Function And Mechanism Of H19 Gene In The Differentiation Of Porcine Skeletal Muscle Satellite Cells

The growth rate and quality of pig muscle are important traits in pig genetics and breeding,and these traits mainly depends on the growth and development of skeletal muscle.Porcine skeletal muscle satellite cells(PSCs)can rapidly proliferate,differentiate and fuse to form muscle fibers after activation as skeletal muscle stem cells.Long noncoding RNA(lnc RNA)is a type of RNA with transcriptional length greater than 200 nt,which can play its role in many biological processes through different regulatory mechanisms,including the growth and development of skeletal muscle.H19,as an important member of lnc RNA,its function and regulatory mechanism in the growth and development of pig skeletal muscle and PSCs differentiation have not been reported.In this study,PSCs were used as research materials to explore the role of H19 in the differentiation process of PSCs.Moreover,we also explored its regulatory mechanism at the cellular and molecular levels.The molecular model of H19 in regulating the differentiation of PSCs was preliminarily constructed.The following results were obtained:1.We successfully isolated PSCs,and the PSCs were detected by Paired box 7(PAX7)immunofluorescence assay.Meanwhile,the expression levels of myogenin(MYOG),myogenic differentiation(MYOD)and myosin heavy chain(MYHC)were detected in PSCs at different proliferation and differentiation time points.The results showed that the cell purity reached more than 94% after separation and differential adherence,and the marker genes expression level of the PSCs was consistent with the reports.2.QPCR was used to detect the expression level of H19 in PSCs at different proliferation and differentiation points and different tissues.The results showed that H19 was highly expressed in skeletal muscles and continued to increase during PSCs differentiation periods.RACE assay was used to identify the full length of H19 and showed that H19 was an lnc RNA with 2280 nucleotides,a poly(A)tail.The subcellular localization of H19 was identified by nuclear-cytoplasmic RNA fractionation and FISH assay.The results showed that H19 expressed in both the nucleus and cytoplasm.3.H19 siRNA were transfected into PSCs to detect the effect of H19 on the differentiation of PSCs.The results showed that transfection of H19 siRNA significantly inhibited the expression level of differentiation marker genes MYOG,MYOD and MYHC.Meanwhile,the results of MYHC immunofluorescence experiment showed that H19 siRNA significantly inhibited the formation of myotubes and resulted in fewer and thinner myotubes.4.RNA-seq and miRNA-seq sequencing were performed at PSCs induced to differentiate 24 h and 36 h after the knockdown of H19.Then,functional enrichment was performed on the differentially expressed genes.It was found that most of the differentially expressed genes were enriched in muscle-related pathways.Moreover,m RNAs and miRNAs related to H19 and PSCs differentiation were screened as research objects in our subsequent experiments.5.miR-140-5p and its target gene SRY-box transcription factor 4(SOX4)were screened through RNA-seq,miRNA-seq data,and online sites prediction.The functional gain-and loss-experiments showed that miR-140-5p inhibited PSCs differentiation,and SOX4 had the opposite effect.The dual-fluorescence reporter system and miR-140-5p IP assay also confirmed the direct binding among H19,miR-140-5p and SOX4.This study found the molecular regulation model among H19,miR-140-5p and SOX4: H19 can act as a molecular sponge of miR-140-5p,and competitively binds miR-140-5p to remove the inhibitory effect of miR-140-5p on SOX4,thus promoting the differentiation of PSCs.6.The RNA pulldown assay was performed to detect the binding protein of H19,and Transactive response DNA-binding protein 43(TDP43)protein was selected as our research object.The RIP and co-localization experiments were used to verify the binding between H19 and TDP43.Functional studies of TDP43 showed that TDP43 could promote the differentiation of PSCs.The Ch IP results of TDP43 and dual-fluorescence experiments of MYOD's promoter showed that TDP43 was significantly enrichment in MYOD's promoter and increased its activity.In this study,it was found that H19 directly binds to TDP43 and recruits TDP43 to MYOD's promoter to increase the promoter activity,thereby promoting the transcription of MYOD and the differentiation of PSCs.7.Pulldown and RIP experiments showed that H19 can combine with drebrin1(DBN1)protein in cytoplasm.Functional studies showed DBN1 also could promote the differentiation of PSCs.The Ch IP assay results of MYOD showed that MYOD binds to the promoter of H19 and DBN1 to regulate the expression of H19 and DBN1.Then H19 and DBN1 were combined to affect the expression of MYOD.We hypothesized that there might be a feedback loop exists in H19,DBN1 and MYOD,thus synergistically promoting the differentiation of PSCs.In summary,this study explored the function of H19 in promoting the differentiation of PSCs,and established a molecular model of H19 regulating the differentiation of PSCs,providing a new genetic material of skeletal muscle growth and development.