Fig 1: ALX1 mediates the tumor-suppressive function of miR-192-5p. Three special cell lines were used in these experiments: EC cell lines transfected with negative control (miR-192-NC), miR-192-5p mimics (miR-192), and both miR-192-5p mimics and a ALX1-expressing vector that encoded the entire coding sequence of ALX1 but lacked the 3'-UTR (miR-192+ALX1). (a) ALX1 expression were examined by western blotting using the indicated antibodies. (b, c ) Cell proliferation was examined by CCK-8 assay (b) and colony formation (c). (d, e) Cell migration and invasion were investigated by migration assays (d) and invasion assays (e), respectively. The number of cells that invaded through the membrane was counted in 10 fields under the x20 objective lens. (f) At 48 hours after transfection, apoptosis was measured by flow cytometric analysis of cells stained with Annexin V-FITC and PI. (g) MMP2, MMP9, VEGF, E-cadherin and Vimentin expression, were subjected to western blotting using the indicated antibodies. Tubulin expression was chosen as a control. (h) In vivo tumorigenicity investigated in in a nude mouse xenograft model. Tumor size and weight were measured 3 weeks after cancer cell injection. All of the results are presented as the means ± s.d. of values obtained in three independent experiments. Statistical significance was calculated using the Tukey's post hoc test. *p<0.05, **p<0.01,***p<0.001.
Fig 2: miR-192-5p downregulates ALX1 expression by directly targeting its 3'-UTR. (a) Human ALX1 3'UTR fragment containing the wild-type or mutant miR-192-5p-binding sequence. (b) 3'-UTR luciferase reporter assays in 293 T cells. 293 T cells were co-transfected with miR-192-5p or its negative control vector and a luciferase reporter construct containing the wild-type or mutant ALX1 3'UTR. For each experiment, the data were normalized to the luciferase activity detected in cells transfected with the control vector. (c, d) Relative ALX1 mRNA expression and protein expression in ISH and KLE cell lines that were stably transfected with miR-192-5p lentiviral vector (miR-192) or its negative control (miR-192-NC). (e) Relative expression of ALX1 mRNA in 56 paired endometrial carcinoma tissues and adjacent non-cancer tissues were assessed by real-time PCR. GAPDH served as an internal control. (f) The correlation between relative miR-192-5p expression and relative ALX1 mRNA expression was evaluated using Spearman's correlation analysis.
Fig 3: ALX1 functions as a onco-protein in endometrial carcinoma. Two endometrial carcinoma cell lines, ISH and KLE, were stably transfected with shALX1 lentiviral vector (shALX1) or its negative control (shALX1-NC), respectively. (a) ALX1 expression were examined by western blotting using the indicated antibodies. (b, c ) Cell proliferation was examined by CCK-8 assay (b) and colony formation (c). (d, e) Cell migration and invasion were investigated by migration assays (d) and invasion assays (e), respectively. The number of cells that invaded through the membrane was counted in 10 fields under the x20 objective lens. (f) At 48 hours after transfection, apoptosis was measured by flow cytometric analysis of cells stained with Annexin V-FITC and PI. (g) MMP2, MMP9, VEGF, E-cadherin and Vimentin expression, were subjected to western blotting using the indicated antibodies. Tubulin expression was chosen as a control. (h) In vivo tumorigenicity investigated in in a nude mouse xenograft model. Tumor size and weight were measured 3 weeks after cancer cell injection. All of the results are presented as the means ± s.d. of values obtained in three independent experiments. Statistical significance was calculated using the Student's t-test. *p<0.05, **p<0.01,***p<0.001.
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