Fig 2: Effects of miR-181a-5p and MEG2 on the growth of gastric cancer xenografted tumours in vivo. a Flowchart of experimental layout and representative images of xenografted tumours from implanted mice. MGC803 cells were divided into four groups and were infected with control lentivirus, miR-181a-5p overexpression lentivirus, MEG2 overexpression plasmid, or miR-181a-5p overexpression lentivirus plus MEG2 overexpression plasmid. These differentially treated MGC803 cells were subcutaneously implanted into 5-week-old SCID mice (1 × 107 cells per mouse and 5 mice per group). After 21 days, tumour growth was evaluated. b Quantitative analysis of xenografted tumour weights. c Quantitative RT-PCR analysis of miR-181a-5p expression levels in xenografted tumours. d Quantitative RT-PCR analysis of MEG2 mRNA expression levels in xenografted tumours. e Representative image of western blot analysis of MEG2 protein expression levels in xenografted tumours. * P < 0.05; ** P < 0.01; *** P < 0.001

Fig 3: 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.

Fig 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.

Fig 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 ß-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.