Expression Of Akirin Gene In Goat Muscle Tissue And Its Regulation Of Proliferation And Differentiation Of Skeletal Muscle Satellite Cells

Meat production as an important economic character has always been an important research direction in the meat goat genetics and breeding.Recent studies have shown that Akirin1 and Akirin2 genes play an important role in the mammalian muscle growth and development,but there is still a lack of systematic understanding on the Akirin1 and Akirin2 genes expression difference in different muscle tissues of goats and the molecular mechanism of regulating the goat skeletal muscle satellite cells proliferation and differentiation.In this study,90 d,180d and 270 d Tianfu goat longissimus dorsi,deltoid,gluteus medius,triceps,biceps femoris,quadriceps femoris,gastrocnemius,semimembrane and semitendinosus muscles as experimental material,using Real-time quantitative PCR and Westen blot analysis the differences of Akirin1 and Akirin2 gene m RNA and protein expression levels;using goat skeletal muscle satellite cells as experimental material,the effects of Akirin1 and Akirin2 gene on goat skeletal muscle satellite cells proliferation and differentiation were studied;and the effects of overexpression Akirin1 and Akirin2 gene on transcriptome expression level in goat skeletal muscle satellite cells were analyzed by RNA-seq technique.The main results as follows:1.Expression analysis of m RNA showed that in 90 d Tianfu goat the expression level of Akirin1 gene in the deltoid and gluteus medius muscle was higher and in the semimembrane muscle was the lowest.The expression level of Akirin2 gene in the deltoid muscle was higher than that in other muscle tissues.In 180 d Tianfu goat,Akirin1 gene was highly expressed in deltoid and semimembrane muscle,and the lowest in quadriceps femoris muscle.The expression level of Akirin2 gene in the semimembrane muscle was higher than that in other muscle tissues.In 270 d Tianfu goat,Akirin1 gene was highly expressed in quadriceps femoris,semimembrane and semitendinosus muscle,with the lowest expression in longissimus dorsi muscle.Akirin2 gene was highly expressed in quadriceps femoris and semimembrane muscle,while the expression in other muscle tissues was low.In longissimus dorsi,deltoid,gluteus medius,triceps,biceps femoris and semimembrane muscles Akirin1 and Akirin2 genes were highly expressed at 180 d;In quadriceps femoris muscles Akirin1 and Akirin2 genes were highly expressed at 270 d.2.Protein expression analysis showed that Tianfu goat at three ages Akirin1 protein was highly expressed in quadriceps femoris,gastrocnemius and semimembrane muscle,while the lowest expression was found in deltoid muscle.In 90 d Tianfu goat Akirin2 protein was highly expressed in longissimus dorsi,biceps femoris and quadriceps femoris muscle.In 180 d Tianfu goat Akirin2 protein was highly expressed in biceps femoris and quadriceps femoris muscle.In 270 d Tianfu goat Akirin2 protein was highly expressed in quadriceps femoris and semimembrane muscle.Akirin2 protein was showed the lowest expression in semitendinosus muscle of three ages Tianfu goat.3.Overexpression of Akirin1 gene during differentiation of goat skeletal muscle satellite cells,after transfection the m RNA expression levels of Myo D and Myf5 genes at24 h and 48 h were significantly increased(P < 0.05)resbectively,and the m RNA expression levels of Myo G gene decreased slowly from 24 h to 72 h after transfection.Overexpression of Akirin1 gene did not significantly increase(P > 0.05)the m RNA and protein expression levels of My HC gene.Akirin1 gene was overexpressed during the proliferation of goat skeletal muscle satellite cells,at 24 h after transfection the cell activity was significantly increased(P < 0.05);m RNA expression levels of MEF2 s gene family(MEF2A,MEF2 B and MEF2 D genes),IGF2 and m TOR genes were significantly increased(P < 0.05).The protein expression levels of m TOR and phospho-m TOR at 24 h after transfection were higher than that in the control group.At 24 h after co-treatment with rapamycin and transfected Akirin1 gene in goat skeletal muscle satellite cells,the cell activity was significantly decreased(P < 0.05)in the rapamycin-treated group.Rapamycin also reduced the m RNA expression levels of IGF2 and m TOR genes as well as the protein expression levels of m TOR and phospho-mtor(Ser 2448).4.Overexpression of Akirin2 gene during differentiation of goat skeletal muscle satellite cells,at 24 h after transfection the m RNA expression levels of Myo D and Myo G genes were significantly increased(P < 0.05),while the m RNA expression levels of Myo D,Myo G and Myf5 genes did not change significantly from 48 h to 72 h after transfection.Protein and m RNA expression levels of My HC gene were significantly increased(P < 0.05)at 48 h after transfection.Akirin2 gene was overexpressed during the proliferation of goat skeletal muscle satellite cells,at 24 h after transfection the cell activity was significantlyincreased(P < 0.05);m RNA expression levels of MEF2 s gene family(MEF2A,MEF2 B and MEF2 D genes),IGF2 and m TOR genes were significantly increased(P < 0.05).The expression of m TOR and phospho-mtor at 24 h after transfection was higher than that of control group.At 24 h after co-treatment with rapamycin and transfected Akirin2 gene in goat skeletal muscle satellite cells,the cell activity was significantly decreased(P < 0.05)in the rapamycin-treated group.Rapamycin also reduced the m RNA expression levels of IGF2 and m TOR genes as well as the protein expression levels of m TOR and phospho-mtor(Ser 2448).5.The 9 sequencing samples in this experiment obtained a total of 58 Gb(gigabases)of clean bases(the bases number filtered from the original data),and the number of clean reads(high-quality reads)that could only be mapping to a unique location in goat genome was 92.66% to 93.77%.Expression differential analysis and enrichment analysis showed that,the overexpression of Akirin1 and Akirin2 gene significantly increased(P < 0.05)the expression of muscle-growth and development related genes in the transcriptome of goat skeletal muscle satellite cells,and the differentially expressed genes were also enriched into the signaling pathways related to muscle growth and development.RT-q PCR was used to verify the differentially expressed genes analyzed by RNA-seq,and the results showed that the m RNA expression of the differentially expressed genes selected by the two experimental combinations were consistent with the results of RNA-seq.In this study,the role of Akirin1 and Akirin2 genes in the proliferation and differentiation of goat skeletal muscle satellite cells was analyzed.It provides reference for further study on the role of Akirin1 and Akirin2 genes in muscle growth and development in mammals.