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miR-211 suppresses epithelial ovarian cancer proliferation and cell-cycle progression by targeting Cyclin D1 and CDK6.

Xia B, Yang S, Liu T, Lou G - Mol. Cancer (2015)

Bottom Line: Its function and loss-of-function have been described in normal and cancer cells and tissues. miR-211 is known to be dysregulated in ovarian cancer: however, its function and the downstream effect of its loss-of-function in ovarian cancer have not been described before.We found that the expression of miR-211 is downregulated in EOC tissues and cell lines compared to normal epithelial ovarian tissue and human ovarian surface epithelial cells, respectively. miR-211 was found to arrest cells in the G0/G1-phase, inhibit proliferation and induce apoptosis.Cyclin D1 and CDK6 were found to be direct targets of miR-211, and when overexpressed in miR-211-expressing EOC cells, could restore proliferative ability.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology, the Affiliated Tumor Hospital, Harbin Medical University, 150 Haping Rd, Nangang, Harbin, 150020, Heilongjiang, China. xiabairong9999@126.com.

ABSTRACT

Background: Epithelial ovarian cancer (EOC) is a significant cause of morbidity and mortality. MicroRNAs play important roles in cancer development and progression. The microRNA miR-211 is localized on intron 6 of the Trpm1 gene at 15q13-q14, a locus that is frequently lost in neoplasms. Its function and loss-of-function have been described in normal and cancer cells and tissues. miR-211 is known to be dysregulated in ovarian cancer: however, its function and the downstream effect of its loss-of-function in ovarian cancer have not been described before.

Methods: We analyzed miR-211 expression in clinical samples of primary EOC tissues compared to normal epithelial ovarian tissues and in the EOC cell lines: OVCAR3, Caov3, OVCA429, SKOV3 and A2780 compared to human ovarian surface epithelial cells. We then investigated the effect of miR-211 on EOC cell proliferation and apoptosis by counting cell numbers, MTT, colony formation, cell cycle, and PI/Annexin V staining assays. A luciferase reporter system was developed to assess miR-211 regulation of the predicted targets. Expression level of discovered targets and correlation with miR-211 expression were analyzed in EOC tissues. Finally, OVCAR3 stably expressing miR-211 or control cells were injected subcutaneously into mice to determine in vivo effect of miR-211 on tumorigenesis.

Results: We found that the expression of miR-211 is downregulated in EOC tissues and cell lines compared to normal epithelial ovarian tissue and human ovarian surface epithelial cells, respectively. miR-211 was found to arrest cells in the G0/G1-phase, inhibit proliferation and induce apoptosis. Cyclin D1 and CDK6 were found to be direct targets of miR-211, and when overexpressed in miR-211-expressing EOC cells, could restore proliferative ability. Finally, in vitro investigation confirmed that miR-211 is a tumor suppressor that controls Cyclin D1 and CDK6 expression.

Conclusions: Our results demonstrate that miR-211 is a tumor suppressor that controls expression of Cyclin D1 and CDK6, and that its downregulation results in overexpression of Cyclin D1 and CDK6 which increases proliferation ability of EOC cells to proliferate compared to normal cells.

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Related in: MedlinePlus

Cyclin D1 and CDK6 are miR-211 targets. A. Schematic representation showing the five predicted miR-211 targets (TCF4, Cyclin D1, Cyclin D2, Cyclin D3 and CDK6) screened using three algorithms (miRNA.org, miRWalk and Targetscan). B. OVCAR3 cells were co-transfected with TCF4, CDK6, Cyclin D1, Cyclin D2 or Cyclin D3 3′UTR pGL plasmid with miR-211. Forty-eight hours later, luciferase assay was performed. *p < 0.05 compared to miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments. C-D. Sequence of miR-211 with the putative binding sites of Cyclin D1 (C) and CDK6 (D) 3′ UTR. E. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. F. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-211 or LV-miR-Ctrl. G-H. The mRNA expression levels of Cyclin D1 (G) or CDK6 (H) correlated inversely with miR-211. I-J. Cyclin D1 (I) and CDK6 (J) expression in ovarian tissues (GDS3592/Cyclin D1, n = 24, p = 0.017; GDS3592/CDK6, n = 24, p = 0.016).
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Fig4: Cyclin D1 and CDK6 are miR-211 targets. A. Schematic representation showing the five predicted miR-211 targets (TCF4, Cyclin D1, Cyclin D2, Cyclin D3 and CDK6) screened using three algorithms (miRNA.org, miRWalk and Targetscan). B. OVCAR3 cells were co-transfected with TCF4, CDK6, Cyclin D1, Cyclin D2 or Cyclin D3 3′UTR pGL plasmid with miR-211. Forty-eight hours later, luciferase assay was performed. *p < 0.05 compared to miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments. C-D. Sequence of miR-211 with the putative binding sites of Cyclin D1 (C) and CDK6 (D) 3′ UTR. E. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. F. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-211 or LV-miR-Ctrl. G-H. The mRNA expression levels of Cyclin D1 (G) or CDK6 (H) correlated inversely with miR-211. I-J. Cyclin D1 (I) and CDK6 (J) expression in ovarian tissues (GDS3592/Cyclin D1, n = 24, p = 0.017; GDS3592/CDK6, n = 24, p = 0.016).

