Mechanism Of MiR-696 And MiR-22 In Regulating Skeletal Muscle Cell Proliferation And Differentiation

MicroRNAs(MiRNAs)are a kind of small noncoding RNAs(-22 nt in length),acting by inducing gene silencing or degradation in animals and plants.A series of studies have shown that miRNA is involved in various developmental and physiological processes,including cell growth and apoptosis,hematopoiesis,virus defense,tumorigenesis,fat metabolism,and so on.Similarly,in skeletal myogenesis,extensive miRNAs have also been confirmed as important regulators.Since the biological process of muscle development is complicated,whieh often needs multiple miRNAs to act together,still requires future studies to investigate individual roles of other vital miRNAs in skeletal myogenesis.miR-696 was previously known as an exercise related miRNA,which could have a function of mitochondrial biogenesis and fatty acid oxidation in skeletal muscle.However,its role in regulating skeletal muscle development is still not clear.While,miR-22 was found to be differentially expressed in different growth stages or different breeds of pigs in longissimus dorsi.However,the mechanism of miR-22 which affect myoblast proliferation or differentiation is also not yet reported.To this end,our laboratory chose miR-696 and miR-22 for the further study.In this study,we first detected the expression of miR-696 and miR-22 in different tissues and skeletal muscle.Then by the using the mouse C2C12 myoblasts model to investigate the role of miR-696 and miR-22 in the proliferation and differentiation of skeletal myoblasts and further analysis the important genes or signaling pathways which regulated by miR-696 and miR-22.The type of muscle fiber is an important factor which affect the meat color.And,in view of the important role of miR-22 which we found to play in the proliferation and differentiation of myoblasts and its differential expression in different types of muscle(red muscle and white muscle),we analyzed the gene sequence of miR-22 and identified the molecular markers of which could affect the variation of meat color at the genome level in Su Huai pigs.The main results are stated as follows:1.The expression patterns of miR-696 in different tissues and different types of muscles in mouseRT-PCR results showed that miR-696 expressed the most in fat and had a higher expression in skeletal muscle,kidney and liver.According to the expression level of miR-696,next follows the order of stomach,heart,lung and spleen.In addition,the expression of miR-696 in the soleus(SOL,red muscle)was significantly lower than that in the extensor digitorum(EDL,white muscle).2.The mechanism of miR-696 in regulating skeletal muscle cell proliferation and differentiationRT-PCR results showed that the expression level of miR-696 in C2C12 cells decreased from 2 days(2 days)to 6 days(6 days).Overexpression of miR-696 reduced the proportion of EdU-positive cells.And significantly blocked cells at G0/G1 phase,resulting in a decrease in the S and G2 phases and the proliferation index.At the same time,it can inhibit the expression of cell cycle related genes:Cyclin D1,Cyclin E and Cdk4.In the process of myoblast differentiation,overexpression of miR-696 significantly inhibited the expression of MyHC and MyoG,which resulted in a decrease of myotube number.On the opposite,the inhibition of miR-696 could promote myoblast proliferation and differentiation.In the C2C12 cell model,the expression of miR-696 and CNTFRa was opposite.miR-696 could specifically target CNTFRa and inhibit its expression.Knockdown of CNTFRa suppressed the proliferation and differentiation of C2C12 cells.Therfore,miR-696 inhibits C2C12 cell proliferation and differentiation by targeting CNTFRa.3.The expression pattern of miR-22 in different tissues and different types of muscle in swine and mouseRT-PCR results shoxved that miR-22 expressed the most in the longissimus dorsi muscle and fat of pigs,followed by the soleus muscle,then highly expressed in heart,liver,stomach,kidney,spleen and lung.In addition,the expression of miR-22 was higher in the muscles which contained higher proportion of fermentation muscle fibers,like longissimus dorsi and biceps femoris muscle.And had a lower expression in the soleus muscle,psoas muscle,and masseter muscle,which had a higher proportion of