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. 2021 Dec 13;9(4):2104759. doi: 10.1002/advs.202104759

Figure 5.

Figure 5

Validation and translational evaluation of miR‐424(322)/503‐regulated target genes. A) The funnel diagram depicts the comprehensive pipeline used to shortlist potential target genes across validation experiments and translational studies. B) Scatter dot plots show the negative correlation between the miR‐424(322) and the expression of the four main miRNA targets, Chordin Like 1 (CHRDL1), γ‐Synuclein (SNCG), TNF Alpha Induced Protein 8 (TNFAIP8), and Uracil DNA Glycosylase (UNG) in human subcutaneous (SC) fat samples (Spearman's rank‐order correlation tests). C) Bean plots illustrating CHRDL1, SNCG, TNFAIP8, and UNG mRNA in normal weight (NW) participants with normal glucose tolerance (NGT) (green, n = 34), and in obese subjects without (orange, n = 31) and with (red, n = 20) impaired glucose tolerance (IGT). D) Expression of SC CHRDL1, SNCG, TNFAIP8, and UNG at the baseline (red) and upon surgery‐induced weight loss (green) (n = 23). Statistical significance was determined by the Tukey's honestly significant difference (HSD) ANOVA's post‐hoc test (cross‐sectional comparisons), and by paired t‐test (longitudinal study) (see also Tables S1–S3, Supporting Information). E) Cloning of the 3'‐UTR regions corresponding to CHRDL1, SNCG, TNFAIP8, and UNG and subsequent luciferase assays show differential targeting activity by the miR‐424(322) and the miR‐503, when compared to a control scrambled non‐targeting RNA. Relative light units (X–axis) are expressed as the normalized luciferase counts to the renilla internal control. F) Predicted miRNA binding site mutagenesis and luciferase assays were performed in the most relevant candidates (TNFAIP8, SNCG, and UNG) from (E) to prove the miR‐424(322) and miR‐503 binding specificity. Data are presented as mean ± S.E.M. (n > 5/group). G) Western blots show that miR‐KO mature adipocytes (MA) have increased gene target protein levels, while H) the 48 h‐lasting induction of the miR‐424(322)/503 cluster triggers a moderate effect on the endogenous levels of adipocyte SNCG, TNFAIP8, and UNG, and I) a more pronounced effect after transient induction with doxycyclin in the 14 day‐lasting adipogenic course assay (n = 2/group). Actin immunobloting was used to ensure equal loading protein amounts. J) Messenger Sncg and Ung RNA expressions in subcutaneous (scWAT) and epididymal (eWAT) white adipose tissue in sex and age‐matched Wt and miR‐KO mice (Tnfaip8 gene expression was undetectable in mouse fat samples.), and K) mouse miR‐322 and Sncg expressions in male and female mice under normal chow (NC) and high‐fat diet (HFD) (n = 4/group; Student's t‐test). r.u. stands for “relative units.” *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant.