Use of mir-431 in the preparation of medicines for the treatment of muscle diseases
A mir-431, muscle disease technology, applied in the field of muscle injury and muscular dystrophy, the treatment of muscle disease, can solve the problems of host immune rejection, survival, self-renewal and poor migration
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Embodiment 1
[0062] Example 1. Myostatin negatively regulates the expression of miR-431 through the Ras / Raf / Mek / Erk signaling pathway
[0063] 1. Screened miR-431 negatively regulated by myostatin
[0064] miR-431, which was upregulated in myostatin knockout mouse skeletal muscle tissue, was selected as a candidate miRNA to study its function and self-expression regulation. During the five stages of skeletal muscle development, the expression of miR-431 in myostatin knockout mice was higher than that in wild-type mice (such as figure 1 ). Different developmental stages (E15.5, 4 weeks, 10 weeks, 16 weeks) from myostatin gene knockout mice (purchased from: Nanjing Institute of Model Animals) and wild-type sibling mice (the littermate control of gene knockout mice) Total RNA was extracted from skeletal muscle tissues of 40 weeks and 40 weeks), and miRNA expression profiling chip method was used to screen and identify miRNAs differentially expressed in skeletal muscle tissues between myosta...
Embodiment 2
[0087] Example 2. miR-431 is highly enriched in skeletal muscle tissue and its expression level increases with the differentiation of C2C12 cells and satellite cells.
[0088] To study the function of miR-431 in skeletal muscle, the expression profile of miR-431 was studied. First, Northern blot was used to measure the expression abundance of miR-431 in various tissues and organs of 8-week-old mice. In order to further determine the expression of miR-431, the expression of miR-431 in different differentiation states of C2C12 cells was detected, and the expression of miR-431 in different stages of skeletal muscle satellite differentiation was also detected.
[0089] miR-431 is highly enriched in skeletal muscle tissue
[0090] Experimental process: 8-week-old mice were selected, and the lung, liver, heart, spleen, skeletal muscle, kidney, pancreas, small intestine, brain, and stomach tissues were collected and RNA was firstly extracted and then Northern Blot hybridization was ...
Embodiment 3
[0113] Example 3. miR-431 promotes C2C12 cell differentiation.
[0114] To study the function of miR-431 in skeletal muscle, the function of miR-431 was detected in C2C12 cells. First, a stable transfection cell line overexpressing miR-431 (about 60 times) was constructed in the C2C12 cell line, and the following functional tests were performed using the established C2C12 cell line overexpressing miR-431.
[0115] Cells stably transfected with mi-431 were first planted at a certain density, and the differentiation medium was changed after 24 hours of proliferation. After 24 hours and 36 hours of differentiation, the early differentiation molecular marker molecule Myogenin (Myogenin) and the late differentiation marker molecule were detected respectively. Expression of MHC. By immunofluorescent staining of cells, it was found that after 24 hours of differentiation, overexpression of miR-431 significantly increased the number of myogenin-positive cells, and at the same time the...
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