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

1,25(OH)2D3 induces miR-145 expression in gastric cancer cells(A) SGC-7901 cells and AGS cells were both treated with 200 nM 1,25(OH)2D3 for 48 hours and total RNA was isolated from the cells and qRT-PCR analysis of miRNAs fold change was performed as described in Materials and Methods. (B) qRT-PCR analysis of miR-145 was quantified, and values are expressed as -fold change (C) miR-145 inhibitor or ASO-NC transfected SGC-7901 cells were treated with 500 nM calcitriol, Cell growth was determined at 24, 48 and 72 hour time points by MTT assay. (D) Empty vector or sh-VDR transfected SGC-7901 cells were treated with 200 nM calcitriol for 24 hours, total RNA was isolated and qRT-PCR analysis was performed. (E) Lists of the putative VDRE sequences and the human miR-145 locus in chromosome5. (F) The in vivo interaction of VDR with miR-145 VDRE was shown. SGC-7901 cells were treated with 500 nM 1,25(OH)2D3 or blank control for 48 hour, and ChIP assays were performed with control (rat IgG), anti-VDR antibody. (G) qRT-PCR analysis was performed with primers spanning predicted VDRE of miR-145. All qRT-PCR results are expressed as mean ± SEM from at least three independent experiments. (*p < 0.05; **p < 0.01.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: 1,25(OH)2D3 induces miR-145 expression in gastric cancer cells(A) SGC-7901 cells and AGS cells were both treated with 200 nM 1,25(OH)2D3 for 48 hours and total RNA was isolated from the cells and qRT-PCR analysis of miRNAs fold change was performed as described in Materials and Methods. (B) qRT-PCR analysis of miR-145 was quantified, and values are expressed as -fold change (C) miR-145 inhibitor or ASO-NC transfected SGC-7901 cells were treated with 500 nM calcitriol, Cell growth was determined at 24, 48 and 72 hour time points by MTT assay. (D) Empty vector or sh-VDR transfected SGC-7901 cells were treated with 200 nM calcitriol for 24 hours, total RNA was isolated and qRT-PCR analysis was performed. (E) Lists of the putative VDRE sequences and the human miR-145 locus in chromosome5. (F) The in vivo interaction of VDR with miR-145 VDRE was shown. SGC-7901 cells were treated with 500 nM 1,25(OH)2D3 or blank control for 48 hour, and ChIP assays were performed with control (rat IgG), anti-VDR antibody. (G) qRT-PCR analysis was performed with primers spanning predicted VDRE of miR-145. All qRT-PCR results are expressed as mean ± SEM from at least three independent experiments. (*p < 0.05; **p < 0.01.)

Mentions: To understand the mechanism involved in 1,25(OH)2D3 cancer growth inhibition, the effects of 1,25(OH)2D3 on miRNA expression in human GC were analyzed. The expression of several miRNAs in RNA samples extracted from SGC-7901 and AGS cells treated with 0.2 μmol 1,25(OH)2D3 or blank control was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) (Figure 2A). Among them, the expression level of miR-145 was significantly increased by three folds (Figure 2B). Therefore we further studied the role of miR-145 in 1,25(OH)2D3 antitumor activity. To validate the cell function affected by the change of miR-145 expression regulated by 1,25(OH)2D3, the MTT assay showed that when miR-145 was inhibited, anti-proliferative effect of 1,25(OH)2D3 decreased (Figure 2C). To determine if VDR was required for miR-145 expression, we transfected a small hairpin RNA against VDR, sh-VDR and a control shRNA into SGC-7901 cells, VDR mRNA and protein expression level were low compared with those of the control shRNA transfected cells (Supplementary Figure 1). As shown in Figure 2D, miR-145 levels were decreased in sh-VDR transfected cells. When sh-VDR transfected cells were treated with 0.2 μmol 1,25(OH)2D3, miR-145 expression level were rescued, but not totally (Figure 2D). We predicted a candidated VDRE at the upstream of miR-145 locus of human chromosome 5 (named as miR-145-VDRE) by bioinformatics based on the known VDRE motif sequences (Figure 2E). To validate our hypothesis that the VDRE interacts with the VDR in vivo, we conducted ChIP assay in SGC-7901 cells. qRT-PCR analysis showed that 1,25(OH)2D3 induced a significant increase of miR-145-VDRE using DNA purified from ChIP assay (Figure 2G). Antibodies against the VDR precipitated genomic DNA fragments miR-145-VDRE in cells treated with 1,25(OH)2D3, and no DNA was detected in IgG precipitates (Figure 2F), showing that the recruitments between VDR and the miR-145-VDRE was induced by 1,25(OH)2D3. Based on these findings, we concluded that miR-145 is induced through VDR and which is critical for 1,25(OH)2D3 actions.


