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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Gastroenterology. 2021 Jan 7;160(5):1662–1678.e18. doi: 10.1053/j.gastro.2020.12.062

Figure 5.

Figure 5.

Target identification and validation of miR-10b-5p in vitro. (A) Pathway analysis of miR-10b-5p and its target genes associated with diabetes mellitus according to Ingenuity Pathway Analysis. (B) The sequence and structure of the mouse miR-10b precursor (pre-miR10b) encoding miR-10b-5p and miR-10b-3p, a synthetic miR-10b-5p molecule (miR-10b-5p mimic) and a synthetic miR-10b-5p antisense molecule (miR-10b-5p inhibitor). (C) Targeting of KLF11, KIT and INS by the miR-10b-5p mimic, miR-10b-5p inhibitor, and KLF11 siRNAs (siKLF11–1 and siKLF11–2 in human Panc.10.05 cells and siKlf11–1 and siKlf11–2 in mouse NIT-1 cells). A non-targeting (scramble) RNA and non-transfection control (NTC) were used as negative controls. A protein marker (M) with corresponding molecular weights (kDa) is shown. (D) Quantification of protein expression levels of KLF11, KIT and INS in Panc.10.05 cells. (E) Diagram of luciferase reporter plasmids with the miR-10b-5p target site (miR-10b-5p mimic binding site) of human and mouse KLF11 (hKLF11 10b TS and mKlf11 10b TS) and a mutant (mKlf11 10b TSM). (F) Target validation of KLF11 with the miR-10b-5p mimic and miR-10b-5p inhibitor in Panc.10.05 cells transfected with luciferase reporter plasmids (Two-way ANOVA). (G) Quantification of miR-10b-5p in NIT-1 cells incubated in media with different glucose concentrations (0, 1.0, and 4.5 mg/L). (H and I) Western blot and quantification of KLF11 and KIT expression at different glucose concentrations. (J) Target effects of KLF11 in NIT-1 and Panc.10.05 cells cultured at different glucose concentrations. n=3 per condition for each experiment. Error bar indicate SEM, One-way ANOVA. *p < 0.05, **p < 0.01.