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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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miR-211 affects EOC cell proliferation through suppression of Cyclin D1 and CDK6. A. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. B. Cell counting of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. C. MTT assay of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. D. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. E. Cell counting of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. F. MTT assay in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. *p < 0.05 compared to LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments.
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Fig5: miR-211 affects EOC cell proliferation through suppression of Cyclin D1 and CDK6. A. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. B. Cell counting of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. C. MTT assay of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. D. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. E. Cell counting of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. F. MTT assay in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. *p < 0.05 compared to LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments.

Mentions: Our previous data suggests that miR-211 inhibits EOC cell proliferation and that Cyclin D1 and CDK6 are direct targets of miR-211. To demonstrate that miR-211 regulates cell proliferation through Cyclin D1 and CDK6, we tested whether Cyclin D1 and CDK6 could rescue the impaired proliferative phenotype in miR-211 overexpressing OVCAR3 and SKOV3 cells. Western blot was performed to examine Cyclin D1 and CDK6 levels in cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 and LV-miR-211 + CDK6 (Figure 5A). Cell counting and MTT assay were performed to analyze cell proliferation. The results indicate that miR-211 inhibited proliferation, while Cyclin D1 or CDK6 partly, and Cyclin D1 + CDK6 almost completely restored cell proliferation (Figure 5B-F). Furthermore, we performed cell cycle assays and found that Cyclin D1 or CDK6 partly rescued the cell proliferation that had been inhibited by miR-211 (Figure 6A). MiR-211 overexpression led to more cell apoptosis, while Cyclin D1 and CDK6 each significantly reduced apoptosis (Figure 6B). When we overexpressed Cyclin D1 and CDK6 in miR-211-overexpressing cells, miR-211-induced cell cycle arrest and apoptosis were completely abrogated (Figure 6C and D). These results together demonstrate that miR-211 affects EOC cell proliferation, at least in part, through suppression of Cyclin D1 and CDK6.Figure 5


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)

miR-211 affects EOC cell proliferation through suppression of Cyclin D1 and CDK6. A. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. B. Cell counting of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. C. MTT assay of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. D. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. E. Cell counting of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. F. MTT assay in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. *p < 0.05 compared to LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments.
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Fig5: miR-211 affects EOC cell proliferation through suppression of Cyclin D1 and CDK6. A. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. B. Cell counting of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. C. MTT assay of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 or LV-miR-211 + CDK6 plasmids. D. Western blot of Cyclin D1 and CDK6 levels in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. E. Cell counting of OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. F. MTT assay in OVCAR3 and SKOV3 cells transfected with LV-miR-Ctrl, LV-miR-211 or LV-miR-211 + Cyclin D1 + CDK6 plasmids. *p < 0.05 compared to LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments.
Mentions: Our previous data suggests that miR-211 inhibits EOC cell proliferation and that Cyclin D1 and CDK6 are direct targets of miR-211. To demonstrate that miR-211 regulates cell proliferation through Cyclin D1 and CDK6, we tested whether Cyclin D1 and CDK6 could rescue the impaired proliferative phenotype in miR-211 overexpressing OVCAR3 and SKOV3 cells. Western blot was performed to examine Cyclin D1 and CDK6 levels in cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 and LV-miR-211 + CDK6 (Figure 5A). Cell counting and MTT assay were performed to analyze cell proliferation. The results indicate that miR-211 inhibited proliferation, while Cyclin D1 or CDK6 partly, and Cyclin D1 + CDK6 almost completely restored cell proliferation (Figure 5B-F). Furthermore, we performed cell cycle assays and found that Cyclin D1 or CDK6 partly rescued the cell proliferation that had been inhibited by miR-211 (Figure 6A). MiR-211 overexpression led to more cell apoptosis, while Cyclin D1 and CDK6 each significantly reduced apoptosis (Figure 6B). When we overexpressed Cyclin D1 and CDK6 in miR-211-overexpressing cells, miR-211-induced cell cycle arrest and apoptosis were completely abrogated (Figure 6C and D). These results together demonstrate that miR-211 affects EOC cell proliferation, at least in part, through suppression of Cyclin D1 and CDK6.Figure 5

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