Study On The Co-regulation Of LncRNA H19 And MyoD During Goat Skeletal Muscle Satellite Cell Differentiation

Muscle growth and development has always been the focus of people's research,its progress contains many complex interlaced regulatory networks.Long noncoding RNA(lncRNA)can participate in biological processes such as muscle cell proliferation,differentiation and apoptosis at different levels of transcription through a variety of factors.LncRNA H19 interacts with MyoD to affect muscle production through the H19/Igf2 locus,however,the mechanism between H19 and MyoD remains unclear.In our laboratory,we used goat longissimus dorsi and skeletal muscle satellite cells(SMSCs)previously isolated as materials,amplified the full length of H19 gene firstly,and detected the expression pattern of H19 and MyoD in different periods or tissues of goat.Overexpression and inhibition assays were used to analyze the effect of the two genes on the differentiated SMSCs.We still carried out the dual luciferase reporter system,chromatin immunoprecipitation,fluorescence in situ hybridization and RNA stability tests to initially investigate the interaction between H19 and MyoD.The major results are as follows.(1)The goat H19 gene was successfully amplified by the rapid amplification of cDNA ends(RACE)for the first time which included two transcripts.We found that H19 was short of protein-coding potential by using online coding capability prediction software and there was a G-quadruplex structure near the transcription start site.(2)Expression patterns showed that the expression of H19 in the longissimus dorsi was remarkably higher than that in other tissues(P<0.01)and the expression level of H19 during the embryonic phase was significantly higher in the muscle tissues of different stages than in the postnatal period(P<0.01)and peaked at E 120.In both muscle tissues and SMSCs at different stages,the expression of H19 and MyoD increased first and then decreased,especially in the cell differentiation phase,the expression levels were significantly higher than those in the proliferative phase(P<0.01).(3)With the overexpression of H19,H19 and MyoD as well as the expression ofdifferentiation marker genes Myomaker,Myomerger and MyoG increased by 4.0,2.1,4.9,2.5,and 2.0 folds,respectively(P<0.01),while inhibiting the expression of H19,the expression levels of these genes were down-regulated by 2.5,5.0,6.2,2.9 and 1.9times,respectively(P<0.01).In addition,overexpression and inhibition of MyoD,H19 and MyoG were also up-regulated and decreased by 6.9,3.0-fold and 4.1,6.7-fold,respectively(P<0.01).Increased expression of H19 and MyoD promoted myogenic differentiation of SMSCs which showed a clear positive regulatory relationship.(4)The binding sites between H19 and MyoD were predicted using JASPAR and IntaRNA software,and dual luciferase reporter systems and chromatin immunoprecipitation experiments were performed based on the distribution of those binding sites.The results demonstrated that MyoD not only can be used as a transcription factor to bind to the H19 promoter region to enhance its activity,but also bind to the H19 exon region to increase its expression.(5)By fluorescence in situ hybridization experiments,we found that the positional relationship between H19 and MyoD,MyoG and MEF2 C at the RNA level was very close and mainly located in the cytoplasm.Further RNA stability assay showed that H19 can affect the expression of MyoD mRNA by changing its stability.In summary,the full length of goat H19 gene was amplified in this study,and it was initially verified that H19 and MyoD can regulate the differentiation of goat skeletal muscle cells both in transcriptional and post-transcriptional levels.