Identification Of Differentially Expressed Lncrnas During Bovine Muscle Development And Function Study Of Lnc＿000100

Long non-coding RNAs(lnc RNAs)are non-coding RNAs with a length of more than 200 nucleotides.Studies have shown that lnc RNAs can participate in the development process of skeletal muscle through various ways.In this study,RNA-seq sequencing was conducted on fetal and adult Angus bovine longissimus dorsi muscle to screen differentially expressed lnc RNAs,and bioinformatics analysis was used to analyze the functional enrichment of differentially expressed lnc RNAs’ target genes.The results showed that the expression level of lnc＿000100 was higher in muscle tissue and lower in other tissues among differentially expressed lnc RNAs.Therefore,lnc＿000100 was selected as a candidate lnc RNA,and the influence of lnc＿000100 on myoblast proliferation and differentiation was explored through RNA interference(RNAi)tests.The main results were as follows:1.RNA-seq results showed that a total of 37,115 candidate lnc RNAs were identified in fetal and adult longissimus dorsi muscle,and a total of 1,998 lnc RNAs were differentially expressed,among which 1,229 lnc RNAs were upregulated and 769 were down-regulated.GO and KEGG enrichment analysis showed that lnc RNA target genes were enriched in pathways related to muscle development,such as muscle cell differentiation,muscle tissue development and myofibril.2.In order to better understand the regulation mode of lnc RNAs,we screened out 4 lnc RNAs with high expression,differential expression and GO analysis showed that they were related to muscle development according to the results of RNA-seq,and analyzed their upstream and downstream 100 kb protein-coding genes.Four lnc RNA-m RNA co-expression networks were constructed.In addition,we predicted the mi RNAs to which three of these lnc RNAs bind,and the results showed that these mi RNAs were related to muscle development.Finally,an interaction network consisting of 3 differentially expressed lnc RNAs,31 differentially expressed m RNAs and 9 mi RNAs was successfully constructed.3.q PCR verified 7 lnc RNAs with differentially expressed and high expression levels,and the results showed that the expression trend of lnc RNAs was consistent with the results of RNA-seq sequencing,among which lnc＿000100 had the highest expression level.The expression of lnc＿000100 in liver,lung,kidney,longissimus dorsi muscle and leg muscle of fetal cattle was analyzed,and the results showed that lnc＿000100 expression level was higher in muscle tissue,but lower in other tissues.Therefore,lnc＿000100 is selected as a candidate lnc RNA.4.RNAi test results of lnc＿000100 showed that lnc＿000100 promoted the proliferation of bovine myoblasts and inhibited their differentiation.The results of Ed U,CCK-8,q PCR and Western Blot showed that lnc＿000100 significantly promoted the proliferation of bovine myoblasts;q PCR,Western Blot and immunofluorescence showed that lnc＿000100 significantly inhibited the differentiation of bovine myoblasts.RNA-FISH results showed that lnc＿000100was mainly distributed in the nucleus.Conclusion: lnc＿000100,which is associated with muscle growth and development,was screened by transcriptome sequencing and bioinformatics analysis of bovine longissimus muscle in fetal and adult stage.The results showed that lnc＿000100 significantly promoted the proliferation of bovine myoblasts and inhibited the differentiation of bovine myoblasts.This study provides theoretical reference for exploring the role of long non-coding RNA in bovine muscle development.