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. 2021 Jan 11;8:596363. doi: 10.3389/fcell.2020.596363

FIGURE 5.

FIGURE 5

MALAT1 bound to miR-320a-5p and miR-2114-5p, and miR-320a-5p directly interacted with Runx2. (A) Ago2 RIP (RNA immunoprecipitation) was performed, and qRT-PCR was used to determine the expression levels of MALAT1 and predicted miRNAs relative to the input. (B) Schematic illustration of the predicted binding sequence of miR-320a-5p in wild-type MALAT1 (MALAT1-wt) and the MALAT1 mutant (MALAT1-mut). (C) Relative luciferase activity of MALAT1-wt and MALAT1-mut in human renal interstitial fibroblasts (hRIFs) treated with the miR-320a-5p mimic (320a-5p-mimic) or mimic negative control (NC-mimic). (D) Schematic illustration of the predicted binding sequence of miR-2114-5p in MALAT1-wt and MALAT1-mut. (E) Relative luciferase activity of MALAT1-wt and MALAT1-mut in hRIFs treated with the miR-2114-5p mimic (2114-5p-mimic) or NC-mimic. (F) The relative mRNA expression level of Runx2 was determined by qRT-PCR in hRIFs treated with the miR-320a-5p mimic or miR-320a-5p inhibitor. (G) The relative mRNA expression level of Runx2 was determined by qRT-PCR in hRIFs treated with the miR-2114-5p mimic or miR-2114-5p inhibitor. (H) The protein expression level of Runx2 was confirmed by WB in hRIFs treated with the miR-320a-5p mimic or miR-320a-5p inhibitor. (I) Schematic illustration of the predicted binding sequence of miR-320a-5p in the wild-type Runx2-3′UTR (Runx2-wt) and mutated Runx2-3’UTR (Runx2-mut). (J) Relative luciferase activity of Runx2-wt and Runx2-mut in hRIFs treated with the miR-320a-5p-mimic or NC-mimic. MicroRNA (miR) expression levels were normalized to those of U6; relative expression levels of other RNAs were normalized to those of GAPDH. *P < 0.05; **P < 0.01.