Figure 7.

MiR-199a-5p targets several components of the WNT signaling pathway in muscle. (a) The schematic of constructs used for transfection of the 3′UTR luciferase reporter constructs with either the miR-199a-5p-binding site or mutated miR-199a-5p seed site. The reporter was transfected with either scrambled-miR (control; white boxes) or miR-199a-5p overexpression constructs (grey boxes) and normalized to luciferase expression levels of the vector alone (black boxes). The ablation of the miR-199a-5p inhibition is observed when the miR-199a-5p site is mutated in the 3′UTR luciferase reporter constructs. (b) Real-time qPCR of endogenous mRNA expression levels of miR-199a-5p WNT signaling target genes (cav1, wnt2, fzd4, jag1b, and gsk3β) in wild-type (WT) and Tg(mylz2-miR-199a-5p) zebrafish at 5 dpf in three separate Tg or control lines. WB analysis of the same five miR-199a-5p WNT targets along with a β-actin loading control. WB densitometries are shown where each miR-199a-5p Tg zebrafish blot is normalized to the corresponding WT blot. (c) Real-time qPCR and WB analysis of endogenous expression levels of miR-199a-5p WNT targets following overexpression of either control (scrambled-miR) or miR-199a-5p in human primary MB. Additional WBs of total β-catenin, phospho-β-catenin (Ser33/37/Thr41), GSK3α, and a β-actin loading control are also shown. WB densitometries are shown where each miR-199a-5p-overexpressing myoblast blot is normalized to the corresponding control blot. (d and e). Inhibition of the WNT (8xTCF/LEF sites) TOPFLASH and FOPFLASH (8xTCF/LEF sites mutated) luciferase reporter constructs by miR-199a-5p in (d) HEK293T and (e) human primary MB. Luciferase values were normalized to empty vector controls. (f) Real-time qPCR expression of the endogenous levels of the WNT/β-catenin downstream target genes axin2, myca, and ccnd1 in WT (black bars) and Tg(mylz2-miR-199a-5p) (white bars) zebrafish at 5 dpf in three separate Tg or control lines (n=3 replicates per individual sample; all expression values are normalized to an ef1α loading control). (g) Real-time qPCR expression of the endogenous levels of WNT/β-catenin downstream target genes AXIN2, CMYC, and CCND1 in scrambled-miR (white bars) and miR-199a-5p (black bars) overexpressing human primary MB. (n=3 replicates per individual sample; all expression values are normalized to an 18sRb loading control). (h) Schematic of miR-199a-5p regulation of WNT signaling during myogenic differentiation. MiR-199a-5p targets several WNT signaling components that affect myoblast proliferation and differentiation capacity. Low levels of miR-199a-5p exist in proliferating MB, whereas an increase in miR-199a-5p levels that occurs during myogenic differentiation is concordant with miR-199a-5p transcriptional activation by SRF and MRTF transcription factors