Mechanisms Of LncRNA SOX6 AU Underlying The Growth And Development Of Bovine Skeletal Muscle

Skeletal muscle is an important part of animal body,and its growth rate and quality play an important role in affecting animal production performance.Muscle growth and development are regulated by various transcription factors and epigenetic factors.Therefore,it is necessary to analyze the regulation of muscle growth and development.Long non-coding RNA(lncRNA),as a novel non-coding RNA with a length greater than 200 nt,plays a key role in various biological processes.Recent studies have revealed that lncRNA regulates skeletal muscle growth and development by participating in transcriptional regulation,fiber type conversion,and skeletal muscle cell proliferation and differentiation.Currently,many lncRNAs in muscle tissue have been discovered,but the functions and regulatory mechanisms of these lncRNAs remain unclear.SRY-box transcription factor 6(SOX6)is an important member of the Sox D gene subfamily in the Sox superfamily and is involved in the regulation of skeletal muscle development.In this study,a lncRNA LOC101904740 reversely transcribed from upstream of the bovine SOX6 gene sequence was found in the NCBI database,which was named SOX6 AU(SRY-box transcription factor 6 antisense upstream).The previous works proved the existence of SOX6 AU,and the expression levels were significantly different in the muscle tissues of Xianan cattle among different developmental stages.In order to further reveal its role in the growth and development overexpression and interference techniques were used to study the effect of SOX6 AU on the proliferation,apoptosis and differentiation of bovine skeletal muscle cell and then the RNA-seq and other techniques were applied to reveal its molecular regulation mechanisms.The results are as follows:(1)Firstly,through bioinformatics analysis,it was found that SOX6 AU mainly existed in the cytoplasm and had no protein coding ability.There were many stem-ring structures in its secondary structure,and the intermediate region of SOX6 AU had high thermal stability.Secondly,the presence of SOX6 AU in the cytoplasm was verified by FISH.The tissue expression of SOX6 AU in Xianan cattle showed that SOX6 AU was expressed in all tissues at different developmental stages,and the relative expression level in leg muscle tissue was relatively high.With the increase of age,the expression of SOX6 AU in leg muscle gradually increased,and the expression of SOX6 AU was significantly different between newborn cattle and 24-month-old cattle(P < 0.05).(2)The effects of SOX6 AU on cell proliferation were detected by RT-qPCR,Western blot,CCK-8,EdU and flow cytometry.The results showed that after overexpression of SOX6 AU,the mRNA and protein expression levels of proliferation marker genes PCNA and CDK4 were significantly increased(P < 0.05),the number of cells in S phase was significantly increased(P < 0.01),and the activity of DNA replication and cell proliferation were significantly enhanced(P < 0.05).However,the proliferation process was inhibited after interference of SOX6 AU expression.Also,the effects of SOX6 AU on the expression of MYOD and MYOG markers of muscle differentiation were detected by RT-qPCR and Western blot.The results showed that the expression of marker genes of muscle differentiation was significantly up-regulated after overexpression of SOX6 AU,while the results were opposite after interference of SOX6 AU.Meanwhile,after overexpression of SOX6 AU,the protein expression levels of proapoptotic genes Caspase3 and Bax were significantly decreased,and the expression level of Caspase3 was significantly down-regulated,while the mRNA expression level of anti-apoptotic gene Bcl-2 was up-regulated.After interference of SOX6 AU,the effects were reserved.In a conclusion,SOX6 AU promoted the proliferation,differentiation and apoptosis of bovine skeletal muscle cells.(3)In C2C12 cells,SOX6 AU was overexpressed and the expression levels of cell proliferation-related genes and proteins were detected by RT-qPCR and Western Blot.It was found that overexpression of SOX6 AU significantly decreased the mRNA and protein expression levels of cell proliferation marker genes PCNA and CDK4(P < 0.05),and the expression level of P21 protein was significantly increased(P < 0.05).At the same time,EdU,CCK-8 and flow cytometry showed that the cell proliferation activity decreased.Overexpression of SOX6 AU in differentiated C2C12 cells significantly increased the expression of muscle differentiation-related genes MYOD and MYOG(P< 0.05),and their expression levels of protein were also significantly increased(P <0.001).Immunofluorescence results showed that the muscle tubes of C2C12 cells became thicker and longer after overexpression of SOX6 AU.In addition,after overexpression of SOX6 AU in C2C12 cells,the effect of SOX6 AU on apoptosis of C2C12 cells was detected by RT-qPCR and Western Blot.The results showed that the mRNA and protein expression levels of pro-apoptotic gene Bax were significantly increased(P < 0.05),the mRNA expression of Caspase3 was significantly increased(P< 0.05),and the mRNA and protein expression levels of anti-apoptotic gene P21 were significantly decreased(P < 0.05).Altogether,SOX6 AU inhibited proliferation of C2C12 cell and promoted differentiation and apoptosis of C2C12 cell.The function of SOX6 AU in myogenic differentiation and apoptosis may be conserved between cattle and mice.(4)In this study,the promoter region of bovine SOX6 AU was successfully cloned,and the dual-luciferase reporter vectors with different lengths were obtained by truncation method.The core promoter region of SOX6 AU(-2016 ～-1767 bp)was determined by dual-luciferase activity assay.The online website predicted the transcription factor SRF(-1826 ～-1811 bp)that may bind to it,which may be involved in the transcriptional regulation of SOX6 AU.(5)A total of 483 differentially expressed genes were identified by transcriptomic sequencing analysis of bovine skeletal muscle cells overexpressing SOX6 AU,including224 up-regulated and 259 down-regulated differentially expressed genes.GO enrichment and KEGG analysis of differential genes showed that differential genes were mainly involved in biological processes related to muscle development,such as muscle structure development,muscle cell proliferation,myocardial tissue growth and ossification regulation,and were significantly enriched in PI3K-Akt and MAPK signaling pathways.It was further suggested that SOX6 AU played an important regulatory role in the development of bovine skeletal muscle,and it was found that SOX6 AU can negatively regulate the expression of SOX6 gene through cis-regulation.In summary,this study revealed the tissue expression patterns of SOX6 AU and clarified that SOX6 AU had an important regulatory role in promoting the proliferation,differentiation and apoptosis of bovine primary skeletal muscle cells.Additionally,the molecular mechanisms of SOX6 AU during muscle growth and development was primally revealed.It was found that SOX6 AU can negatively regulate the expression of SOX6 gene.Our results lay a theoretical foundation for improving beef yield and meat quality,and provide new ideas for cattle genetic breeding.