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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

miR-211 inhibits EOC cell proliferation. A. miR-211 levels in OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. B. OVCAR3 and SKOV3 cells were transfected with miR-211 or miR-Ctrl for 48 hours, then seeded in 24-well plates (0.25 × 104 cells/well). Viable cell numbers were counted at indicated time points. C. MTT assay of OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. D. Expression of miR-211 in LV-miR-211 or LV-miR-Ctrl transfected OVCAR3 and SKOV3 cells. E. Colony formation assay in OVCAR3 and SKOV3 cells stably expressing miR-211 compared to control cells. *p < 0.05, **p < 0.01 compared to miR-Ctrl or LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments.
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Fig2: miR-211 inhibits EOC cell proliferation. A. miR-211 levels in OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. B. OVCAR3 and SKOV3 cells were transfected with miR-211 or miR-Ctrl for 48 hours, then seeded in 24-well plates (0.25 × 104 cells/well). Viable cell numbers were counted at indicated time points. C. MTT assay of OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. D. Expression of miR-211 in LV-miR-211 or LV-miR-Ctrl transfected OVCAR3 and SKOV3 cells. E. Colony formation assay in OVCAR3 and SKOV3 cells stably expressing miR-211 compared to control cells. *p < 0.05, **p < 0.01 compared to miR-Ctrl or LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments.

Mentions: To investigate the function of miR-211 in EOC tumorigenesis, we transfected miR-211 or miR-Ctrl in OVCAR3 and SKOV3 cell lines and determined their miR-211 levels 48 hours after transfection. Results showed increased miR-211 levels in both miR-211 transfected cell lines compared to miR-Ctrl transfected cells (Figure 2A). We then investigated the effect of miR-211 on OVCAR3 and SKOV3 cell proliferation. As shown in Figure 2B, miR-211 significantly inhibited EOC proliferation. MTT assay further confirmed that miR-211 had a negative effect on EOC cell proliferation (Figure 2C). To examine the effect of miR-211 on long-term EOC cell proliferation, we performed colony formation assays. We first constructed a miR-211 lentiviral vector (LV-miR-211), then infected OVCAR3 and SKOV3 cells with LV-miR-211 to establish stably expressing miR-211 cells (Figure 2D). LV-miR-211 and LV-miR-Ctrl cells were subjected to colony formation assay for two weeks. As expected, miR-211 significantly reduced colony numbers to 20% in both EOC cell lines (Figure 2E).Figure 2


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 inhibits EOC cell proliferation. A. miR-211 levels in OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. B. OVCAR3 and SKOV3 cells were transfected with miR-211 or miR-Ctrl for 48 hours, then seeded in 24-well plates (0.25 × 104 cells/well). Viable cell numbers were counted at indicated time points. C. MTT assay of OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. D. Expression of miR-211 in LV-miR-211 or LV-miR-Ctrl transfected OVCAR3 and SKOV3 cells. E. Colony formation assay in OVCAR3 and SKOV3 cells stably expressing miR-211 compared to control cells. *p < 0.05, **p < 0.01 compared to miR-Ctrl or LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments.
© Copyright Policy - open-access
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Fig2: miR-211 inhibits EOC cell proliferation. A. miR-211 levels in OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. B. OVCAR3 and SKOV3 cells were transfected with miR-211 or miR-Ctrl for 48 hours, then seeded in 24-well plates (0.25 × 104 cells/well). Viable cell numbers were counted at indicated time points. C. MTT assay of OVCAR3 and SKOV3 cells transfected with miR-211 or miR-Ctrl. D. Expression of miR-211 in LV-miR-211 or LV-miR-Ctrl transfected OVCAR3 and SKOV3 cells. E. Colony formation assay in OVCAR3 and SKOV3 cells stably expressing miR-211 compared to control cells. *p < 0.05, **p < 0.01 compared to miR-Ctrl or LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM of three independent experiments.
Mentions: To investigate the function of miR-211 in EOC tumorigenesis, we transfected miR-211 or miR-Ctrl in OVCAR3 and SKOV3 cell lines and determined their miR-211 levels 48 hours after transfection. Results showed increased miR-211 levels in both miR-211 transfected cell lines compared to miR-Ctrl transfected cells (Figure 2A). We then investigated the effect of miR-211 on OVCAR3 and SKOV3 cell proliferation. As shown in Figure 2B, miR-211 significantly inhibited EOC proliferation. MTT assay further confirmed that miR-211 had a negative effect on EOC cell proliferation (Figure 2C). To examine the effect of miR-211 on long-term EOC cell proliferation, we performed colony formation assays. We first constructed a miR-211 lentiviral vector (LV-miR-211), then infected OVCAR3 and SKOV3 cells with LV-miR-211 to establish stably expressing miR-211 cells (Figure 2D). LV-miR-211 and LV-miR-Ctrl cells were subjected to colony formation assay for two weeks. As expected, miR-211 significantly reduced colony numbers to 20% in both EOC cell lines (Figure 2E).Figure 2

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