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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 is downregulated in epithelial ovarian cancer (EOC) tissues and cell lines. A. Expression of miR-211 was lower in clear cell ovarian carcinomas (CCOC, n = 9) and high-grade serous ovarian carcinomas (HGSC, n = 12) compared to ovarian surface epithelial cells (OSEC, n = 9) (GSE47841, p < 0.001). B. Levels of miR-211 were lower in 60 EOC tissues compared to 20 normal epithelial tissues samples. **p < 0.01. Relative levels of miRNA expression normalized to U6 snRNA was determined by setting the miRNA expression levels of normal epithelial tissue samples to 1. C. miR-211 expression was downregulated in six EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780 and COV644) compared to human ovarian surface epithelial (HOSE) cells. Relative levels of miRNA expression normalized to U6 snRNA was determined by setting the miRNA expression levels of HOSE cells to 1. *p < 0.05, **p < 0.01. Data are presented as mean ± SEM of three independent experiments.
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Fig1: miR-211 is downregulated in epithelial ovarian cancer (EOC) tissues and cell lines. A. Expression of miR-211 was lower in clear cell ovarian carcinomas (CCOC, n = 9) and high-grade serous ovarian carcinomas (HGSC, n = 12) compared to ovarian surface epithelial cells (OSEC, n = 9) (GSE47841, p < 0.001). B. Levels of miR-211 were lower in 60 EOC tissues compared to 20 normal epithelial tissues samples. **p < 0.01. Relative levels of miRNA expression normalized to U6 snRNA was determined by setting the miRNA expression levels of normal epithelial tissue samples to 1. C. miR-211 expression was downregulated in six EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780 and COV644) compared to human ovarian surface epithelial (HOSE) cells. Relative levels of miRNA expression normalized to U6 snRNA was determined by setting the miRNA expression levels of HOSE cells to 1. *p < 0.05, **p < 0.01. Data are presented as mean ± SEM of three independent experiments.

Mentions: Searching the literature, we found that miR-211 is downregulated in OC tissues [9]. We further used a public data base to investigate miR-211 expression in EOC tissues and found that the of miR-211 expression was significantly lower in clear-cell OC (CCOC, n = 9) and high-grade serous ovarian carcinomas (HGSC, n = 12) than in ovarian surface epithelial cells (OSES, n = 9) (Figure 1A, GSE47841, p < 0.001) [27]. Next, we explored this finding in clinical samples by comparing miR-211 expression in normal epithelial ovarian tissues with primary EOC tissues. Consistent with the mentioned literature, miR-211 expression was significantly lower in EOC tissues than in normal epithelial ovarian tissues (Figure 1B, p < 0.01). We extended our investigations to six EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780 and COV644) and found that their miR-211 expression levels were significantly lower than those of human ovarian surface epithelial (HOSE) cells (Figure 1C). These findings suggest that downregulation of miR-221 may affect EOC development.Figure 1


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 is downregulated in epithelial ovarian cancer (EOC) tissues and cell lines. A. Expression of miR-211 was lower in clear cell ovarian carcinomas (CCOC, n = 9) and high-grade serous ovarian carcinomas (HGSC, n = 12) compared to ovarian surface epithelial cells (OSEC, n = 9) (GSE47841, p < 0.001). B. Levels of miR-211 were lower in 60 EOC tissues compared to 20 normal epithelial tissues samples. **p < 0.01. Relative levels of miRNA expression normalized to U6 snRNA was determined by setting the miRNA expression levels of normal epithelial tissue samples to 1. C. miR-211 expression was downregulated in six EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780 and COV644) compared to human ovarian surface epithelial (HOSE) cells. Relative levels of miRNA expression normalized to U6 snRNA was determined by setting the miRNA expression levels of HOSE cells to 1. *p < 0.05, **p < 0.01. Data are presented as mean ± SEM of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: miR-211 is downregulated in epithelial ovarian cancer (EOC) tissues and cell lines. A. Expression of miR-211 was lower in clear cell ovarian carcinomas (CCOC, n = 9) and high-grade serous ovarian carcinomas (HGSC, n = 12) compared to ovarian surface epithelial cells (OSEC, n = 9) (GSE47841, p < 0.001). B. Levels of miR-211 were lower in 60 EOC tissues compared to 20 normal epithelial tissues samples. **p < 0.01. Relative levels of miRNA expression normalized to U6 snRNA was determined by setting the miRNA expression levels of normal epithelial tissue samples to 1. C. miR-211 expression was downregulated in six EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780 and COV644) compared to human ovarian surface epithelial (HOSE) cells. Relative levels of miRNA expression normalized to U6 snRNA was determined by setting the miRNA expression levels of HOSE cells to 1. *p < 0.05, **p < 0.01. Data are presented as mean ± SEM of three independent experiments.
Mentions: Searching the literature, we found that miR-211 is downregulated in OC tissues [9]. We further used a public data base to investigate miR-211 expression in EOC tissues and found that the of miR-211 expression was significantly lower in clear-cell OC (CCOC, n = 9) and high-grade serous ovarian carcinomas (HGSC, n = 12) than in ovarian surface epithelial cells (OSES, n = 9) (Figure 1A, GSE47841, p < 0.001) [27]. Next, we explored this finding in clinical samples by comparing miR-211 expression in normal epithelial ovarian tissues with primary EOC tissues. Consistent with the mentioned literature, miR-211 expression was significantly lower in EOC tissues than in normal epithelial ovarian tissues (Figure 1B, p < 0.01). We extended our investigations to six EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780 and COV644) and found that their miR-211 expression levels were significantly lower than those of human ovarian surface epithelial (HOSE) cells (Figure 1C). These findings suggest that downregulation of miR-221 may affect EOC development.Figure 1

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