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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 reduces EOC tumorigenesisin vivo.A. Nude mice were implanted with OVCAR3 cells stably expressing miR-Ctrl or miR-211. Tumor volume was measured every five days. B. Tumor weight determined at day 25. *p < 0.05, **p < 0.01 compared to LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM. C. Representative images of formed tumors. D. Immunohistochemistry of Cyclin D1 and CDK6 staining in LV-miR-Ctrl and LV-miR-211 groups.
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Fig7: miR-211 reduces EOC tumorigenesisin vivo.A. Nude mice were implanted with OVCAR3 cells stably expressing miR-Ctrl or miR-211. Tumor volume was measured every five days. B. Tumor weight determined at day 25. *p < 0.05, **p < 0.01 compared to LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM. C. Representative images of formed tumors. D. Immunohistochemistry of Cyclin D1 and CDK6 staining in LV-miR-Ctrl and LV-miR-211 groups.

Mentions: We performed in vivo experiments to confirm our in vitro results that suggested that miR-211 inhibited EOC cell proliferation by targeting Cyclin D1 and CDK6. Sixteen mice were randomly divided into two groups. OVCAR3 cells stably expressing miR-211 or control cells were injected subcutaneously into mice in each group. We found that tumor growth was slower in the LV-miR-211 group compared to the LV-miR-Ctrl group (Figure 7A). The tumor weights and sizes were smaller in LV-miR-211 group compared to LV-miR-Ctrl group (Figure 7B, C). Finally, these tumor tissues were assessed with immunohistochemistry. We observed that Cyclin D1 and CDK6 staining in LV-miR-211 group was weaker than in the control group (Figure 7D). These in vivo results further indicated that miR-211 inhibits EOC growth and reduces Cyclin D1 and CDK6 expression.Figure 7


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 reduces EOC tumorigenesisin vivo.A. Nude mice were implanted with OVCAR3 cells stably expressing miR-Ctrl or miR-211. Tumor volume was measured every five days. B. Tumor weight determined at day 25. *p < 0.05, **p < 0.01 compared to LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM. C. Representative images of formed tumors. D. Immunohistochemistry of Cyclin D1 and CDK6 staining in LV-miR-Ctrl and LV-miR-211 groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359570&req=5

Fig7: miR-211 reduces EOC tumorigenesisin vivo.A. Nude mice were implanted with OVCAR3 cells stably expressing miR-Ctrl or miR-211. Tumor volume was measured every five days. B. Tumor weight determined at day 25. *p < 0.05, **p < 0.01 compared to LV-miR-Ctrl transfected cells. Data are presented as mean ± SEM. C. Representative images of formed tumors. D. Immunohistochemistry of Cyclin D1 and CDK6 staining in LV-miR-Ctrl and LV-miR-211 groups.
Mentions: We performed in vivo experiments to confirm our in vitro results that suggested that miR-211 inhibited EOC cell proliferation by targeting Cyclin D1 and CDK6. Sixteen mice were randomly divided into two groups. OVCAR3 cells stably expressing miR-211 or control cells were injected subcutaneously into mice in each group. We found that tumor growth was slower in the LV-miR-211 group compared to the LV-miR-Ctrl group (Figure 7A). The tumor weights and sizes were smaller in LV-miR-211 group compared to LV-miR-Ctrl group (Figure 7B, C). Finally, these tumor tissues were assessed with immunohistochemistry. We observed that Cyclin D1 and CDK6 staining in LV-miR-211 group was weaker than in the control group (Figure 7D). These in vivo results further indicated that miR-211 inhibits EOC growth and reduces Cyclin D1 and CDK6 expression.Figure 7

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