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. 2016 Nov 18;6:37421.
doi: 10.1038/srep37421.

miR-96 Promotes Cell Proliferation, Migration and Invasion by Targeting PTPN9 in Breast Cancer

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Free PMC article

miR-96 Promotes Cell Proliferation, Migration and Invasion by Targeting PTPN9 in Breast Cancer

Yeting Hong et al. Sci Rep. .
Free PMC article

Abstract

microRNAs (miRNAs) have emerged as major regulators of the initiation and progression of human cancers, including breast cancer. The aim of this study is to determine the expression pattern of miR-96 in breast cancer and to investigate its biological role during tumorigenesis. We showed that miR-96 was significantly upregulated in breast cancer. We then investigated its function and found that miR-96 significantly promoted cell proliferation, migration and invasion in vitro and enhanced tumor growth in vivo. Furthermore, we explored the molecular mechanisms by which miR-96 contributes to breast cancer progression and identified PTPN9 (protein tyrosine phosphatase, non-receptor type 9) as a direct target gene of miR-96. Finally, we showed that PTPN9 had opposite effects to those of miR-96 on breast cancer cells, suggesting that miR-96 may promote breast tumorigenesis by silencing PTPN9. Taken together, this study highlights an important role for miR-96 in the regulation of PTPN9 in breast cancer cells and may provide insight into the molecular mechanisms of breast carcinogenesis.

