LncRNA HIF1A-AS2 accelerates malignant phenotypes of renal carcinoma by modulating miR-30a-5p/SOX4 axis as a ceRNA

Knockdown or overexpression of HIF1A-AS2 suppressed or promoted cell progression. The relative expression level of HIF1A-AS2 was significantly downregulated by si-HIF1A-AS2 (A) and pcDNA3.1-HIF1A-AS2 (B). Analysis of variance was used for the comparison of curves of cell proliferation. Cell proliferation was detected in both renal carcinoma cells after transfection of si-RNA [C (a-c)] and pcDNA3.1-HIF1A-AS2 [C (d)]. Representative images of EdU assays and the relative fold changes of EdU positive cells were detected by si-RNA [D (a–c)] and pcDNA3.1-HIF1A-AS2 [D (d)]. The relative cell migration was suppressed after transfection of si-RNA, and the representative images were as follow [E (a–c)]. The relative cell migration was promoted after transfection of pcDNA3.1-HIF1A-AS2, and the representative images are shown in [E (d)]. Apoptotic cells were measured after transfection of si-RNA [F (a–c)] and pcDNA3.1 [F (d)] by flow cytometry analyses. (^*P < 0.05; ^**P < 0.01).

The miR-30a-5p acted as an anti-oncogene. The relative expression level of miR-30a-5p was significantly downregulated by the miR-30a-5p inhibitor and increased by miR-30a-5p mimics (A). We detected cell proliferation after miR-30a-5p inhibitor and miR-30a-5p mimic transfection [B (a-c)] in renal carcinomas cells. Representative images of EdU assays and the relative fold changes of EdU positive cells were detected by the miR-30a-5p inhibitor and miR-30a-5p mimics [C (a–c)]. The relative cell migration was inhibited or accelerated after miR-30a-5p mimics or inhibitor transfection in renal cancer [D (a–c)] cell lines. Flow cytometry analysis was used to measured apoptotic cells after miR-30a-5p mimics or inhibitor transfection in the renal cancer [E (a–c)] cell lines.(^*P < 0.05; ^**P < 0.01).

HIF1A-AS2 was a target of miR-30a-5p. The relative expression of miR-30a-5p was increased by si-HIF1A-AS2 (A). Dual-luciferase reporter assays were performed in 786-O and OS-RC-2 cells co-transfected with HIF1A-AS2-Wt or HIF1A-AS2-Mut and miR-30a-5p mimics or normal control (NC) (B). We detected cell proliferation after co-transfection with si-NC+NC in both renal carcinoma cell lines, si-HIF1A-AS2+miR-30a-5p inhibitor or si-HIF1A-AS2+miR-30a-5p mimics by CCK-8 and Edu assays (C-F). The relative cell migration after co-transfection with si-NC+NC, si-HIF1A-AS2+miR-30a-5p inhibitor or si-HIF1A-AS2+miR-30a-5p mimics, and the representative images are shown (G and H). The apoptotic cells were measured by flow cytometry analysis after co-transfection with si-NC+NC, si-HIF1A-AS2+miR-30a-5p inhibitor or si-HIF1A-AS2+miR-30a-5p mimics (I and J). (^*P < 0.05; ^**P < 0.01).

HIF1A-AS2 positively regulates SOX4 expression via sponging miR-30a-5p. SOX4-Wt and miR-30a-5p mimics co-transfection inhibited luciferase activity (A) as shown using the dual-luciferase reporter assay. The relative expression level of SOX4 was decreased by si-HIF1A-AS2 (B). The relative expression level of SOX4 was regulated by miR-30a-5p mimics and inhibition (C). Overexpressing miR-30a-5p downregulated SOX4 expression and knockdown of miR-30a-5p upregulated SOX4 expression in renal carcinomas cells (D). Overexpression of HIF1A-AS2 upregulated SOX4 expression in 293T cells (E). Knockdown of HIF1A-AS2 downregulated SOX4 expression in renal carcinomas cells (F). Knockdown of HIF1A-AS2 downregulated transcriptional activity of the Wnt/β-catenin pathway as determined by the TOP flash/FOP flash reporter luciferase activity assay (G). CCK-8 assay (H), migration (I), flow cytometric analysis (J), Western blot and qPCR (K) showed that IWR-1 inhibited and even reversed the effect of pcDNA3.1-HIF1A-AS2 on cell progression in 786-O cells. Knockdown of SOX4 downregulated β-catenin expression in renal carcinomas cells (M). Knockdown of SOX4 reversed malignant phenotypic promotion of renal carcinomas cells induced by HIF1A-AS2. Si-SOX4 reversed SOX4 expression promotion induced by overexpression of HIF1A-AS2 in renal carcinomas cells (N). The relative expression level of SOX4 was decreased by si-SOX4 (L). (^*P < 0.05; ^**P < 0.01).

HIF1A-AS2 positively regulates SOX4 expression via sponging miR-30a-5p. Knockdown SOX4 reversed cell proliferation promotion induced by overexpression of HIF1A-AS2 (EdU, A and B; CCK8, C and D). Knockdown of SOX4 reversed cell migration promotion induced by overexpression of HIF1A-AS2 (E and F). Knockdown SOX4 reversed cell apoptosis inhibition induced by overexpression of HIF1A-AS2 (G and H). (^*P < 0.05; ^**P < 0.01).