Mentions: We used microRNA.org, Targetscan and miRWalk databases to predict potential miR-211 targets. Hundreds of potential targets were found, but we selected transcription factor 4 (TCF4), Cyclin D1, Cyclin D2, Cyclin D3 and Cyclin dependent kinase 6 (CDK6) for further analysis since these genes have previously been reported to affect EOC cell proliferation [28-31] (Figure 4A). We inserted TCF4, CDK6, Cyclin D1, Cyclin D2 and Cyclin D3 3′UTR into luciferase reporter vectors and co-transfected with miR-211 expression plasmid into OVCAR3 and SKOV3 cells (Figure 4A). Forty-eight hours after transfection, only cells transfected with CDK6 and Cyclin D1 3′UTR plasmids had lower luciferase activity than their controls. Luciferase activity in both of these cell lines was 70% less than that of the miR-Ctrl groups (Figure 4B). Next, we compared the 3′UTR sequence of Cyclin D1 and CDK6 with miR-211. Both genes have two potential complementary miR-211 binding sites (Figure 4C, D). We next constructed Cyclin D1 and CDK6 3′UTR mutant plasmid (Additional file 1: Figure S1A-B), cotransfected with miR-211 and then performed luciferase assay. The data revealed that miR-211 targets two sites in Cyclin D1 and CDK6 (Additional file 1: Figure S1C-D). To confirm that Cyclin D1 and CDK6 are specifically targeted by miR-211, OVCAR3 and SKOV3 cells transfected with miR-211 or infected with LV-miR-211 were subjected to western blot analysis. We found that the protein levels of Cyclin D1 and CDK6 were lower in miR-211 and LV-miR-211 transfected cells compared to control (Figure 4E, F). The mRNA level of Cyclin D1 and CDK6 and the levels of miR-211 were measured in 60 EOC tissues using qRT-PCR to analyze their correlation. Spearman correlation analysis revealed a reverse correlation between miR-211 expression and Cyclin D1 or miR-211 and CDK6 expression (Figure 4G, H). The high expression of Cyclin D1 and CDK6 in OEC tissues compared to normal tissue was consistent with results from the public database (Figure 4I, J) [32]. Taken together, these results indicate that miR-211 can repress the expression of Cyclin D1 and CDK6 in EOC by directly targeting the 3′UTR of Cyclin D1 and CDK6 mRNA.Figure 4


miR-211 suppresses epithelial ovarian cancer proliferation and cell-cycle progression by targeting Cyclin D1 and CDK6.

Xia B, Yang S, Liu T, Lou G - Mol. Cancer (2015)

Cyclin D1 and CDK6 are miR-211 targets. A. Schematic representation showing the five predicted miR-211 targets (TCF4, Cyclin D1, Cyclin D2, Cyclin D3 and CDK6) screened using three algorithms (miRNA.org, miRWalk and Targetscan). B. OVCAR3 cells were co-transfected with TCF4, CDK6, Cyclin D1, Cyclin D2 or Cyclin D3 3′UTR pGL plasmid with miR-211. Forty-eight hours later, luciferase assay was performed. *p < 0.05 compared to miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments. C-D. Sequence of miR-211 with the putative binding sites of Cyclin D1 (C) and CDK6 (D) 3′ UTR. E. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. F. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-211 or LV-miR-Ctrl. G-H. The mRNA expression levels of Cyclin D1 (G) or CDK6 (H) correlated inversely with miR-211. I-J. Cyclin D1 (I) and CDK6 (J) expression in ovarian tissues (GDS3592/Cyclin D1, n = 24, p = 0.017; GDS3592/CDK6, n = 24, p = 0.016).
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Fig4: Cyclin D1 and CDK6 are miR-211 targets. A. Schematic representation showing the five predicted miR-211 targets (TCF4, Cyclin D1, Cyclin D2, Cyclin D3 and CDK6) screened using three algorithms (miRNA.org, miRWalk and Targetscan). B. OVCAR3 cells were co-transfected with TCF4, CDK6, Cyclin D1, Cyclin D2 or Cyclin D3 3′UTR pGL plasmid with miR-211. Forty-eight hours later, luciferase assay was performed. *p < 0.05 compared to miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments. C-D. Sequence of miR-211 with the putative binding sites of Cyclin D1 (C) and CDK6 (D) 3′ UTR. E. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. F. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-211 or LV-miR-Ctrl. G-H. The mRNA expression levels of Cyclin D1 (G) or CDK6 (H) correlated inversely with miR-211. I-J. Cyclin D1 (I) and CDK6 (J) expression in ovarian tissues (GDS3592/Cyclin D1, n = 24, p = 0.017; GDS3592/CDK6, n = 24, p = 0.016).
Mentions: We used microRNA.org, Targetscan and miRWalk databases to predict potential miR-211 targets. Hundreds of potential targets were found, but we selected transcription factor 4 (TCF4), Cyclin D1, Cyclin D2, Cyclin D3 and Cyclin dependent kinase 6 (CDK6) for further analysis since these genes have previously been reported to affect EOC cell proliferation [28-31] (Figure 4A). We inserted TCF4, CDK6, Cyclin D1, Cyclin D2 and Cyclin D3 3′UTR into luciferase reporter vectors and co-transfected with miR-211 expression plasmid into OVCAR3 and SKOV3 cells (Figure 4A). Forty-eight hours after transfection, only cells transfected with CDK6 and Cyclin D1 3′UTR plasmids had lower luciferase activity than their controls. Luciferase activity in both of these cell lines was 70% less than that of the miR-Ctrl groups (Figure 4B). Next, we compared the 3′UTR sequence of Cyclin D1 and CDK6 with miR-211. Both genes have two potential complementary miR-211 binding sites (Figure 4C, D). We next constructed Cyclin D1 and CDK6 3′UTR mutant plasmid (Additional file 1: Figure S1A-B), cotransfected with miR-211 and then performed luciferase assay. The data revealed that miR-211 targets two sites in Cyclin D1 and CDK6 (Additional file 1: Figure S1C-D). To confirm that Cyclin D1 and CDK6 are specifically targeted by miR-211, OVCAR3 and SKOV3 cells transfected with miR-211 or infected with LV-miR-211 were subjected to western blot analysis. We found that the protein levels of Cyclin D1 and CDK6 were lower in miR-211 and LV-miR-211 transfected cells compared to control (Figure 4E, F). The mRNA level of Cyclin D1 and CDK6 and the levels of miR-211 were measured in 60 EOC tissues using qRT-PCR to analyze their correlation. Spearman correlation analysis revealed a reverse correlation between miR-211 expression and Cyclin D1 or miR-211 and CDK6 expression (Figure 4G, H). The high expression of Cyclin D1 and CDK6 in OEC tissues compared to normal tissue was consistent with results from the public database (Figure 4I, J) [32]. Taken together, these results indicate that miR-211 can repress the expression of Cyclin D1 and CDK6 in EOC by directly targeting the 3′UTR of Cyclin D1 and CDK6 mRNA.Figure 4

