Fig 1: The regulation of SMAD4 on ESCC cells is mediated by miR-574-5p. (A) Expression of SMAD4 was confirmed by qRT-PCR and western blot in co-transfected ESCC cell lines. (B–E) CCK-8 assays, colony formation assays, and EdU assays were used to detect the cell proliferation after co-transfecting ESCC cells with miR-574-5p + Ctl and miR-574-5p + SMAD4 (scale bar: 100 μm for Edu assay). (F) Western blot analysis confirmed that MTX2-6 inhibits cell cycle process and promotes cell apoptosis through regulating miR-574-5p of ESCC. (G) The IHC of CDK4, Cyclin D1, Caspase3, and Bax in the high expression SMAD4 of tumors and low expression SMAD4 of tumors. (H) Proposed model of lnc MTX2-6 suppresses proliferation by acting as ceRNA of miR-574-5p to modulate SMAD4 in ESCC cells. ∗∗p < 0.01, ∗∗∗p < 0.001.
Fig 2: The effects of MTX2-6 on ESCC cells are mediated by miR-574-5p. (A) Expression of miR-574-5p was verified in co-transfected ESCC cell lines by qRT-PCR. (B–E) CCK-8 assays, colony formation assays, and the Edu assays confirmed that overexpressed miR-574-5p abrogated the effects of upregulated MTX2-6 on proliferation of ESCC cells (scale bar: 100 μm for Edu assay). (F) miR-574-5p restoration promoted cell cycle from G0/G1 phase stepped to S phase of MTX2-6-overexpressing of ESCC cells. (G) miR-574-5p restoration inhibited cell apoptosis of MTX2-6-overexpressing of ESCC cells. (H) The IHC of CDK4, Caspase3, and Bax in the high expression miR-574-5p of tumors and low expression miR-574-5p of tumors. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Fig 3: Overexpressed MTX2-6 inhibits ESCC cell proliferation in vitro. (A) qRT-PCR analysis of MTX2-6 in EC-1 and EC-109 cells treated with LV-MTX2-6. (B) CCK-8 assay was applied to detect cell viability. (C–E) Colony formation assays and the Edu assays were conducted to measure cell proliferation ability in EC-1 and EC-109 cells (scale bar: 100 μm for Edu assay). (F) Effects of MTX2-6 on regulating cell cycle were detected by flow cytometry. (G) Flow cytometry analysis of EC-1 and EC-109 cells apoptosis rates. (H) The IHC of CDK4, Caspase3 and Bax in the high expression MTX2-6 of tumors and low expression MTX2-6 of tumors. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
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