miR-191 affects skeletal muscle differentiation by regulating Wwp1 in mouse myoblasts

Fig 1. Screening of candidate target genes regulated by miR-191. (A) Analysis and screening of differentially expressed miRNAs in the serum of patients with chronic obstructive pulmonary disease. (B) GO enrichment analysis of differentially expressed genes induced by miR-191-5p overexpression (Top10 for each ontology). The y-axis shows GO terms, and the x-axis represents enrichment score, and the color represents -log10(p-value). (C) KEGG enrichment analysis of differentially expressed genes induced by miR-191-5p overexpression (Top20). The y-axis indicates KEGG pathways, the x-axis shows the number of differentially expressed genes enriched in each pathway, and the color represents -log10(p-value). (D) Prediction of target genes of miR-191-5p using RDB, TargetScan Mouse 8.0 (TS8.0), and ENCORI databases, and identification of candidate target genes through Venn diagram analysis. (E) Venn diagram analysis of overlapping differentially expressed genes and candidate target genes.

Fig 2. Expression of Wwp1 gene in skeletal muscle. (A) Expression of WWP1 gene across human tissues (databases: The Human Protein Atlas). (B) Analysis of WWP1 gene expression in different skeletal muscle cell types based on single-cell sequencing of skeletal muscle. (C)-(D) RT-qPCR was used to verify the expression of miR-191 and Wwp1 at 1 d and 3 d after CTX or PBS injection. n = 3 (CTX injection), n = 5 (PBS injection). **: p < 0.01. (E) Wwp1 expression in myocyte cells. Bubble size indicates the percentage of Wwp1-positive cells; color indicates average expression level.

Fig 3. Transfection with miR-191 mimics affects myoblast differentiation. (A) RT-qPCR was used to verify the expression of miR-191 after transfection with miR-191 mimics (n = 3), **: p < 0.01. (B)-(C) miR-191 mimics were transfected before cell differentiation, and differentiation was induced when changing the culture medium after transfection. On day 3 (B) or day 5 (C) of differentiation, RT-qPCR was used to verify the expression of myosin heavy chain (Myh1, Myh7, Myh8) (n = 3), *: p < 0.05, **: p < 0.01. (D) miR-191 mimics were transfected before cell differentiation, and differentiation was induced when changing the culture medium after transfection. On day 3 of differentiation, RT-qPCR was used to verify the expression of Myog, PI3K (Pik3ca), and mTOR (Mtor) (n = 3), **: p < 0.01. (E) miR-191 mimics were transfected on day 3 of cell differentiation. On day 5 of differentiation, RT-qPCR was used to verify the expression of myosin heavy chain (Myh1, Myh7, Myh8) (n = 3), **: p < 0.01. (F) miR-191 mimics were transfected on day 3 of cell differentiation. On day 5 of differentiation, Western blot was used to verify the expression of MyHC. n = 4, shown is a representative result.

Fig 4. The effect of miR-191 on myoblast proliferation. (A)-(B) The expression of Pax7 and Ccne1 in myoblasts was detected by RT-qPCR 24 h or 36 h after transfection with miR-191 mimics (n = 3), *: p < 0.05, **: p < 0.01. (C)-(D) The expression of Cdk4, Cdk6, and Ccnd1 in myoblasts was detected by RT-qPCR 24 h (C) or 36 h (D) after transfection with miR-191 mimics (n = 3), **: p < 0.01. (E) Flow cytometry was used to analyze the percentage of cells in the G0/G1, S, and G2/M phases (n = 3).

Fig 5. The effect of Wwp1 on myoblast proliferation. (A)-(B) The expression of Pax7 and Ccne1 in myoblasts was detected by RT-qPCR 24 h (A) or 36 h (B) after transfection with Wwp1 siRNA (n = 3), *: p < 0.05, **: p < 0.01. (C)-(D) The expression of Cdk4, Cdk6, and Ccnd1 in myoblasts was detected by RT-qPCR 24 h (C) or 36 h (D) after transfection with Wwp1 siRNA (n = 3).

Fig 6. miR-191 directly regulates the Wwp1 gene. (A) Analysis of 3’-UTRs of Wwp1 gene and miR-191 binding sites across multiple species. (B) Schematic diagram of psi-wt-Wwp1 (wild-type) and psi-m-Wwp1 (mutant) vector construction. (C) Detection and relative activity analysis of Firefly luciferase and Renilla luciferase 24 h after co-transfection with miR-191 mimics and psiCHECK-2 vector (n = 3), *: p < 0.05. (D) The expression of Wwp1 in myoblasts was detected by RT-qPCR 12 h after transfection with miR-191 mimics (n = 3), *: p < 0.05.

Fig 7. Graphical abstract.