Research Progress Of The Interaction Mechanism Between LncYYW And MiRNA Regulating The Differentiation Process Of Bovine Skeletal Muscle Satellite Cells

Skeletal muscle development is a complex biological process.The differentiation of skeletal muscle satellite cells is an important part of skeletal muscle development.In recent years,Studies have found that lncRNAs and miRNAs play a role in biological processes through various mechanisms to affect skeletal muscle development.At present,many lncRNAs and miRNAs that have an important influence on the maturation of muscle fibers have been identified,but the research on the interaction between lncRNA and miRNA regulating skeletal muscle development is rare.In the early stage of this research group,a high-throughput sequencing screened a lncRNA-lncYYW highly expressed in bovine skeletal muscle satellite cells.In this study,the full length of 5’RACE of lncYYW was amplified.Partial sequences of lncYYW were first used to construct an overexpression vector to transfect mouse C2C12 cells to detect the effect of high expression of lncYYW on the proliferation of C2C12 cells.Then we constructed an overexpression vector to study the effect of lncYYW on the proliferation and differentiation of bovine skeletal muscle cells.Finally we collected cell samples overexpressing lncYYW and the control group for miRNA sequencing,in order to deeply explore the interaction mechanism between lncYYW and miRNA.The main results are as follow:1.The 5’RACE full-length of lncYYW was amplified by RACE technology.It was predicted by online analysis that lncYYW had non-coding potential and was distributed on chromosome 5,which was reverse complementary to the partial sequence of Myf6 3’UTR region.2.Using the sequence of 420 bp lncYYW obtained in the previous stage of the research group,it was constructed on p CDNA3.1-EGFP vector and transfected into C2C12 cells.It was found that overexpression of lncYYW had no significant effect on the proliferation of C2C12 cells,this result is inconsistent with the previous verification results on cattle,we initially speculated that lncYYW has different functions in different species.3.Real-time quantitative PCR and Ed U were used to verify the effect of overexpression of lncYYW on the proliferation of bovine skeletal muscle satellite cells.At the same time,fluorescent quantitative PCR and Western blot were used to detect the effect of overexpression of lncYYW on cell differentiation.The results showed that lncYYW had no significant effect on the proliferation of cells.But overexpression of lncYYW can inhibit the differentiation of bovine skeletal muscle satellite cells.Using the same method as the differentiation effect test,the expression of lncYYW’s sense m RNA（Myf6）was detected.The results showed that after overexpression of lncYYW,Myf6 was significantly down-regulated at the m RNA and protein levels,We assumed lncYYW is an antisense lncRNA.4.Using miRNA sequencing technology,over-expressed lncYYW and control cell samples were detected,and 51 differentially expressed miRNAs were screened,of which 30 were significantly up-regulated and 21 were significantly down-regulated.KEGG enrichment analysis was performed on the differential miRNA target genes obtained by sequencing.It was found that miRNA target genes are mainly enriched in signaling pathways such as cytoskeletal regulation,Ras,MAPK,and Rap.Verification of sequencing results by fluorescent quantitative PCR confirmed that miR-1 and miR-206 were significantly down-regulated after overexpression of lncYYW.5.Overexpression of miR-1 found that lncYYW was significantly down-regulated during the proliferation phase,and there was no significant change the next day of differentiation;overexpression of miR-206,lncYYW did not change significantly during the proliferation and differentiation phases;subsequent selection of miR-1 as the research object,overexpression lncYYW detected the expression of miR-1 target gene.6.After overexpressing lncYYW,the known miR-1 target genes（CX43,CCDN1,SFRP1,ZNF281,SRSF9,FN1）and miR-1 predicted the expression of target gene ITGB3.The test results showed that SFRP1 and SRSF9 were significantly up-regulated,and FN1 and ITGB3 were significantly decreased（P<0.05）.In summary,this study preliminarily determined that lncYYW is a Myf6 antisense lncRNA that can inhibit the myogenic differentiation of bovine skeletal muscle satellite cells,and its function is not the same in cattle and mice.It was further found that miR-1 was a miRNA interacting with lncYYW,and it was initially confirmed that lncYYW inhibited the expression of miR-1 by promoting the upregulation of miR-1 target gene SRSF1,and on the other hand,overexpressing lncYYW downregulated the miR-1 Expression inhibits the expression of its target genes ITGB3 and FN1,and ultimately inhibits the differentiation of bovine skeletal muscle satellite cells.