Abstract
Simple SummaryThe meat production of Hu sheep affects the mutton supply, and how to increase the meat production is the primary focus of genetic breeding. Therefore, in order to explore the key factors affecting the proliferation and differentiation of Hu sheep skeletal muscle cells, we performed functional verification of miRNA at the cellular level. Our findings are helpful to clarify the molecular regulation mechanism of proliferation and differentiation of skeletal muscle cells, which will benefit the molecular breeding of Hu sheep.The growth and development of skeletal muscle require a series of regulatory factors. MiRNA is a non-coding RNA with a length of about 22 nt, which can inhibit the expression of mRNA and plays an important role in the growth and development of muscle cells. The role of miR-22-3p in C2C12 cells and porcine skeletal muscle has been reported, but it has not been verified in Hu sheep skeletal muscle. Through qPCR, CCK-8, EdU and cell cycle studies, we found that overexpression of miR-22-3p inhibited proliferation of skeletal muscle cells (p < 0.01). The results of qPCR and immunofluorescence showed that overexpression of miR-22-3p promoted differentiation of skeletal muscle cells (p < 0.01), while the results of inhibiting the expression of miR-22-3p were the opposite. These results suggested that miR-22-3p functions in growth and development of sheep skeletal muscle cells. Bioinformatic analysis with mirDIP, miRTargets, and RNAhybrid software suggested IGFBP3 was the target of miR-22-3p, which was confirmed by dual-luciferase reporter system assay. IGFBP3 is highly expressed in sheep skeletal muscle cells. Overexpression of IGFBP3 was found to promote proliferation of skeletal muscle cells indicated by qPCR, CCK-8, EdU, and cell cycle studies (p < 0.01). The results of qPCR and immunofluorescence experiments proved that overexpression of IGFBP3 inhibited differentiation of skeletal muscle cells (p < 0.01), while the results of interfering IGFBP3 with siRNA were the opposite. These results indicate that miR-22-3p is involved in proliferation and differentiation of skeletal muscle cells by targeting IGFBP3.
Highlights
Hu sheep is a Chinese local breed, famous for its high reproductivity worldwide.Today, improving meat production of Hu sheep is necessary for the sheep industry inChina
Our results suggested that miR-22-3p plays a significant role in the growth process of skeletal muscle cells by targeting IGFBP3 in Hu sheep, making miR-22-3p as a molecular marker for breeding
We focused on IGFBP3 because the function of this gene in sheep skeletal muscle cells is unknown yet, and it is differentially expressed during skeletal muscle development
Summary
Hu sheep is a Chinese local breed, famous for its high reproductivity worldwide.Today, improving meat production of Hu sheep is necessary for the sheep industry inChina. Probing the molecular mechanism underlying the proliferation and differentiation of skeletal muscle cells can provide useful clue for this problem. The seed region of miRNA can inhibit transcription and translation by targeting and binding mRNA, affecting cell proliferation, apoptosis at different developmental stages [1]. Several important miRNAs have been identified for regulating muscle growth and development, such as miR-205, miR-126, miR-60, miR-75, miR-133, miR-499, etc. MyoD1, a marker gene of muscle differentiation, is regulated by miR-1 and miR-206 [3,4]. MiR-192 targets myogenic regulator RB1, inhibiting the proliferation and promoting the differentiation of Hu sheep skeletal muscle cells [5]. MiR-128 participates in the regulation of the CDS region of myostatin and inhibits the proliferation and promotes differentiation of C2C12 cells [6]
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