Regulatory Effect Of MiR-206 Interaction LncRNA On Myogenic Differentiation Of Bovine Skeletal Muscle Satellite Cells

Muscle development and differentiation is an important part of developmental biology research.Previous studies had shown that non-coding RNAs including micro RNAs(miRNAs)and long non-coding RNAs(lncRNAs)play an important regulatory role in muscle development.In our previous studies,we found that miR-206 acted as a critical role in the myogenic differentiation of bovine skeletal muscle satellite cells(MSCs),and lncRNAs interacted with miRNAs to exert regulatory functions.Based on the preliminary work,this study screened and identified miR-206 interaction lncRNAs,analyzed the function of lncRNAs,and studied the interaction mechanism with miR-206.In order to provide reference for the regulation mechanism of non-coding RNA related to bovine muscle development and differentiation.1.In this study,high throughput sequencing(RNA-seq)was used to screen and identify differentially expressed lncRNAs in bovine MSCs before and after differentiation.We identified 2348 lncRNAs,among which 301 lncRNAs were differentially expressed,213 lncRNAs were up-regulated in differentiated myotubes,and 88 lncRNAs were down-regulated.Through prediction and functional enrichment analysis,the target genes of differentially expressed lncRNAs were discovered,and they were enriched in GO and KEGG pathways which is related to cell proliferation and differentiation and muscle development differentiation.These results indicated that identified lncRNAs may modulate muscle development and differentiation.2.Previous studies had found that miR-206 could regulate the myogenic differentiation of bovine MSCs.In this study,we further analyzed the interaction between miR-206 and differentially expressed lncRNAs,and predicted 22 lncRNAs related to miR-206.According to the differential multiples of lncRNAs,we preliminarily screened 3 lncRNAs(lnc4351,lnc0803,lnc8554)which can be regulated by miR-206.We designed their bovine MSCs interference models separately.By EDU staining,it was confirmed that the interference of lnc4351 and lnc0803 could significantly inhibit the proliferation of bovine MSCs;q RT-PCR and Western Blot detected the expression of differentiation marker My OG and My HC,indicating that the interference of lnc4351 and lnc0803 promoted myogenic differentiation of bovine MSCs.Similarly,the interference of lnc8554 promoted the proliferation of bovine MSCs and inhibits their myogenic differentiation.We chose to follow the study of lnc8554,which showed different trends in miR-206 expression regulation.After bioinformatics analysis,subcellular localization and prokaryotic expression experiments,it was found that lnc8554 was a long non-coding RNA located on bovine chromosome 2,both in nucleus and cytoplasm.Further overexpression test confirmed that lnc8554 promoted myogenic differentiation of bovine MSCs.3.This study further analyzed the interaction mechanism between miR-206 and lnc8554.Through online software predicted a binding site between miR-206 and lnc8554.It was verified by double luciferase assay that lnc8554 was able to bind to miR-206.Immediately after interference or overexpression of lnc8554,q RT-PCR and Western Blot were used to detect the expression of miR-206 target genes Pax7 and HDAC4,and it was found that lnc8554 inhibited their expression.These results showed that miR-206 and lnc8554 was not mediated by ce RNA.The binding protein of miR-206 and lnc8554 was analyzed by RNA-pull down technique,and two co-binding proteins HNRNPA2B1 and DHX9 were obtained.The interaction regulation analysis showed that both lnc8554 and miR-206 could affect the expression of binding proteins HNRNPA2B1 and DHX9.HNRNPA2B1 and DHX9 could also promote the expression of lnc8554 and miR-206,respectively.It is presumed that miR-206 and lnc8554 can form RNA-protein complexes with HNRNPA2B1 and DHX9,thereby affecting the expression of miR-206 and lnc8554,and ultimately regulating the myogenic differentiation of bovine MSCs by affecting the expression of target genes of miR-206.In this study,we identified an lncRNA-lnc8554 that interacts with miR-206 and promotes myogenic differentiation of bovine MSCs,and preliminarily analyzed its interaction with miR-206.These results can accumulate basic data for the mechanism of the co-regulation of muscle development and differentiation by miRNAs and interacting lncRNAs,and deeply understand the molecular genetic basis of muscle development and growth in theory.