Study On Regulation And Function Of LincRNA TCONS₀0026800 In Proliferation And Differentiation Of Porcine Skeletal Muscle Satellite Cell

With the progress of society and the increase of national income,people's demand for pork production and quality is increasing.It is of great significance to improve the yield and quality of pork through genetic breeding.The study of the genetic and epigenetic basis of skeletal muscle development is an important aspect and a research hotspot in this field.Skeletal muscle is the main metabolic tissue in pigs and is abundant in content.There are some important regulatory networks in the development of skeletal muscle,involving many important regulatory genes.These regulatory genes include coding genes and a large number of lncRNAs.It has been reported that lncRNAs play an important role in embryonic development,tissue differentiation,organogenesis,etc.However,there are relatively few studies on the function of lncRNAs during muscle growth and development.This study systematically revealled the expression pattern of a newly identified lincRNA TCONS₀0026800 in the proliferation and differentiation process of skeletal muscle satellite cells,and the methylation level of promoter of TCONS₀0026800 and transcription regulation were studied during muscle development.In order to obtain the apparent regulation mode of the lincRNA on the proliferation and differentiation of porcine skeletal muscle satellite cells,and lay a theoretical foundation for its functional study on skeletal muscle development in pigs.The main results are as follows:?1?By analyzing of RNA-Seq data of domestic and foreign pig breeds,a differentially expressed lincRNA TCONS₀0026800 was screened out.The results of Q-PCR showed that the expression level of TCONS₀0026800 in embryonic 35 D brain tissue was significantly higher than that in heart,liver,kidney and muscle tissues.TCONS₀0026800 gradually increased in the proliferative phase of porcine skeletal muscle satellite cells?6 h–42 h?;during the differentiation period?6 h–24 h?,its expression level gradually decreased.?2?The full length sequence of TCONS₀0026800 was obtained using RACE technology and showed that the lincRNA had two transcripts,V1 and V2.V1 had a full length of 1638 bp and contained three exons.V2 had a full length of 1798 bp and contained two exons.The lincRNA had no protein coding ability.The nuclear distribution results indicated that TCONS₀0026800 was mainly distributed in the cytoplasm.?3?The results of the double fluorescence experiment indicated that the core promoter of TCONS₀0026800 was 33 bp–376 bp upstream of its first exon.The transcription factor binding prediction showed that the core promoter binds to the muscle-associated transcription factor MyoD and MyoG;mutation experiments showed that the fluorescence values of the three mutation sites were significantly reduced.The results of double fluorescence and quantitative analysis showed that the fluorescence value and expression level of TCONS₀0026800 were significantly increased after transfection of MyoD and MyoG,respectively.?4?In the skeletal muscles of adult Meishan pigs?n=3?and adult White pigs?n=3?,there were 44 CpG sites in the core promoter region of TCONS₀0026800,of which the last CpG site was methylated and the remaining 43 CpGs were no methylation?no difference between cultivars?;the methylation pattern of this gene was the same in the fetus 45 D and adult Larger pig tissues?heart,liver,kidney,brain,cerebellum,muscle?.?5?Real-time quantitative PCR was used to study the effects of knockdown of TCONS₀0026800 gene on MSTN and TBX3,TCONS₀0026800 adjacent genes and proliferation and differentiation of skeletal muscle satellite cells.The results showed that after knocking down TCONS₀0026800,the expression level of MSTN was significantly increased at 24 h and 36 h after satellite cell differentiation.The expression level of TBX3 was significantly decreased when satellite cells proliferated for 30 h and 36 h after differentiation;The expression level of the adjacent gene COMMD5 of TCONS₀0026800 was significantly decreased when satellite cells proliferated for 24 h and 36 h after differentiation.TCONS₀0026800 could inhibit the proliferation of skeletal muscle satellite cells,but has little effect on the differentiation of skeletal muscle satellite cells.Conclusions: 1.TCONS₀0026800 had 2 transcripts V1 and V2.Among them,V1 had a full length of 1638 bp and contained three exons;V2 had a full length of 1798 bp and contained two exons.2.TCONS₀0026800 was mainly distributed in the cytoplasm.3.MyoD and MyoG could synergistically regulate the expression of TCONS₀0026800.4.TCONS₀0026800 could inhibits the proliferation of porcine skeletal muscle satellite cells.