Study On The Action And Regulation Mechanism Of Lnc23 On Myogenic Differentiation Of Bovine Skeletal Muscle Satellite Cells

Accumulating evidence suggests that long non-coding RNA(lnc RNA)plays a crucial role in regulating the growth and development of skeletal muscle,a highly coordinated multistep biological process.Up to now,most studies on muscle development related lnc RNA are mainly focused on humans and mouse.However,there are relatively few studies on bovine muscle development related lnc RNA.Hence,it is helpful to understand the process of bovine muscle development by obtaining the lnc RNA which could regulate bovine skeletal muscle development and analyzing its mechanism.And a new target reference for molecular breeding of cattle would be provided.1.A muscle highly expressed lnc RNA,named lnc23,located on bovine genome chromosome 23,was screened from the previous lnc RNAs sequencing results which from different stages of embryonic development of muscle tissues.The q RT-PCR showed that the expression of lnc23 increased firstly and then decreased during the differentiation of muscle satellite cells,and the expression level reached the peak on the second day of differentiation(DM2).Subcellular localization indicated that lnc23 was localized in the nucleus.Both prediction of coding potential and in vitro translation experiments showed that lnc23 could not encode proteins and was proved as a non-coding RNA.2.The inhibited and over-expressed model of lnc23 in bovine skeletal muscle satellite cells were successfully constructed by si-ASO-lnc23 and pc DNA-lnc23,respectively.The q RT-PCR and Western blot were performed to detect the expression level of cell proliferation and cell differentiation marker genes.And the effects of lnc23 on bovine skeletal muscle satellite cell proliferation and differentiation were further examined by Ed U cell proliferation assay and immunofluorescence staining.It was found that the down-regulated expression of lnc23 promoted cell proliferation and inhibited cell myogenic differentiation;and the cell myogenic differentiation was promoted,with no effect on cell proliferation when lnc23 was up-regulated.Suggesting that lnc23 had a positively regulated effect on myogenic differentiation of bovine skeletal muscle satellite cells.3.The TMT 10-plex labeling quantitative proteomics was performed to analyze the potentially regulatory proteins,which were from the lnc23 inhibited bovine skeletal muscle satellite cells and control cells.Under the standard of P≤0.05 and cut-off at 0.446,a total of312 differentially expressed proteins were screened.Bioinformatics analysis revealed that these differential proteins were significantly enriched into the key processes of myogenic differentiation,such as skeletal muscle development,actin cytoskeleton and cell fusion,suggesting that down regulation of lnc23 may inhibit myogenic differentiation by reducing signal transduction and cell fusion among cells,and further illustrating a positive regulation of lnc23 on myogenic differentiation.4.A total of 140 potential binding proteins of lnc23 were identified by RNA pulldown/LC-MS.Bioinformatics analysis demonstrated that these potential binding proteins were significantly enriched into Arp2/3 complex mediated actin nucleation and RNA binding biological process,indicating that the potential binding proteins of lnc23 may be involved in myogenic differentiation.Further,the RIP technology was performed to verify the binding of lnc23 to the candidate proteins.It was found that the enrichment of lnc23 in PFN1 immunoprecipitates was 31.4 times higher than the negative control Ig G,confirming that PFN1 was a binding protein of lnc23.5.The effect of PFN1 on myogenic differentiation was further detected by q RT-PCR and showed that down regulation of PFN1 could promote the expression of cell differentiation marker genes Myo G and My HC,indicating that PFN1 may have a negatively regulated effect on myogenic differentiation.It was preliminarily found that down regulation of PFN1 could promote the expression of its interaction proteins Rho A and Rac1.Meanwhile,lnc23 has been proved to be a positive regulator of the proteins expression of Rho A and Rac1.It was speculated that lnc23 may reduce the inhibited effect of PFN1 on Rho A and Rac1 by binding to PFN1,thereby promoting myogenic differentiation.In summary,we discovered a novel bovine muscle highly expressed lnc RNA–lnc23,which could promote the myogenic differentiation of bovine skeletal muscle satellite cells.Further study demonstrated that PFN1 was a binding protein of lnc23,and we initially speculated that lnc23 might regulate the myogenic differentiation via affecting the role of PFN1 on Rho A/Rac1.