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This study aimed to identify the target genes of IGFBP3（insulin growth factor binding protein）protein and to investigate its target genes effects on the proliferation and differentiation of Hu sheep skeletal muscle cells. IGFBP3 was an RNA-binding protein that regulates mRNA stability. Previous studies have reported that IGFBP3 promotes the proliferation of Hu sheep skeletal muscle cells and inhibits differentiation, but the downstream genes that bind to it have not been reported yet. We predicted the target genes of IGFBP3 through RNAct and sequencing data, and verified by qPCR and RIP（RNA Immunoprecipitation）experiments, and demonstrated GNAI2（G protein subunit alpha i2）as one of the target gene of IGFBP3. After interference with siRNA, we carried out qPCR, CCK8, EdU, and immunofluorescence experiments, and found that GNAI2 can promote the proliferation and inhibit differentiation of Hu sheep skeletal muscle cells. This study revealed the effects of GNAI2 and provided one of the regulatory mechanisms of IGFBP3 protein underlying sheep muscle development. [ABSTRACT FROM AUTHOR]

This study aimed to understand the expression level of YAP1 in the skeletal muscle of Hu sheep and to reveal the regulatory mechanism of YAP1 on Hu sheep skeletal muscle satellite cells (SMSCs). Previous research by our group has found that YAP1 may affect the growth and development of Hu sheep skeletal muscle. In the present study, we found the expression of YAP1 in the skeletal muscle is higher than in other tissues of Hu sheep. Then, we detected the effect of YAP1 on proliferation and differentiation in Hu sheep SMSCs. According to the results of qPCR, CCK-8, EDU, and Western blot, compared to the group of negative control, overexpression of YAP1 promoted the proliferation and inhibited the differentiation of SMSCs according to the results of qPCR, CCK-8, EDU, Western blot, while the interference of YAP1 was on the contrary. Overall, our study suggests that YAP1 is an important functional molecule in the growth and development of skeletal muscle by regulating the proliferation and differentiation of SMSCs. These findings are of great use for understanding the roles of YAP1 in the skeletal muscle of Hu sheep. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) is of great importance to muscle growth and development, including the regulation of the proliferation and differentiation of skeletal muscle satellite cells (SMSCs). In our research group's previous study, we found that miR-181a is differentially expressed in the longissimus dorsi muscle of Hu sheep at different stages. We speculated that miR-181a may participate in the growth and development process of Hu sheep. To understand the mechanism of miR-181a regulating the growth and development of Hu sheep skeletal muscle, we extracted skeletal muscle satellite cells from the longissimus dorsi muscle of 3-month-old Hu sheep fetuses and performed a series of experiments. Our results showed that miR-181a suppressed SMSCs' proliferation using QRT-PCR, Western blot, CCK-8, EDU, and Flow cytometry cycle tests. In addition, QRT-PCR, Western blot, and immunofluorescence indicated that miR-181a facilitated the differentiation of SMSCs. Then, we used dual-luciferase reporter gene detection, QRT-PCR, and Western blot to find that the Yes1-related transcription regulator (YAP1) is the target gene of miR-181a. Our study supplies a research basis for understanding the regulation mechanism of miR-181a on the growth of Hu sheep skeletal muscle. [ABSTRACT FROM AUTHOR]

Simple Summary: The 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. [ABSTRACT FROM AUTHOR]