Effect of Pig (Sus scrofa) SELW Gene on the Differentiation of Skeletal Muscle Satellite Cells
HU Rong-Bin, WU Xing-Feng, PAN Zhi-Hong, LI Li, HE Yi-Yi, XU E*
Key Laboratory of Genetic Breeding and Reproduction of Plateau Mountain Animals, Ministry of Education/Institute of Animal Nutrition and Feed, School of Animal Science, Guizhou University, Guiyang 550025, China
Abstract Selenoprotein W (SELW) is one of the important selenoproteins in skeletal muscle, which has important roles in cell differentiation and other biological functions. To investigate the effect of SELW gene on the differentiation of skeletal muscle satellite cells in pigs (Sus scrofa), three 7-day-old healthy white pigs were selected, and 7 tissue samples of heart, liver, spleen, lung, kidney, dorsal muscle and leg muscle were collected to extract RNA. qPCR was performed to detect the relative expression of SELW gene in different tissues. Bioinformatics analysis of porcine SELW proteins was performed using online software. The full-length sequence of the CDS region of porcine SELW gene (GenBank No. NM_213977) was cloned, a recombinant lentiviral vector overexpressing SELW gene was constructed and infected with skeletal muscle satellite cells, SELW stably expressed cell lines were obtained by puromycin screening, SELW protein expression sites was determined by subcellular localization, and SELW gene and its protein expression levels were detected by qPCR and Western blot. Cell samples were collected at 0 and 48 h of induction differentiation, and qPCR was performed to detect the expression of paired box 3 (PAX3), PAX7, myogenic factor 5 (MYF5), myogenic determinant (MYOD), and myocyte generating factor (MYOG) genes. The results showed that SELW genes were expressed in 7 different tissues of pigs, and the expression was extremely significant differenct in heart and dorsal muscle than in other tissues (P<0.01). The molecular formula of SELW protein was C421H677N109O119S3Se1, the relative molecular weight was 9 344.81 D, and the theoretical isoelectric point was 9.15, which was a stable protein. Genetic evolutionary analysis showed that pig SELW gene had the highest similarity with human (Homo sapiens), rhesus monkey (Macaca mulatta) and Sumatran orangutan (Pongo abelii). Subcellular localization results indicated that SELW proteins were localized in the nucleus and cytoplasm of cells. qPCR and Western blot results showed that the expression of SELW-OE group was very significant higher than that of SELW-NC group (P<0.01), indicating the successful construction of SELW gene stably expressing cell lines. qPCR results showed that the expression of PAX7 and MYF5 genes in SELW-OE group were extremely significantly upregulated at 0 h of induction differentiation, while MYOD gene expression was significantly down-regulated (P<0.05), and MYOG gene expression was extremely significantly down-regulated (P<0.01). The expression of PAX3 gene was significantly upregulated (P<0.05) and the expression of PAX7, MYF5, MYOD and MYOG genes were extremely significantly upregulated (P<0.01) after 48 h of induction of differentiation. It was shown that overexpression of SELW gene could promote the expression of genes related to skeletal muscle cell differentiation. It provides basic information for further study of the molecular regulation mechanism of SELW gene on muscle growth and development in pigs.
HU Rong-Bin,WU Xing-Feng,PAN Zhi-Hong, et al. Effect of Pig (Sus scrofa) SELW Gene on the Differentiation of Skeletal Muscle Satellite Cells[J]. 农业生物技术学报, 2024, 32(1): 168-179.
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