empty vector, miR-145 inhibitor, and control oligos into the SGC-7901 and AGS cell lines. qRT-PCR was then performed to validate miR-145 expression after transfection. miR-145 expression level was increased 200-fold 48 h after transfection of the miR-145 expression vector and almostly no fold change after miR-145 inhibitor tranefection (Figure 4A). The MTT assay and colony formation assay dispalyed a significant inhibition of cell growth and colony formation after miR-145 transfection when compared with cells transfected with the empty vector (control) in both cell lines, but increased by transfecting miR-145 inhibitor (Figure 4B and 4C; Supplementary Figure 2A–2D). We then study the impact of miR-145 on cell cycle progression. Both cell lines showed overexpression of miR-145 caused the accumulation of cells in the S phase and a corresponding reduction of cells in the G2/M phase, it's consistent there was a decrease percentage of cells in S phase when transfected miR-145 inhibitor (Figure 4D; Supplementary Figure 2E and 2F). These results suggested that miR-145 blocked the S/G2 transition in GC cells in vitro.

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