The Effects Of MiR-7 And KLF4 Gene On Muscle Growth Development Of Jinghai Yellow Chicken

Muscle is the most important component in the carcass of livestock and poultry.Understanding the mechanism of regulating muscle growth and development is of great significance to livestock and poultry production.Muscle content is mainly determined by three factors:the number of muscle fibers,the diameter of muscle fibers,and the type of muscle fibers.Current studies have found that the number of muscle fibers in poultry is largely fixed during embryonic development.Primary myoblasts are a kind of precursor cells of muscle development,which play a key role in the growth and development of skeletal muscle in the embryonic stage,while non coding RNA is a key regulatory factor involved in the development and development of primary myoblasts.Our previous analysis by RNA-seq revealed that miR-7 was highly expressed during the growth medium(GM)of primary myoblasts and continued to decline during the differentiation medium(DM),while the expression trend of KLF4 and FSTL1 genes was exactly opposite to miR-7.They may play an important role in embryonic skeletal muscle development.This study focused on the relationship between miR-7 and KLF4 gene,and the role of miR-7 and KLF4 gene in the growth and development of chicken primary myoblasts(CPMs),The main results are as follows:1.The expression trend of miR-7 in embryonic pectoral muscle and leg muscle was consistent,and the expression level first decreased and then continued to increase,reaching the highest level at one-day-old.In one-day-old tissues,the expression level of miR-7 in lung and intestine was higher than that in pectoral muscle(P<0.05).The expression level of miR-7 increased first and then decreased in induced differentiation CPMs,and the expression level of miR-7 increased significantly from DM1 to DM3(P<0.05).CCK-8,flow cytometry,and Edu tests revealed that overexpression of miR-7 significantly reduced the proliferation efficiency of CPMs(P<0.01),increased the number of cells entering the G0G1 phase,and reduced the number of cells entering the S phase,and inhibited the proliferation of CPMs.Interference with miR-7 significantly increased the proliferation efficiency of CPMs(P<0.01),reduced the number of cells entering the G0G1 phase,increased the number of cells entering the S phase,and promoted the proliferation of CPMs.RT-PCR,western blot and indirect immunofluorescence tests showed that overexpression of miR-7 significantly decreased the expression level of differentiation marker genes MyHC,MyoG and MyoD1,up-regulated the expression of MSTN gene,inhibited the expression level of MyHC and MyoD1 protein,significantly reduced myotube area(P<0.01),and inhibited the differentiation of CPMs.While interference with miR-7 had the opposite effect To sum up,miR-7 expression can inhibit the growth and development of skeletal muscle fibers in embryonic stage,and play an important role in the process of muscle development,providing reference for the later study of muscle growth of Jinghai yellow chicken.2.The expression changes of apoptosis marker genes(RAF1,Cytc,Fas,Caspase 3,Caspase 8 and Caspase 9)after interference and overexpression miR-7 were detected by RT-PCR.The results showed that overexpression of miR-7 significantly increased the expression level of RAF1,Cytc,Caspa 3,Caspase 8 and Caspase 9(P<0.05).In contrast,interference with miR-7 significantly down-regulated the expression level the six apoptosis marker genes.Flow cytometry detection of CPMs apoptosis showed that overexpression of miR-7 could promote the occurrence of myoblast apoptosis,and interference with miR-7 could inhibit the occurrence of myoblast apoptosis.In summary,miR-7 could promote the apoptosis of CPMs.3.Through RNA-seq data analysis and prediction using miRanda software and miRBase database,two candidate target genes FSTL1 and KLF4 of miR-7 were found.The results of double luciferase test showed that there was no targeting relationship between miR-7 and FSTL1 gene(P>0.05),and there was a targeting relationship between miR-7 and KLF4 genes(P<0.01).RT-PCR results showed that there was a negative correlation between the expression trend of miR-7 and KLF4 gene in embryonic skeletal muscle Overexpression of miR-7 down-regulated the expression of KLF4 gene,and interference with miR-7 up-regulated the expression of KLF4 gene.In addition,in the rescue experiment,CCK-8 detection results showed that the transfection efficiency of the miR-7 mimic and pcDNA3.1-KLF4 group was highest.At the same time,RT-PCR was used to detect the expression of 6 apoptosis marker genes,and the results showed that miR-7 and KLF4 gene had a negative regulatory relationship.In summary,KLF4 gene is a target gene of miR-7,and there is a negative correlation between the two in the process of chicken myoblast proliferation and apoptosis.4.The expression trend of KLF4 gene in the chest and leg muscles of the embryonic phase was the same.The KLF4 expression level decreased first and then increased slightly,and the expression level was highest at E12.In one-old-day tissues,the expression of KLF4 gene in the chest and leg muscles was significantly higher than that in other tissues(P<0.05).Compared with the proliferation period(50%),the expression level of KLF4 gene increased significantly during the induced differentiation of CPMs.Interference with KLF4 gene significantly reduced the proliferation efficiency of CPMs(P<0.01),increased the number of cells entering the G0G1 phase,reduced the number of cells entering the S phase,inhibited the expression of differentiation marker genes MyHC,MyoG and MyoD1(P<0.05),increased the expression of MSTN gene(P<0.05),inhibited the expression of MYHC and MYOD1 proteins,significantly reduced the myotube area(P<0.01),and inhibited the proliferation and differentiation of CPMs.while overexpression of KLF4 gene had the opposite effects.In conclusion,KLF4 gene expression can promote the development of skeletal muscle fiber in chicken embryo,and it is an important regulatory factor in the process of muscle growth and development.