miR-9-5p promotes myogenic differentiation via the Dlx3/Myf5 axis

Abstract

MicroRNAs play an important role in myogenic differentiation, they bind to target genes and regulate muscle formation. We previously found that miR-9-5p, which is related to bone formation, was increased over time during the process of myogenic differentiation. However, the mechanism by which miR-9-5p regulates myogenic differentiation remains largely unknown. In the present study, we first examined myotube formation and miR-9-5p, myogenesis-related genes including Dlx3, Myod1, Mef2c, Desmin, MyoG and Myf5 expression under myogenic induction. Then, we detected the expression of myogenic transcription factors after overexpression or knockdown of miR-9-5p or Dlx3 in the mouse premyoblast cell line C2C12 by qPCR, western blot and myotube formation under myogenic induction. A luciferase assay was performed to confirm the regulatory relationships between not only miR-9-5p and Dlx3 but also Dlx3 and its downstream gene, Myf5, which is an essential transcription factor of myogenic differentiation. The results showed that miR-9-5p promoted myogenic differentiation by increasing myogenic transcription factor expression and promoting myotube formation, but Dlx3 exerted the opposite effect. Moreover, the luciferase assay showed that miR-9-5p bound to the 3'UTR of Dlx3 and downregulated Dlx3 expression. Dlx3 in turn suppressed Myf5 expression by binding to the Myf5 promoter, ultimately inhibiting the process of myogenic differentiation. In conclusion, the miR-9-5p/Dlx3/Myf5 axis is a novel pathway for the regulation of myogenic differentiation, and can be a potential target to treat the diseases related to muscle dysfunction.

Keywords: Dlx3; MiR-9-5p; Myf5; Myogenic differentiation.

Figure 1. Myogenic induction downregulates Dlx3 and upregulates miR-9-5p expression.

(A) The expression of myogenic differentiation related genes as well as Dlx3 and miR-9-5p were detected by qPCR after primary mouse myoblast cells were induced in myogenic medium at the indicated days. The internal controls were Rps18 for mRNA and U6 for miR-9-5p, respectively. (B) Myotubes formation was observed under microscope, Bar = 200 µm for 4× images, and Bar = 40 µm for 20× images. (C–E) The expression of miR-9-5p, Dlx3 and myogenic differentiation related genes mRNAs were confirmed by qPCR (C & D) and western blot (E) in C2C12 cells, respectively. The numbers above each band of western blot were quantified by ImageJ software and represent the relative expression level of each protein in different groups. RPS18 protein served as the loading control. (∗)P < 0.05, (∗∗)P < 0.01, (∗∗ ∗)P < 0.001, (∗∗ ∗ ∗)P < 0.0001.

Figure 2. MiR-9-5p negatively regulates Dlx3 expression via directly targeting Dlx3 3′UTR.

(A & B) C2C12 cells were transiently transfected with mimics (50 nM) or inhibitor miR-9-5p (100 nM) and their negative control, respectively, then incubated in myogenic medium for 2 days and examined by qPCR (A), and western blot analysis (B). (C) After 6 days, the numbers of myotubes per field were counted from four randomly chosen fields under microscope. Bar = 245 µm. (D) Immunofluorescence staining of Desmin in C2C12 cells after overexpression or knockdown of miR-9-5p. Bar = 200 µm. (E) The luciferase report plasmids were constructed as (E top panel) in pGL3B vector. Luciferase assay verifies that DLX3 is the target of miR-9-5p (E bottom panel). (∗)P < 0.05, (∗∗)P < 0.01, (∗∗ ∗)P < 0.001.

Figure 3. Dlx3 inhibits myogenic differentiation.

(A–C) C2C12 cells were transiently transfected with Dlx3 overexpression plasmid (OE-Dlx3) and empty vector (EV), respectively, then incubated in myogenic medium for 2 days and Dlx3, Myod1 and Myf5 expression was examined by real-time PCR (A), and western blot (B). 6 days after myogenic induction, the numbers of myotubes per field were counted from four randomly chosen fields under a microscope (C). Bar = 100 µm. (D-F) C2C12 cells were transiently transfected with siDlx3 and siRNA negative control (siNC), respectively, then followed the above assays using qPCR (D), western blot (E) and myotube formation (F). (G) Immunofluorescence staining of desmin in C2C12 cells transfected OE-Dlx3, siDlx3 and their controls. Bar = 100 µm. (∗)P < 0.05, (∗∗)P < 0.01, (∗∗ ∗)P < 0.001.

Figure 4. Dlx3 downregulates Myf5 by binding to its promoter and verified by luciferase assay.

(A) Schematic diagram showing the constructed luciferase report plasmids in pGL3B vector. (B) Luciferase assay confirmed that Dlx3 regulates the expression of Myf5. (C & D) C2C12 cells were co-transfected with siDlx3, and either siMyf5 or siRNA negative control, as well as pTK-RL plasmid with Renilla luciferase expression cassette. Myogenic differentiation-related genes expression was detected by qPCR and western blot at mRNA (C) and protein (D) levels. (E) The numbers of myotubes per field were counted from four randomly chosen fields under a microscope. Bar = 200 µm. (F) Fluorescence micrographs to detect Desmin in C2C12 cells were performed. Bar = 100 µm. (∗)P < 0.05, (∗∗)P < 0.01, (∗∗ ∗)P < 0.001, (∗∗ ∗ ∗)P < 0.0001.

Figure 5. The mechanism of miR-9-5p regulating myogenic differentiation through DLX3/Myf5 axis.