| In his study, we aim to investigate the effect of mi RNAs on myoblast differentiation, and explore the molecular mechanism of mi RNAs on regulating myoblast differentiation and the formation of skeletal muscle.In addition,we also study the relation between the myoblast differentiation and its apoptosis.According to the previous studying on mi R-449 a, we found that mi R-449 a promote cell differentiation and apoptosis through inhibit some genes which regulate cell proliferation. There is a little research about the function of mi R-449 a on myoblast differentiation and apoptosis. However, study have shown that mi R-449 a is decreased in the mdx mice,but they didn’t do further research on it. So, here we emphatically study on the effect of the mi R-449 a on C2C12 myoblast differentiation and apoptosis. First of all,we use the biological web site for getting the mature sequence of mi R-449 a, and then synthesis of mi R-449 a mimics and mi R-449 a inhibitor。We respectively transfect mi R-449 a mimics and mi R-449 a inhibitor into C2C12 myoblast, so it can increase the number of mi R-449 a or reduce cell endogenous mi R-449 a.Finally, we collected these cells for using QPCR and Western blotting to detect the effect of mi R-449 a on myoblast differentiation marker genes. The results of the study as follows:1.The analysis of mi R-449 a by bioinformaticsWe found that the mi R-449 a of human and the mi R-449 a of mouse are the same, suggesting that mi R-449 a is conservative between mouse and human., we select several potential target genes of mi R-449 a,through the target gene prediction database of mi RNAs. They are Hdac1, Cdc25 a, Bcl2 and Cdk6 genes which exist the sequence for combining mi R-449 a.2. The relative expression of mi R-449 a and related genes during C2C12 myoblast differentiationWe collect the C2C12 myoblast which differentate zero, the second, the third and the fifth day, then we detected the relative expression of mi R-449 a by QPCR. We found that the relative expression of mi R-449 a upregulate in the process of differentiation.There is a significant difference between zero day with the second the third and fifth day. Next, we also chected the relative expression of the five gene which are Cdk6, Cdc25 a, Hdac1, Bcl2 and E2F1, and the relative expression of these genes which including Cdk6, Cdc25 a,Hdac1 and E2F1,are decreased in C2C12 myoblast differentiation,but the difference of the relative expression of Bcl2 gene is not obvious.3. The effects of transfecting miR-449 a mimics on C2C12 myoblast differentiationthe vector and mi-449 a mimics in cell together, and detectded the relative activity of fluorescence.we found the relative activity of Hdac1 3 ’UTR fluorescence is similar with the relative activity mutant.When we transfected mi R-449 a mimics into C2C12 myoblast, we found that the relative expression of Hdac1 m RNA and its protein have no obvious change.This suggest that Hdac1 gene were not targeted by mi R-449 a in mice.Here we also detected the predicted target genes of mi R-449 a which named Cdc25 a and Cdk6 genes. we found that the relative expression of Cdc25 a and Cdk6 m RNA are decreased significantly(p<0.01), this suggest that mi R-449 a can effectively inhibit the expression of Cdc25 a and Cdk6 genes m RNA.In addition, we chected the transcriptional factor E2F1 gene which control cell proliferation by QPCR.the results of the QPCR show that the m RNA level oftranscriptional factor E2F1 gene also was decreased significantlyt(P < 0.05). This means that mi R-449 a can inhibit the transcriptional factor E2F1 gene through target Cdk6 gene and Cdc25 a gene.We detected the Bcl2 gene which also is the predict gene of mi R-449 a from QPCR detection and western blotting detection. The results showed that the relative expression of Bcl2 m RNA decreased significantly(P < 0.05), the protein expression of Bcl2 genes is also reduced obvious. This means that mi R-449 a can inhibit the exepression of Bcl2 genes.Here we have completed the detection of the muscle differentiation marker gene,which include MHC, Myog, Myod and Myomaker. first of all, we checked the protein of MHC(myosion heavy chain) gene by western blotting, and we found that the exepression of MHC protein is increased in the C2C12 myoblast which were transfected with mi R-449 a mimics.Interesting,we noticed that the protein of Myog gene protein with a little changed during our western blotting. At the same time, we also detected the m RNA exepression of Myod gene and Myomaker gene, found that the two genes m RNA levels have little change in transfection with mi R-449 a mimics. The results show that mi R-449 a can promote the differentiation of C2C12 myoblast are not depented on Myod gene,Myomake and Myog.There must be other ways to regulate the differentiation of C2C12 myoblast by mi R-449 a.In order to further study on how mi R-449 a affects C2C12 myoblast differentiation, we detected the other two genes which can regulate C2C12 myoblas differentiation,and called Caspase-3 and P21 genes by western blotting, we found that transfection mi R-449 a mimics can promote the expression of Caspase-3 and p21 protein.4. The effects of transfecting mi R-449 a inhibitor on C2C12 myoblast differentiationWe detected Hdac1, Cdc25 a, Bcl2 and Cdk6 genes which are the forecast target gene of mi R-449 a,with transfecting mi R-449 a inhibitor in into C2C12 myoblast, and we also detected Myomaker gene, which is the maker of C2C12 myoblast differentiation. We found that he relative expression of Cdc25 a gene and Cdk6 m RNA decreased obviously(P < 0.05), but the relative expression of Hdac1 gene and Myomaker are not abvious. Which are similar with the result of trensfenting mi R-449 a mimics. it is further illustrate that mi R-449 a likely control the process of C2C12 myoblast differentiation by target Cdc25 a gene and Cdk6. |
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