miR-762 can negatively regulate menin in ovarian cancer

Abstract

Ovarian cancer accounts for the major part of the mortality attributable to female reproductive system malignant tumors worldwide. Recently, the incidence of ovarian cancer has been increasing annually, and there remains a lack of suitable treatment methods that can significantly improve the 5-year survival rates of patients. Therefore, it is necessary to identify more effective treatments for ovarian cancer. It is established that microRNAs (miRNAs) have important roles in the diagnosis and treatment of ovarian cancer and a specific miRNA, miR-762, can promote the development of a variety of tumors. Menin is encoded by MEN1, a tumor suppressor gene, that is usually downregulated in ovarian cancer. In this study, we evaluated the expression levels of miR-762 and menin in ovarian cancer tissues and demonstrated that they were correlated. In addition, we found that miR-762 can downregulate the expression of menin through a binding site in its 3'-UTR and consequently upregulate the Wnt cell signaling pathway to promote the development of ovarian cancer. These results indicate that miR-762 is a promising potential target for the treatment of ovarian cancer.

Keywords: menin; miR-762; ovarian cancer.

Figure 1

The relationship between miR-762 and menin in ovarian cancer. Notes: (A) The levels of miR-762 were detected by real-time PCR in 60 samples of ovarian cancer and adjacent tissues. There was higher expression of miR-762 in ovarian cancer tissues. Data are presented as mean ± SEM. (B) The relationship between miR-762 expression levels and patient survival. The survival time of patients with high expression of miR-762 was shorter than that of patients with low expression. (C) Menin protein levels in ovarian cancer (C) and adjacent tissues (N) were detected by Western blotting. The expression level of menin was lower in the ovarian cancer tissues than in the control tissues. Quantitative data (right) are presented as mean ± SEM; **P<0.01 tumor tissue group vs adjacent control tissue group. (D) mRNA expression of MEN1 in 60 samples of ovarian cancer and adjacent tissues was detected by real-time PCR. The expression of menin was lower in ovarian cancer tissues than in the control tissues. Data are presented as mean ± SEM. **P<0.01 for the comparison of tumor tissue group vs adjacent control tissue group. (E) Correlation between the expression levels of miR-762 and menin. The expression of miR-762 was negatively correlated with that of menin. (F) Prediction that miR-762 can specifically bind to the menin 3′-UTR using miRDB. (G) The interaction between miR-762 and the menin 3′-UTR was evaluated using luciferase reporter assays. Assays were performed by the cotransfection of miR-762 or miR-762 antisense and menin-wt or menin-del. Data are presented as mean ± SEM. **P<0.01 vs TK-menin group. Abbreviations: SEM, standard error of mean; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated regions.

Figure 2

miR-762 can promote the proliferation of ovarian cancer cells by targeting menin. Notes: (A) After transfecting with miR-762 mimic, the proliferation rates of SKOV3 and CAOV cells were determined using MTT assays. Data are presented as mean ± SEM. (B) After transfection with miR-762 inhibitor, the levels of proliferation of SKOV3 and CAOV cells were determined using MTT assays. Data are presented as mean ± SEM. (C and D) After transfecting SKOV3 cells with miR-762 mimic, the protein and mRNA levels of menin, β-catenin, and cyclin D1 were detected by Western blotting and real-time PCR, respectively. Data are presented as mean ± SEM. **P<0.01. (E and F) After transfection of SKOV3 cells with miR-762 inhibitor, the protein and mRNA levels of menin, β-catenin, and cyclin D1 were detected by Western blotting and real-time PCR. Data are presented as mean ± SEM. **P<0.01. Abbreviation: MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 2

miR-762 can promote the proliferation of ovarian cancer cells by targeting menin. Notes: (A) After transfecting with miR-762 mimic, the proliferation rates of SKOV3 and CAOV cells were determined using MTT assays. Data are presented as mean ± SEM. (B) After transfection with miR-762 inhibitor, the levels of proliferation of SKOV3 and CAOV cells were determined using MTT assays. Data are presented as mean ± SEM. (C and D) After transfecting SKOV3 cells with miR-762 mimic, the protein and mRNA levels of menin, β-catenin, and cyclin D1 were detected by Western blotting and real-time PCR, respectively. Data are presented as mean ± SEM. **P<0.01. (E and F) After transfection of SKOV3 cells with miR-762 inhibitor, the protein and mRNA levels of menin, β-catenin, and cyclin D1 were detected by Western blotting and real-time PCR. Data are presented as mean ± SEM. **P<0.01. Abbreviation: MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 3

miR-762 inhibits the apoptosis of ovarian cancer cells by suppressing the Wnt/β-catenin pathway. Notes: (A and B) After transfection of SKOV3 cells with miR-762 mimic, the protein and mRNA levels of menin, β-catenin, BAX, and BCL-2 were determined by Western blotting and real-time PCR. Data are presented as mean ± SEM. **P<0.01. (C and D) After transfection of SKOV3 cells with miR-762 inhibitor, the protein and mRNA levels of menin, β-catenin, BAX, and BCL-2 were detected by Western blotting and real-time PCR. Data are presented as mean ± SEM. **P<0.01. Abbreviation: SEM, standard error of mean.

Figure 4

miR-762 promotes the metastasis of ovarian cancer cells by the suppression of menin. Notes: (A and B) After overexpression of miR-762 in SKOV3 and CAOV cells, transwell assays with or without Matrigel were performed. Cells were counted and results represent the means ± SD of three experiments. **P<0.01. (C and D) After transfecting miR-762 mimic into SKOV3 cells, the protein and mRNA levels of menin, β-catenin, and MMP7 were detected by Western blotting and real-time PCR. Data are presented as mean ± SEM. **P<0.01. (E and F) After downregulation of miR-762 in SKOV3 and CAOV cells, transwell assays with or without Matrigel were performed. Cells were counted, and the results represent the mean ± SD of three experiments. **P<0.01. (G and H) After transfection of miR-762 inhibitor into SKOV3 cells, the protein and mRNA levels of menin, β-catenin, and MMP7 were detected by Western blotting and real-time PCR. Data are presented as mean ± SEM. **P<0.01. Abbreviations: SEM, standard error of mean; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

miR-762 promotes the metastasis of ovarian cancer cells by the suppression of menin. Notes: (A and B) After overexpression of miR-762 in SKOV3 and CAOV cells, transwell assays with or without Matrigel were performed. Cells were counted and results represent the means ± SD of three experiments. **P<0.01. (C and D) After transfecting miR-762 mimic into SKOV3 cells, the protein and mRNA levels of menin, β-catenin, and MMP7 were detected by Western blotting and real-time PCR. Data are presented as mean ± SEM. **P<0.01. (E and F) After downregulation of miR-762 in SKOV3 and CAOV cells, transwell assays with or without Matrigel were performed. Cells were counted, and the results represent the mean ± SD of three experiments. **P<0.01. (G and H) After transfection of miR-762 inhibitor into SKOV3 cells, the protein and mRNA levels of menin, β-catenin, and MMP7 were detected by Western blotting and real-time PCR. Data are presented as mean ± SEM. **P<0.01. Abbreviations: SEM, standard error of mean; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.