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)

1,25(OH)2D3 induces miR-145 expression in gastric cancer cells(A) SGC-7901 cells and AGS cells were both treated with 200 nM 1,25(OH)2D3 for 48 hours and total RNA was isolated from the cells and qRT-PCR analysis of miRNAs fold change was performed as described in Materials and Methods. (B) qRT-PCR analysis of miR-145 was quantified, and values are expressed as -fold change (C) miR-145 inhibitor or ASO-NC transfected SGC-7901 cells were treated with 500 nM calcitriol, Cell growth was determined at 24, 48 and 72 hour time points by MTT assay. (D) Empty vector or sh-VDR transfected SGC-7901 cells were treated with 200 nM calcitriol for 24 hours, total RNA was isolated and qRT-PCR analysis was performed. (E) Lists of the putative VDRE sequences and the human miR-145 locus in chromosome5. (F) The in vivo interaction of VDR with miR-145 VDRE was shown. SGC-7901 cells were treated with 500 nM 1,25(OH)2D3 or blank control for 48 hour, and ChIP assays were performed with control (rat IgG), anti-VDR antibody. (G) qRT-PCR analysis was performed with primers spanning predicted VDRE of miR-145. All qRT-PCR results are expressed as mean ± SEM from at least three independent experiments. (*p < 0.05; **p < 0.01.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: 1,25(OH)2D3 induces miR-145 expression in gastric cancer cells(A) SGC-7901 cells and AGS cells were both treated with 200 nM 1,25(OH)2D3 for 48 hours and total RNA was isolated from the cells and qRT-PCR analysis of miRNAs fold change was performed as described in Materials and Methods. (B) qRT-PCR analysis of miR-145 was quantified, and values are expressed as -fold change (C) miR-145 inhibitor or ASO-NC transfected SGC-7901 cells were treated with 500 nM calcitriol, Cell growth was determined at 24, 48 and 72 hour time points by MTT assay. (D) Empty vector or sh-VDR transfected SGC-7901 cells were treated with 200 nM calcitriol for 24 hours, total RNA was isolated and qRT-PCR analysis was performed. (E) Lists of the putative VDRE sequences and the human miR-145 locus in chromosome5. (F) The in vivo interaction of VDR with miR-145 VDRE was shown. SGC-7901 cells were treated with 500 nM 1,25(OH)2D3 or blank control for 48 hour, and ChIP assays were performed with control (rat IgG), anti-VDR antibody. (G) qRT-PCR analysis was performed with primers spanning predicted VDRE of miR-145. All qRT-PCR results are expressed as mean ± SEM from at least three independent experiments. (*p < 0.05; **p < 0.01.)
Mentions: To understand the mechanism involved in 1,25(OH)2D3 cancer growth inhibition, the effects of 1,25(OH)2D3 on miRNA expression in human GC were analyzed. The expression of several miRNAs in RNA samples extracted from SGC-7901 and AGS cells treated with 0.2 μmol 1,25(OH)2D3 or blank control was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) (Figure 2A). Among them, the expression level of miR-145 was significantly increased by three folds (Figure 2B). Therefore we further studied the role of miR-145 in 1,25(OH)2D3 antitumor activity. To validate the cell function affected by the change of miR-145 expression regulated by 1,25(OH)2D3, the MTT assay showed that when miR-145 was inhibited, anti-proliferative effect of 1,25(OH)2D3 decreased (Figure 2C). To determine if VDR was required for miR-145 expression, we transfected a small hairpin RNA against VDR, sh-VDR and a control shRNA into SGC-7901 cells, VDR mRNA and protein expression level were low compared with those of the control shRNA transfected cells (Supplementary Figure 1). As shown in Figure 2D, miR-145 levels were decreased in sh-VDR transfected cells. When sh-VDR transfected cells were treated with 0.2 μmol 1,25(OH)2D3, miR-145 expression level were rescued, but not totally (Figure 2D). We predicted a candidated VDRE at the upstream of miR-145 locus of human chromosome 5 (named as miR-145-VDRE) by bioinformatics based on the known VDRE motif sequences (Figure 2E). To validate our hypothesis that the VDRE interacts with the VDR in vivo, we conducted ChIP assay in SGC-7901 cells. qRT-PCR analysis showed that 1,25(OH)2D3 induced a significant increase of miR-145-VDRE using DNA purified from ChIP assay (Figure 2G). Antibodies against the VDR precipitated genomic DNA fragments miR-145-VDRE in cells treated with 1,25(OH)2D3, and no DNA was detected in IgG precipitates (Figure 2F), showing that the recruitments between VDR and the miR-145-VDRE was induced by 1,25(OH)2D3. Based on these findings, we concluded that miR-145 is induced through VDR and which is critical for 1,25(OH)2D3 actions.

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