| Under an identical condition, meat production depends on the number of muscle fibers and cross sectional area. Whereas the number of muscle fibers has been fixed in the embryonic period and will not change after birth, the only change are diameter and length. Therefore, the growth of skeletal muscle during embryonic is one of the important factors of meat production yiels and quality. However, the growth of skeletal muscle is a very complex biological process regulated by many factors, and microRNA (miRNA) was reported to play a significant role in this regulation. MicroRNA is a class of endogenous non-coding small molecules with regulatory functions in plants and animals, about18to25nucleotides length. It’s mainly involved in gene transcriptional regulation and plays an important role in living things growth, development, aging, death and other biological processes. Our research group found that miR-21differentially expressed during skeletal muscle development and abundant expression in a particular stage by high-throughput sequencing. Based on its important role in medical disease and our earlier found, miR-21regulation in porcine skeletal muscle development was studied in this research to provide some reference information in mechanism of pig skeletal muscle development and genetic breeding.Like many microRNA, miR-21regulated skeletal muscle development in the same way by targeting gene expression to take an extensively participation in numerous physiological and disease processes. In this study the target TGFBI gene was identified by comparative analysis of bioinformatics, and its primers and miR-21minics was synthetized. The3’UTR of TGFBI gene was cloned in Psi-Check2-TGFBI vector and was transfected into PIEC and PK15cells to detect luciferase activity. RT-PCR and Western blot were performed to test the miR-21RNA and its protein levels changes. The results found that the luciferase activity was lower in miR-21minics than in the control. miR-21repressed the expression of TGFBI appeared no significant difference at mRNA level (P>0.05), while significant difference at protein level (P<0.05). Tissue expression pattern of the miR-21and its candidate target gene TGFBI in prenatal and postnatal skeletal muscle development (28developmental stage) in Landrace pigs and Tongcheng pigs were detected with quantitative PCR. The results showed that miR-21showed a different temporal and spatial expression profile between Landrace pigs and Tongcheng pigs. As shown in tissue expression profile, miR-21was expressed relatively more abundant in the ovary (P<0.05) than that in other tissues (heart, liver, lung, kidney, small intestine, uterus) in Tongcheng sows. While, miR-21expression was significantly higher in liver than in other tissues (P<0.05) and that in the other tissues were relatively balanced, and no specific expression was found in Landrace pigs. Significant difference was found in same tissue from Tongcheng and Landrace pigs. miR-21expression level in heart, liver, lung, kidney, small intestine, uterus and longissimus dorsi muscle of Landrace pigs were more abundant than that in those tissues from Tongcheng pigs (P<0.05). Other tissues (spleen, uterus) were barely observed difference. Significant differneces of skeletal muscle expression in the whole growth periods were found between Togcheng and Landrance pigs. As shown in the temporal and spatial expression profile, miR-21was expressed through the embryonic period33d to adult and was most abundant in embryonic period90d in Tongcheng pig. While, miR-21level was almost undetectable in the embryonic period33d and reached a peak level in embryonic period105d, with lower expression in the adult dorsal muscle than in the other period. The significant difference of miR-21expression at55d,80, d90d, and95d embryos were found between two breeds. The candidate target gene TGFBI had similar tissue expression pattern between adult Tongcheng pig and Landrace pig at mRNA level. The expression of TGFBI was the most abundant in spleen and significantly higher than that in the other tissues (P<0.05) and that in lung was a little shift. But the mRNA expression was the lowest in the adult dorsal muscle and low as well in cardiac muscle, liver, small intestine, uterus and kidney. The expression of TGFBI was up-regulated from33d to50d and peaked at50d embryos. Its expression was down-regulated through the skeletal muscle development and was almost undetectable in the adult skeletal muscle. The expression of TGFBI was significantly different in the two breeds, at prenatal55d,100d and postnatal9d,180d and adult. The correlation of miR-21and TGFBI expression in skeletal muscle development was found to be negative in Tongcheng pigs (-0.421,P=0.026<0.05), while no significant effect in Landrace pigs (-0.123, P=0.40>0.05). Results suggested that TGFBI may be related to the skeletal muscle development and muscle phenotypic differences between breeds of pig, and it may be affected under miR-21to regulate skeletal muscle development. |
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