Research Paper Volume 13, Issue 9 pp 12780—12799

CircRNA_30032 promotes renal fibrosis in UUO model mice via miRNA-96-5p/HBEGF/KRAS axis

MiRNA-96-5p directly binds to the 3’UTR of HBEGF and KRAS mRNAs in BUMPT cells. (A) TargetScan database analysis shows putative miR-96-5p binding sites in the 3’UTR of HBEGF and KRAS mRNAs. (B, C) Dual luciferase reporter assay results show relative luciferase activities in BUMPT cells co-transfected with luciferase reporter vectors carrying wild-type (WT) or mutant (MUT) 3’ UTR’s of (B) HBEGF and (C) KRAS (C) plus miR-96-5p or miR-NC. (D, E) RT-qPCR analysis shows (D) HBEGF and (E) KRAS mRNA levels in BUMPT cells transfected with 100 nM miR-96-5p mimics or miR-NC, and then treated with TGF-β1 for 24h. (F) Representative western blot images and (G, H) densitometric measurements show the levels of (G) HBEGF and (H) KRAS proteins in BUMPT cells transfected with 100 nM miR-96-5p mimics or miR-NC, and then treated with TGF-β1 for 24h. Note: The data are expressed as means ± SD (n = 6). # denotes p  when comparing miR-NC plus TGF-β1or miR-96-5p mimic with saline groups vs. miR-NC plus saline group; * denotes p  when comparing miR-96-5p mimics plus TGF-β1group vs. miR-NC plus TGF-β1 group and KRAS-WT or HBEGF-WT plus miR-96-5p mimic vs. other groups.

Figure 7. MiRNA-96-5p directly binds to the 3’UTR of HBEGF and KRAS mRNAs in BUMPT cells. (A) TargetScan database analysis shows putative miR-96-5p binding sites in the 3’UTR of HBEGF and KRAS mRNAs. (B, C) Dual luciferase reporter assay results show relative luciferase activities in BUMPT cells co-transfected with luciferase reporter vectors carrying wild-type (WT) or mutant (MUT) 3’ UTR’s of (B) HBEGF and (C) KRAS (C) plus miR-96-5p or miR-NC. (D, E) RT-qPCR analysis shows (D) HBEGF and (E) KRAS mRNA levels in BUMPT cells transfected with 100 nM miR-96-5p mimics or miR-NC, and then treated with TGF-β1 for 24h. (F) Representative western blot images and (G, H) densitometric measurements show the levels of (G) HBEGF and (H) KRAS proteins in BUMPT cells transfected with 100 nM miR-96-5p mimics or miR-NC, and then treated with TGF-β1 for 24h. Note: The data are expressed as means ± SD (n = 6). # denotes p < 0.05 when comparing miR-NC plus TGF-β1or miR-96-5p mimic with saline groups vs. miR-NC plus saline group; * denotes p < 0.05 when comparing miR-96-5p mimics plus TGF-β1group vs. miR-NC plus TGF-β1 group and KRAS-WT or HBEGF-WT plus miR-96-5p mimic vs. other groups.