Functional Analysis Of Porcine Embryonic Skeletal Muscle Lincrna And The Effect Of Promoter Methylation On Its Expression

A large number of studies have shown that the total amount of pig muscle fibers is finalized during the embryonic period,so the development of skeletal muscle during the embryonic period of pigs has an important impact on the level of meat production after birth.This study analyzed the transcriptome data and methylation data of 4 time periods in embryonic period 33-50 days,50-70 days,70-105 days,105-less than 1 day(0 day)after birth.The purpose is to explore the potential effects of linc RNA on porcine skeletal muscle development at different stages of porcine embryonic stage.The main results of this study are as follows:1.A total of 15 transcriptome data at 5 time points during the embryonic stage of pigs are used as materials.According to the existing procedures in the laboratory,418 linc RNAs were obtained,of which 141 were newly identified linc RNAs and 277 were known linc RNAs.Through the different developmental stages of porcine embryonic stage linc RNA and protein coding gene expression level difference analysis.In the 33-50 days of embryonic development,101 differentially expressed linc RNAs(DEL)and 2885 differentially expressed protein-coding RNAs(DEm RNA)were obtained.In the 50-70 days of embryonic development,88 DELs and 1183 DEm RNAs were obtained.152 DELs and 2978 DEm RNAs were analyzed during the developmental stage of embryonic stage 70-105 days.From the 105 th day of embryonic stage to 0th day after birth,132 DELs and 3788 DEm RNAs were obtained.2.Since linc RNA can regulate its potential target genes in trans,we calculated the Pearson correlation coefficient between the expression levels of the four developmental stages of pig embryonic period based on the expression levels of DEL and DEm RNA(embryonic period 33-50 days,Embryonic stage 50-70 days,embryo stage 70-105 days,and embryo stage 105 days to 0 days after birth).Subsequently,functional enrichment analysis was performed on the potential target genes of DEL at each developmental stage.Later,by consulting the literature,we learned about the detailed regulatory function of DEL’s potential target genes in skeletal muscle development.As a result,several linc RNAs related to porcine skeletal muscle development were found in different developmental stages of pig embryonic period.3.Use the DEL and DEm RNA expression matrix to construct a co-expression network through the WGCNA package in the R software,and find the key modules.Then use cytoscape software to calculate the core linc RNA(hub linc RNA)in the key module.As a result,20 hub linc RNAs were obtained in 4 key modules(turquoise,red,blue and brown).It was found that they were co-expressed with protein-coding genes related to skeletal muscle development,such as Mstn,Myf5,Myo G,and Sox6 in their respective modules.4.A total of 15 methylation data at 5 time points in the embryonic stage are used as materials.Use bismark,bedtools software,and the DSS package in the R software to identify the differentially methylated regions(DMR)of the DEL promoter,and calculate the methylation level of the DEL promoter region.According to the DEL expression level from high to low,it is divided into 5 expression levels(highest,highly,moderately,lowly and silent).Using deeptools software to compare the methylation levels of DEL promoter regions of different expression levels,it is found that there are 5 developmental times in the embryonic stage the DEL with the lowest expression level has the highest methylation level in its promoter region.5.Joint analysis of transcriptome data and methylation data.Calculate the Pearson correlation coefficient between the expression level of DEL at different developmental stages and the methylation level of its promoter region.Results the expression levels of multiple DELs were found to be significantly negatively correlated with their promoter methylation levels in the four developmental stages,including multiple linc RNAs that are presumed to be related to skeletal muscle development.In summary,by analyzing transcriptome data and methylation data,we have obtained a number of linc RNAs that have potential regulatory effects on skeletal muscle development at different developmental stages of the pig embryo.At the same time,we analyzed the effect of methylation level of linc RNA promoter region on linc RNA expression level.It provides valuable materials for studying the effects of linc RNA on porcine embryonic skeletal muscle development and methylation on the transcription level of non-coding RNA.