oxidized.In mice,miR-22 also expressed the most in skeletal muscle and fat,followed by lung,heart,spleen,stomach,kidney and liver.In the soleus muscle(SOL,red muscle),the expression level of miR-22 was significantly lower than that in the extensor digitorum(EDL,white muscle).4.The mechanism of miR-22 in regulating skeletal muscle cell proliferation and differentiationThere was no significant difference in the expression of miR-22 during C2C12 cell proliferation.In the cell differentiation stage,the miR-22 expression level gradually increased from day 2 to day 6.Overexpression miR-22 significantly reduced the proportion of EdU-positive cells and blocked cells in GO/G1 phase,accompanied by a significant reduction in proliferation index.Overexpression miR-22 also significantly inhibited the expression of cyclin D1 and Cdk4 in cell cycle.In contrast,inhibition of miR-22 significantly increased EdU positive cells and cells in S phase.The expression of cell cycle-related genes also increased significantly.In the cell differentiation stage,overexpression of miR-22 significantly promoted the expression of MyHC and MyoG and increased the number of myotubes.While inhibition of miR-22 expression showed the opposite results.Through target gene prediction and dual-luciferase report gene system,we found that TGFBRI was the direct target gene of miR-22.In the process of cell proliferation and differentiation,overexpression of miR-22 could inhibit the expression of TGFBR1 and smad3 signaling pathway.But when the endogenous miR-22 was inhibited,the expression level of TGFBR1 and smad3 signaling pathway significantly increased.In addition,by transfection of TGFBR1 siRNA or TGFBR1 pcDNA 3.1,to inhibit or promote its expression,revealed that TGFBR1 could promote myoblast proliferation and inhibit differentiation,which showed the same results as miR-22.Treated C2C12 cells with different concentration of TGF-β1(0,10,20 ng/ml)significantly inhibited the expression of miR-22.MiR-22 was able to inhibit the expression of TGFBR1 protein in C2C12 cells when treated with TGF-pl.In conclusion,miR-22 inhibits myoblast proliferation and promotes differentiation by repressing the expression of TGFBR1.At the same time,TGF-β1 inhibits miR-22 expression in C2C12 cells.5.The SNP in the ssc-miR-22 gene of Su Huai pigsEight individuals were randomly selected from 300 Suhuai pigs.By using specific primers,688 bp long fragments containing ssc-miR-22 precursor sequences were amplified.After sequencing,it was found that there were multiple mutations,and one G/A mutation site which closed to the miR-22 precursor sequence was selected.Then,we genotyped the 300 Suhuai pigs.According to the association analysis of meat color brightness value(L value),red value(a value)and yellow value(b value)at 2 hours and 24 hours after slaughter in Suhuai pig,we found the G/A mutation site was significantly associated with the meat colour redness(a value)at 2 hours after slaughter in Suhuai pigs(P=0.0008).Quantitative analysis shows that,this site mutation can affect the expression of miR-22 in skeletal muscle.In AA genotype,the expression of miR-22 was the lowest.And it was significantly lower than that of GG and GA genotype.However,there was no significant difference between GG genotype and GA genotype.As stated above,here we draw the following conclusions:1.miR-696 expressed in different tissues and highly expressed in fat and skeletal muscle.In different types of muscle(red muscle and white muscle),the expression of miR-696 was different.2.miR-696 could inhibit myoblast proliferation and differentiation by targeting the expression of CNTFRa.3.miR-22 expressed in different tissues and highly expressed in skeletal muscle and fat.In different types of muscle(red muscle and white muscle),the expression of miR-696 was different.4.miR-22 could repress the expression of TGFBR1,thereby inhibiting myoblast proliferation and promoting differentiation.TGF-β1 could inhibit the expression of miR-22,thus taking part in an important negative feedback regulation network.5.The G/A mutation site on ssc-miR-22 gene was significantly associated with the meat color traits of Su Huai pigs,and it could be used as a molecular marker for meat color selection of Su Huai pigs.Meanwhile,miR-22 might play an important role in the regulation of meat color.