Figures

Figure 1
Figure 1. Expression levels of miR-96 in breast cancer tissues and xenograft tumors in mice.
(A) Quantitative RT-PCR analysis of the relative expression levels of miR-96 in 10 pairs of breast cancer tissues and matched adjacent noncancerous tissues. (B and C) MCF-7 cells were infected with a control lentivirus (pre-miR-NC-LV) or a lentivirus to overexpress miR-96 (pre-miR-96-LV) and then implanted subcutaneously into 4-week-old nude mice. Tumor growth was evaluated at day 21 after cell implantation. Mice implanted with wide-type MCF-7 cells (Mock) serve as the negative control. (B) Representative images of the nude mice excised tumors; (C) Relative tumor weight. (D) Quantitative RT-PCR analysis of miR-96 levels in the tumors from implanted mice. (E) Representative H&E-stained sections of the tumors from implanted mice. (F) Proliferative activity assessed by anti-PCNA and anti-Ki-67 monoclonal antibody in the tumors from implanted mice. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2
Figure 2. The effect of miR-96 on the proliferation, migration and invasion of breast cancer cells.
(A–C) The EdU proliferation assay was performed 24 h after the transfection of MCF-7 cells with equal doses of miR-NC, miR-96, anti-miR-NC or anti-miR-96. The untransfected cells (Mock) serve as the negative control. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (A and B) representative images, Scale bar = 300 μm; (C) quantitative analysis of EdU-positive MCF-7 cells. (D–F) Cell migration ability was analyzed using wound healing assays after the transfection of MCF-7 cells with equal doses of miR-NC, miR-96, anti-miR-NC or anti-miR-96. (D and E) Representative images, Scale bar = 100 μm; (F) quantitative analysis s of wound closure. (G–J) Cell invasion ability was analyzed using transwell assays after the transfection of MCF-7 cells with equal doses of miR-NC, miR-96, anti-miR-NC or anti-miR-96. (G and I) Representative images, Scale bar = 250 μm; (H and J) quantitative analysis. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3
Figure 3. The effect of miR-96 on cell cycle progression in breast cancer cells.
(A–D) Cell cycle profiles were analyzed using flow cytometry after the transfection of MCF-7 cells with equal doses of miR-NC, miR-96, anti-miR-NC or anti-miR-96. The untransfected cells (Mock) serve as the negative control. The panel shows histograms of cell numbers (y axis) against DNA content (x axis) determined by measuring fluorescence intensity. Numbers denote the percentages of cells in the G1/G0, S and G2/M phases. (A and B) Representative images; (C and D) quantitative analysis. (E and F) Quantitative RT-PCR analysis of the relative expression levels of Cyclin D1, CDK6, CDK4 and p21 mRNA in MCF-7 cells transfected with equal doses of miR-NC, miR-96, anti-miR-NC or anti-miR-96. The untransfected cells (Mock) serve as the negative control. (G) Western blot analysis of Cyclin D1, CDK6, CDK4 and p21 protein levels in MCF-7 cells transfected with equal doses of miR-NC, miR-96, anti-miR-NC or anti-miR-96. The untransfected cells (Mock) serve as the negative control. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4
Figure 4. PTPN9 was predicted as a target of miR-96 and was downregulated in breast cancer tissues.
(A) Schematic description of the hypothesized duplex formed by interaction between the PTPN9 3′-UTR binding site and miR-96. The predicted free energy value of the hybrid is indicated. The seed recognition site is denoted, and all nucleotides in this region are highly conserved across species, including human, mouse and rat. (B and C) Western blotting analysis of the expression levels of PTPN9 protein in 10 pairs of breast cancer tissues and matched adjacent noncancerous tissues. (B) Representative image; (C) quantitative analysis. (D) Quantitative RT-PCR analysis of the relative expression levels of PTPN9 mRNA in 10 pairs of breast cancer tissues and matched adjacent noncancerous tissues. (E) Pearson’s correlation scatter plot of the fold-change of miR-96 and PTPN9 protein in human breast cancer tissues. (F) Pearson’s correlation scatter plot of the fold change of miR-96 and PTPN9 mRNA in human breast cancer tissues. (G–I) MCF-7 cells were infected with a control lentivirus (pre-miR-NC-LV) or a lentivirus to overexpress miR-96 (pre-miR-96-LV) and then implanted subcutaneously into 4-week-old nude mice. Tumor growth was evaluated at day 21 after cell implantation. Mice implanted with wide-type MCF-7 cells (Mock) serve as the negative control. (G) Western blotting analysis of PTPN9 protein levels in the tumors from implanted mice. (H) Quantitative RT-PCR analysis of PTPN9 mRNA levels in the tumors from implanted mice. (I) Immunohistochemical analysis of PTPN9 protein levels in the tumors from implanted mice. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 5
Figure 5. PTPN9 is a direct target of miR-96.