Bottom Line: Its function and loss-of-function have been described in normal and cancer cells and tissues. miR-211 is known to be dysregulated in ovarian cancer: however, its function and the downstream effect of its loss-of-function in ovarian cancer have not been described before.We found that the expression of miR-211 is downregulated in EOC tissues and cell lines compared to normal epithelial ovarian tissue and human ovarian surface epithelial cells, respectively. miR-211 was found to arrest cells in the G0/G1-phase, inhibit proliferation and induce apoptosis.Cyclin D1 and CDK6 were found to be direct targets of miR-211, and when overexpressed in miR-211-expressing EOC cells, could restore proliferative ability.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology, the Affiliated Tumor Hospital, Harbin Medical University, 150 Haping Rd, Nangang, Harbin, 150020, Heilongjiang, China. xiabairong9999@126.com.

ABSTRACT

Background: Epithelial ovarian cancer (EOC) is a significant cause of morbidity and mortality. MicroRNAs play important roles in cancer development and progression. The microRNA miR-211 is localized on intron 6 of the Trpm1 gene at 15q13-q14, a locus that is frequently lost in neoplasms. Its function and loss-of-function have been described in normal and cancer cells and tissues. miR-211 is known to be dysregulated in ovarian cancer: however, its function and the downstream effect of its loss-of-function in ovarian cancer have not been described before.

Methods: We analyzed miR-211 expression in clinical samples of primary EOC tissues compared to normal epithelial ovarian tissues and in the EOC cell lines: OVCAR3, Caov3, OVCA429, SKOV3 and A2780 compared to human ovarian surface epithelial cells. We then investigated the effect of miR-211 on EOC cell proliferation and apoptosis by counting cell numbers, MTT, colony formation, cell cycle, and PI/Annexin V staining assays. A luciferase reporter system was developed to assess miR-211 regulation of the predicted targets. Expression level of discovered targets and correlation with miR-211 expression were analyzed in EOC tissues. Finally, OVCAR3 stably expressing miR-211 or control cells were injected subcutaneously into mice to determine in vivo effect of miR-211 on tumorigenesis.

Results: We found that the expression of miR-211 is downregulated in EOC tissues and cell lines compared to normal epithelial ovarian tissue and human ovarian surface epithelial cells, respectively. miR-211 was found to arrest cells in the G0/G1-phase, inhibit proliferation and induce apoptosis. Cyclin D1 and CDK6 were found to be direct targets of miR-211, and when overexpressed in miR-211-expressing EOC cells, could restore proliferative ability. Finally, in vitro investigation confirmed that miR-211 is a tumor suppressor that controls Cyclin D1 and CDK6 expression.

Conclusions: Our results demonstrate that miR-211 is a tumor suppressor that controls expression of Cyclin D1 and CDK6, and that its downregulation results in overexpression of Cyclin D1 and CDK6 which increases proliferation ability of EOC cells to proliferate compared to normal cells.

Show MeSH
Related in: MedlinePlus