Mechanisms Of LncRNA-MEG3 In Regulating The Differentiation Of Porcine Satellite Cells

The number of skeletal muscle fibers has been determined in most mammals after birth,and the increase of muscle mass is largely dependent on the growth potential of skeletal muscle fibers after birth.Satellite cells,a kind of myogenic progenitor cells,have the characteristics of stem cells and form skeletal muscle fibers through proliferation and differentiation after activation,which is of great significance for the development of skeletal muscle and the repair of injury.The development of skeletal muscle is regulated by a variety of functional molecules,such as MRFs,MEF2 family,IGFs,SRF,and non-coding RNA.In recent years,the research of long non-coding RNA(lnc RNA)as a functional regulatory molecule involved in the differentiation and development of porcine skeletal muscle has attracted much attention.At present,thousands of lnc RNAs have been excavated,but their regulatory functions still need to be further explored.Among them,some single nucleotide polymorphisms of long non-coding RNA MEG3(maternally expressed gene 3)have been confirmed to be correlated with backfat thickness in pigs(Yu et al.2018),however,the function and molecular mechanism of MEG3 gene regulating skeletal muscle differentiation and development in pigs is still unclear.In this study,5' and 3' RACE,nuclear and cytoplasmic RNA fractionation,western blot,flow cytometry,RIP,biotin-labeled mi RNA pull-down,Ch IP,dual luciferase report analysis and other techniques were used to explain the molecular mechanism of MEG3 in regulating the differentiation of porcine satellite cells.The main findings are as follows:1.The MEG3 core transcript was regularly expressed during the proliferation and differentiation of porcine satellite cellsIn this study,the basic characteristics of porcine MEG3 were identified using 5?and 3? rapid amplification of c DNA ends assays,tissue expression profiling and nuclear and cytoplasmic RNA fractionation.We identified two polyadenylated MEG3 transcripts: MEG3 variant 1(MEG3 v1),1430 nt in length,and MEG3 variant 2(MEG3 v2),1380 nt in length.Notably,MEG3 v2 was the core transcript in porcine satellite cells and was highly expressed in porcine muscle tissue.Furthermore,it was upregulated in the proliferation and early stage of differentiation in porcine satellite cells,but gradually decreased in the later stage of differentiation.The nuclear and cytoplasmic RNA fractionation experiments demonstrated that MEG3 was mainly located in the nuclear compartment of proliferating myoblasts(77%)and differentiated myotubes(60%).Interestingly,its proportion in the cytoplasm had increased in myotubes with a range from 23% to 40%.2.MEG3 inhibits myoblast proliferation and promotes myoblast dierentiation in porcine satellite cellsCCK-8,Ed U and flow cytometry assay were used to validate the function of MEG3 during the stage of porcine satellite cells proliferation.The results showed a considerable reduction of cell quantity in the G0/G1 phase,a remarkable increase of cell quantity in the S phase and cell proliferation and DNA replication activities after MEG3 knockdown.The propidium iodide flow cytometry assays indicated a considerable reduction of cell quantity in the G0/G1 phase and a remarkable increase of cell quantity in the S phase after MEG3 knockdown.In contrast,the results of overexpression of MEG3 showed an opposite effect and the proliferation process of porcine satellite cells was inhibited.During the differentiation stage of porcine satellite cells,we detected the expression regulation of Myo D,Myo G and My HC with q RT-PCR,western blot,and immunofluorescence staining.The results showed that both the expression of myogenic regulatory factors and number of Myo G and My HC positive myotubes was down-regulated after MEG3 knockdown,which indicated that the differentiation process of porcine satellite cells was inhibited.However,MEG3 overexpression resulted in the opposite effects.3.Transcriptome analysis suggests that MEG3 could regulate skeletal muscle myogenesis in pigsWe performed the transcriptome sequencing analysis and respectively identified 273 and 207 differentially expressed protein-coding genes from MEG3 knockdown for the 30 h and 40 h groups.The differentially expressed genes were performed gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.The results showed that differentially expressed genes were enriched in the biological processes related to structural constituent of muscle,skeletal muscle contraction and skeletal muscle cell differentiation.KEGG pathway analysis showed these differentially expressed genes were mainly involved in PI3K-Akt signaling pathway,focal adhesion,MAPK signaling pathway and so on.Overall,these results further indicated that MEG3 has important regulatory functions in porcine myogenesis at the transcriptional level.4.MEG3 promotes differentiation of porcine satellite cells by sponging mi R-423-5p to regulate the expression of SRFThe complementary seed regions of mi R-423-5p with MEG3 core transcript and another potential target gene(i.e.,SRF)of mi R-423-5p,a crucial transcription factor for myogenesis process,were obtained using Target Scan and RNAhybrid software.With the knockdown or overexpression of MEG3,SRF showed the same expression trend at m RNA and protein levels,but the expression of mi R-423-5p showed the opposite trend.The RIP analysis results indicated that Ago2 protein could bind to MEG3,suggesting that MEG3 has the ability to competitively bind to mi RNA.Biotin-labeled mi R-423-5p pull-down analysis showed that mi R-423-5p could significantly capture MEG3 and SRF,but the mutation type mi R-423-5p with mutational binding site lost this ability.Further analysis of dual luciferase reports showed that mi R-423-5p has a competitive binding relationship with MEG3,which could attenuate the decrease of luciferase activity of SRF induced by mi R-423-5p in a dose-dependent manner.Afterwards,the results of transfection with overexpression plasmid and RNAi of SRF verified the promoting role of SRF for porcine satellite cell differentiation.In addition,Western blot and immunofluorescence results showed that overexpression of MEG3 can reduce the inhibitory effect of mi R-423-5p on SRF protein expression and the number reduction of Myo G and My HC positive myotubes.Overall,MEG3,as a competing endogenous RNA(ce RNA),can promote the differentiation of porcine satellite cells by sponging mi R-423-5p to relieve the inhibiting effect on SRF.5.SRF promotes differentiation of porcine satellite cells by regulating MEG3 transcriptionWe analyzed and identified the MEG3 core promoter region(MEG3-CP)(the region from-558 bp to-226 bp upstream of the transcription start site)using dual luciferase reporter assay.Subsequently,transcription factor binding in MEG3 core promoter region was predicted online through Animal TFDB 3.0 and Jaspar.The results showed that there were two identical conservative binding sites of SRF(CCCAGC),which were respectively located in the region from-365 bp to-360 bp and from-33 bp to-28 bp upstream of the transcription start site.To seek the direct evidence of the interaction between SRF and MEG3 core promoter region,we performed Ch IP and dual luciferase report assay.Ch IP-PCR results showed that SRF was significantly enriched in this region.Dual luciferase reporter assay was performed after constructing the mutant binding sites vectors of MEG3 core promoter(MEG3-CP-mutant).The luciferase activity of MEG3-CP showed decrease or increase with the interference or overexpression of SRF,whereas,that of MEG3-CP-mutant was not affected by SRF expression.In combination with the research results of SRF promoting porcine satellite cell differentiation,we infer that SRF may regulate MEG3 transcription by binding to MEG3 core promoter,thus promoting the differentiation of porcine satellite cells.This study clarified the important regulatory role of MEG3 in inhibiting proliferation and promoting differentiation during the development of porcine satellite cells.And our research initially established two molecular mechanisms of MEG3 regulating satellite cell differentiation,which provides a theory basis for the study of porcine skeletal muscle development and molecular breeding.