miR-885-5p通过靶向COL1A1促进猪原代成肌细胞增殖并抑制其分化OA

This study aims to investigate the effect of miR-885-5p on the proliferation and differentiation of porcine primary myoblasts by targeting COL1A1 and explore its underlying mechanisms.High-throughput sequencing was first performed to analyze the miRNA transcriptome in muscle samples of Shaziling pigs at different ages.Subsequently,RT-qPCR was used to detect the expression levels of miR-885-5p in various tissues and primary myoblasts of the pig.An in vitro model of porcine primary myoblasts was established,and miR-885-5p was overexpressed or inhibited.The effects of miR-885-5p on cell proliferation and differentiation were evaluated through cell cycle analysis,EdU labeling,CCK-8 assays,flow cytometry and immunofluorescence experiments.Bioinformatics tools were employed to predict the target genes of miR-885-5p,followed by dual-luciferase reporter assays to verify its targeting effect on COL1A1.Furthermore,the expression of COL1A1 in various tissues and porcine primary myoblasts was examined,and COL1A1 was interfered to assess whether miR-885-5p regulates myoblast proliferation and differentiation via COL1A1.Finally,Western blot was performed to analyze the effects of miR-885-5p on muscle fiber type conversion and its relationship with the PI3K/AKT signaling pathway.The results showed:1)The expression of miR-885-5p significantly increased with age in the longissimus dorsi and soleus muscles.In porcine primary myoblasts,miR-885-5p expression was gradually elevated during the proliferative phase and significantly decreased during the differentiation phase.Overexpression of miR-885-5p significantly promoted myoblast proliferation,enhanced cell viability,and upregulated the expression of related genes and proteins,but inhibited cell differentiation,leading to a decrease in myotube formation and lower levels of differentiation markers and proteins.Inhibition of miR-885-5p yielded the opposite effects.2)Sequence prediction and dual-luciferase reporter assays confirmed that miR-885-5p directly targeted the 3'UTR of COL1A1,and their expressions were significantly negatively correlated in the muscle tissue and primary myoblasts of Shaziling pigs.Furthermore,interference with COL1A1 significantly promoted myoblast proliferation and inhibited differentiation.3)Overexpression of miR-885-5p and interference with COL1A1 reduced the protein expression of MyHCI and PPARβ/8 while upregulating MyHC-Ⅱb expression.Inhibition of miR-885-5p caused the opposite effects,indicating that miR-885-5p regulates muscle fiber type conversion by targeting COL1A1.Additionally,overexpression of miR-885-5p and interference with COL1A1 significantly decreased the phosphorylation levels of PI3K and AKT,while inhibition of miR-885-5p increased phosphorylation levels.This study suggests that miR-885-5p promotes the proliferation and inhibits the differentiation of porcine primary myoblasts by targeting COL1A1 and regulating the PI3K/AKT signaling pathway,while promoting the conversion of muscle fibers towards a glycolytic type.