The Role And Molecular Mechanism Of Long Noncoding RNA LncMGPF In The Regulation Of Skeletal Muscle Growth And Development

Skeletal muscle is the largest tissue of animal organisms.The muscle growth rate and quantity of muscle is one of the important traits characteristics in animal production.The process of skeletal muscle growth and development is very complex,which is regulated by lots of transcription factors and epigenetic regulatory.Thus,analyzing the regulatory network of muscle growth and development has important theoretical and practical value.Long noncoding RNAs(lnc RNAs)is defined sequences as more than200 nt and with no protein-coding capacity,and play an important role in many biological processes.Although a large number of lnc RNAs have been identified in muscle,most of their roles in myogenesis are still unclear,and more lnc RNAs need to be identified.We analysed and identified a promoting factor of muscle growth and regeneration lnc RNA lncMGPF(lnc RNA muscle growth promoting factor).In present study,we investigated the role and molecular mechanism of lnc RNA lncMGPF in the regulation of skeletal muscle growth and conservation analysis of lncMGPF in mouse,pig and human genome.The results are as follows:1.qRT-PCR results showed lncMGPF was highly expressed in muscle tissue,including leg muscle,longissimus dorsi and heart.lncMGPF was significantly increased during C2C12 cell differentiation.Promoter activity assay and Ch IP-q RT-PCR results found that Myo D could specifically bind to the E-box between-345 bp and +200bp of lncMGPF,suggesting that Myo D could regulate the expression of lncMGPF.2.In order to study the function of lncMGPF in muscle development in vivo,we performed CRISPR-Cas9 to generate lncMGPF knockout mice.The founder heterozygous mice were randomly hybridized to get lncMGPF homozygous knockout mice(lncMGPF KO mice).lncMGPF KO mice were significant decreases in weight and growth rate compared with WT mice.Likewise,the weights of the whole leg,gastrocnemius(Gas),tibialis anterior(TA),quadriceps(Qu)muscles and cross?sectional areas of individual myofibres of KO mice were significantly lower than those of WT mice.And found that lncMGPF KO significantly decreased primary myoblasts differentiation.We used CTX-induced TA muscles injury with WT and KO mice and found that lncMGPF promotes muscle regeneration.Finally,we injected the lentivirus-mediated overexpression of lncMGPF(LV-lncMGPF)vector and empty control(LV-control)vector intramuscularly into the left and right legs of mice.We found that overexpression of lncMGPF can promote muscle growth of WT mice.3.We using bioinformatics prediction and luciferase activity assay found that the targeting of five mi RNAs by lncMGPF.lncMGPF knockdown and overexpression significantly decreased and increased expression of MEF2C(the target gene of mi R?135a-5p),respectively,but no significant changes were observed in the other target genes.Transfection of mi R?135a?5p suppressed the luciferase activity of Luc?lncMGPF.We performed cotransfection experiments using lncMGPF and mi R?135a?5p overexpression vectors in C2C12 myoblasts and WT myogenic progenitor cells.Overexpression of mi R?135a?5p decreased m RNA and protein expression of MEF2 C,Myo D,Myo G and My HC,whereas cotransfection of lncMGPF eliminated mi R?135a?5p activity.4.We used RNA pulldown assay,mass spectrometry and RIP to identify lncMGPF?specific binding proteins,and found Hu R interacted with lncMGPF.To identify the Hu R binding region of lncMGPF,we constructed three truncated fragments of lncMGPF and used performed RNA pulldown experiments.The results showed that fragment 1630–1795 bp was the core region involved in the interaction between lncMGPF and Hu R.We performed Hu R RIP assays in C2C12 cells after overexpression or knockdown of lncMGPF.Overexpression and knockdown of lncMGPF significantly increased and decreased the capacity of Hu R protein to bind to Myo D and Myo G m RNAs,respectively.We performed cotransfection experiments with the lncMGPF overexpression vector and Hu R si RNAs in C2C12 myoblasts to explore the effects of Hu R on lncMGPF?mediated m RNA stabilization.These results suggest that lncMGPF regulation of target m RNA stability depends on Hu R protein.5.Conservation analysis of lncMGPF in pig genome,we found that pig lnc RNA AK394747 has conserved genomic position with mouse lncMGPF.We performed RACE assays to obtain the full-length c DNA sequence of AK394747.AK394747 was highly expressed in muscle tissue,and the expression of AK394747 increased during myogenic differentiation.Cell fractionation assays demonstrated that AK394747 mainly in the nuclei of proliferating cells and in the cytoplasm of differentiated cells.Results of q RT?PCR,western blotting and immunofluorescence staining for differentiated myogenic progenitor cells showed that lentivirus?mediated AK394747 knockdown(LV?sh?AK394747)inhibited myogenic progenitor cells differentiation and fusion,whereas lentivirus?mediated overexpression of AK394747(LV-AK394747)promoted myogenic progenitor cells differentiation.Consistent with the functions of lncMGPF in the mouse,AK394747 overexpression significantly promoted muscle growth in mice.At last,AK394747 has conserved genomic position with mouse lncMGPF and contains potential mi R?135a?5p binding site and Hu R protein binding motifs.Luciferase activity assay and cotransfection experiments found that the AK394747 target with mi R?135a?5p and increased expression of MEF2 C.RNA pull down assay and RNA stability analyses suggest that AK394747 regulation of target m RNA stability depends on Hu R protein.6.Conservation analysis of lncMGPF in human genome,we found that human lnc RNA MT510647(hlncMGPF)has conserved genomic position with mouse lncMGPF.We performed RACE assays to obtain the full-length c DNA sequence of hlncMGPF.Overexpression of hlncMGPF significantly increased the expression of Myo D,Myo G,My HC,MEF2 C,and ??actin and promoted human skeletal muscle myoblast differentiation.Consistent with the functions of lncMGPF in the mouse and pig,hlncMGPF overexpression significantly promoted muscle growth in mice.At last,hlncMGPF has conserved genomic position with mouse lncMGPF and contains potential mi R?135a?5p binding site and Hu R protein binding motifs.In conclusion,we have found that lncMGPF is a novel positive regulator of myogenesis in mice and pigs that regulates muscle differentiation at the post?transcriptional level.The Myo D?activated lnc RNA lncMGPF is transferred from the nucleus to the cytoplasm during cell differentiation and promotes myogenesis mainly through post?transcriptional regulation.lncMGPF functions as a mi RNA sponge of mi R?135a?5p,weakening the inhibitory effects of mi R?135a?5p on MEF2 C and thereby increasing expression of the MEF2 C gene.Meanwhile,lncMGPF increases Hu R accumulation in the cytoplasm and enhances stability of Myo D and Myo G m RNAs via recruitment of Hu R to AREs in the 3? UTRs of Myo D and Myo G m RNAs.The lncMGPF/mi R?135a?5p/MEF2 C and lncMGPF/Hu R/Myo D/Myo G pathways together constitute a novel MRF?mediated regulatory network for myogenesis.