| Skeletal muscle development is a key determinant of growth rate and meat yield traits in pigs.As myogenic stem cells,porcine skeletal muscle satellite cell(PSC)are closely related to muscle growth and development,and therefore it is crucial to investigate the key factors that influence satellite cell behavior and fate transition.With the advancement of various sequencing and experimental techniques,long non-coding RNAs(lnc RNAs)have become one of the most important areas to be explored in the biological processes affecting skeletal muscle growth and development.In this study,we explored a specific and highly expressed lnc RNA-TCONS＿00323213 in porcine skeletal muscle tissue,and used PSC as the study material,and analyzed it by quantitative PCR(q PCR),RACE,RNA-seq,5-ethynyl-20-deoxyuridine(Ed U),protein immunoblotting(WB),immunofluorescence staining(IF),RNA pulldown assay,mass spectrometry,RNA immunoprecipitation assay and CUT & Tag experiments were performed to resolve the mechanism of action of TCONS＿00323213 in regulating the differentiation of PSC at the molecular and cellular levels,and the following results were obtained:1.TCONS＿00323213 is highly expressed in PSC differentiation and not have coding abilityIn this study,the expression pattern of TCONS＿00323213 was detected in porcine satellite cells and its molecular characterization was performed.The q PCR results showed that the expression of TCONS＿00323213 in PSC was very low in proliferative phase and high in differentiation phase,and the expression was very significantly higher than that in proliferative phase;the full-length sequence of TCONS＿00323213 was obtained at 7321 bp in 5′/3′ RACE experiment;the subcellular localization experiment found that TCONS＿00323213 was mainly localized in The subcellular localization assay revealed that TCONS＿00323213 was mainly localized in the nucleus,with a small amount expressed in the cytoplasm;in vitro translation assay determined that TCONS＿00323213did not have the ability to translate the protein.2.TCONS＿00323213 inhibits the proliferation and promotes the differentiation of PSCIn this study,the effects of TCONS＿00323213 on cell proliferation were examined by CCK-8 and Ed U assays,which showed that knockdown of TCONS＿00323213significantly enhanced satellite cell proliferation and increased DNA replication activity,while overexpression of TCONS＿00323213 diminished satellite cell proliferation and decreased DNA replication activity.The effects of TCONS＿00323213 on the expression of Myo G,MEF2 C and My HC,marker genes of myogenic differentiation,were examined by q PCR,WB and IF techniques during the period of PSC differentiation,and the results showed that knockdown of TCONS＿00323213 significantly down-regulated the expression of myogenic differentiation marker genes and reduced the number of Myo G and My HC expression-positive myotubes.This inhibited satellite cell differentiation and myotube formation,while overexpression of TCONS＿00323213 significantly upregulated myogenic differentiation marker gene expression and promoted satellite cell differentiation and myotube formation.3.Transcriptomic analysis reveals that TCONS＿00323213 regulates PSC differentiationIn this study,RNA-seq and data analysis were performed on PSC induced to differentiate at 24 h and 36 h after knockdown of TCONS＿00323213,and a total of 4,298 and 3,597 genes with significant expression differences were obtained,respectively.GO and KEGG functional enrichment showed that the differential genes were mainly involved in biological processes such as cellular life activities,biological regulation and metabolism The enrichment of GO and KEGG functions showed that differential genes were significantly enriched mainly in cellular differentiation,immune response,protein ubiquitination,apoptosis,cell cycle and asymmetric division processes.This suggests that TCONS＿00323213 plays an important regulatory role in the differentiation of PSC.4.TCONS＿00323213 promotes the differentiation of PSC by directly interacting with PKNOX2In this study,the protein PKNOX2 bound to TCONS＿00323213 was identified by biotin-labeled RNA pulldown assay,and the binding of PKNOX2 to TCONS＿00323213was confirmed by RNA RIP assay in reverse;the study of the effect of PKNOX2 on satellite cell differentiation showed that knockdown of PKNOX2 promoted satellite differentiation and myotube formation,while overexpression of PKNOX2 showed the opposite result.In addition,we found that knockdown of TCONS＿00323213 significantly increased the enrichment of PKNOX2 on Myo G promoter but significantly downregulated the promoter activity and significantly inhibited satellite cell differentiation by CUT & Tag,dual luciferase assay,q PCR,WB and IF experiments.In this study,we found that TCONS＿00323213 interacted with PKNOX2 and reduced the enrichment of PKNOX2 on Myo G promoter,which promoted the transcription and protein expression levels of Myo G,and thus promoted satellite cell differentiation and myotube formation.5.SP1 promotes PSC differentiation by activating the transcription of TCONS＿00323213In this study,the core promoter sequence of TCONS＿00323213 was localized at-513 bp to-970 bp upstream of the transcription start site(TSS),as verified by dual luciferase assay.The results showed that there were three SP1 binding sites(GC-box)on the core promoter sequence of TCONS＿00323213.To determine the enrichment of SP1 in the core promoter region of TCONS＿00323213,this study revealed a significant enrichment of SP1 in the TCONS＿00323213-CP region by Ch IP,DNA pulldown and dual luciferase reporter analysis;and we found that knockdown of SP1 diminished the TCONS＿00323213-CP region activity,repressed the transcription of TCONS＿00323213gene and inhibitd Myo G expression and myotubular formation;overexpression of SP1 resulted in the opposite.In this study,the regulatory role of TCONS＿00323213 in inhibiting the proliferation and promoting the differentiation of PSC was elucidated through a series of experiments,and it is proposed that TCONS＿00323213 is involved in the regulation of satellite cell differentiation through two molecular mechanisms,which provides a theoretical basis for the study of porcine skeletal muscle development and porcine molecular breeding. |