(A–D) Western blot analysis of PTPN9 protein levels in MCF-7 and MDA-MB-468 cells transfected with equal doses of miR-NC, miR-96, anti-miR-NC or anti-miR-96. The untransfected cells (Mock) serve as the negative control. (A and C) Representative image; (B and D) Quantitative analysis. (E and F) Quantitative RT-PCR analysis of the relative expression levels of PTPN9 mRNA in MCF-7 and MDA-MB-468 cells transfected with equal doses of miR-NC, miR-96, anti-miR-NC or anti-miR-96. The untransfected cells (Mock) serve as the negative control. (G) Direct recognition of the PTPN9 3′-UTR by miR-96. Firefly luciferase reporters containing wild-type (WT) or mutant (MUT) miR-96 binding sites in the PTPN9 3′-UTR were co-transfected into 293 T cells with equal doses of miR-NC or miR-96. The cells transfected only with luciferase reporters (Mock) serve as the negative control. Twenty-four hours after transfection, luciferase assays were performed. Firefly luciferase values were normalized to β-galactosidase activity and the results were calculated as the ratio of firefly luciferase activity in the transfected cells normalized to the mock cells. **P < 0.01; ***P < 0.001.
Figure 6
Figure 6. The effect of PTPN9 on the proliferation, migration and invasion of breast cancer cells.
(A–C) The EdU proliferation assay was performed 48 h after the transfection of MCF-7 cells with equal doses of siRNA-NC or PTPN9 siRNA, or with equal doses of plasmid-NC or PTPN9 overexpression plasmid. The untransfected cells (Mock) serve as the negative control. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (A and B) Representative images, Scale bar = 300 μm; (C) quantitative analysis of EdU-positive MCF-7 cells. (D and E) Cell migration ability was analyzed using wound healing assays after the transfection of MCF-7 cells with equal doses of siRNA-NC or PTPN9 siRNA, or with equal doses of plasmid-NC or PTPN9 overexpression plasmid. (D) Representative images, Scale bar = 100 μm; (E) quantitative analysis s of wound closure. (F–I) Cell invasion ability was analyzed using transwell assays after the transfection of MCF-7 cells with equal doses of siRNA-NC or PTPN9 siRNA, or with equal doses of plasmid-NC or PTPN9 overexpression plasmid. The untransfected cells (Mock) serve as the negative control. (F and H) representative images, Scale bar = 250 μm; (G and I) quantitative analysis. **P < 0.01; ***P < 0.001.
Figure 7
Figure 7. The effect of PTPN9 on cell cycle progression in breast cancer cells.
(A–D) Cell cycle profiles were analyzed using flow cytometry after the transfection of MCF-7 cells with equal doses of siRNA-NC or PTPN9 siRNA, or with equal doses of plasmid-NC or PTPN9 overexpression plasmid. The untransfected cells (Mock) serve as the negative control. The panel shows histograms of cell numbers (y axis) against DNA content (x axis) determined by measuring fluorescence intensity. Numbers denote the percentages of cells in the G1/G0, S and G2/M phases. (A and B) Representative images; (C and D) quantitative analysis. (E and F) Quantitative RT-PCR analysis of the relative expression levels of Cyclin D1, CDK6, CDK4 and p21 mRNA in MCF-7 cells transfected with equal doses of siRNA-NC or PTPN9 siRNA, or with equal doses of plasmid-NC or PTPN9 overexpression plasmid. The untransfected cells (Mock) serve as the negative control. (G) Western blot analysis of Cyclin D1, CDK6, CDK4 and p21 protein levels in MCF-7 cells transfected with equal doses of siRNA-NC or PTPN9 siRNA, or with equal doses of plasmid-NC or PTPN9 overexpression plasmid. The untransfected cells (Mock) serve as the negative control. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 8
Figure 8. Co-effect of miR-96 and PTPN9 on the proliferation, migration and invasion of breast cancer cells.
(A and B) Western blot analysis of PTPN9 protein levels in MCF-7 cells transfected with equal doses of miR-NC plus plasmid-NC, miR-NC plus PTPN9 overexpression plasmid, miR-96 plus plasmid-NC, or miR-96 plus PTPN9 overexpression plasmid. (A) Representative image; (B) quantitative analysis. (C and D) The EdU proliferation assay was performed 24 h after the transfection of MCF-7 cells with equal doses of miR-NC plus plasmid-NC, miR-NC plus PTPN9 overexpression plasmid, miR-96 plus plasmid-NC, or miR-96 plus PTPN9 overexpression plasmid. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (C) Representative images, Scale bar = 500 μm; (D) quantitative analysis of EdU-positive MCF-7 cells. (E and F) Cell migration ability was analyzed using wound healing assays after the transfection of MCF-7 cells with equal doses of miR-NC plus plasmid-NC, miR-NC plus PTPN9 overexpression plasmid, miR-96 plus plasmid-NC, or miR-96 plus PTPN9 overexpression plasmid. (E) Representative images, Scale bar = 200 μm; (F) quantitative analysis s of wound closure. (G and H) Cell invasion ability was analyzed using transwell assays after the transfection of MCF-7 cells with equal doses of miR-NC plus plasmid-NC, miR-NC plus PTPN9 overexpression plasmid, miR-96 plus plasmid-NC, or miR-96 plus PTPN9 overexpression plasmid. (G) Representative images, Scale bar = 250 μm; (H) quantitative analysis. *P < 0.05; **P < 0.01; ***P < 0.001.

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