Biological Roles And Molecular Mechanisms Of MiR-1 And MiR-206 In Myogenesis

How to repair tissue injury effectively has been considered a hot and difficult issue for a long time. In recent years, cell replacement therapy or gene therapy with adult stem cells(ASCs) has been studied in the treatment of tissue injury. Whether therapy strategies based on ASCs could obtain safe and effective purpose, to some degree, depends on our understanding of the molecular mechanisms that regulate proliferation and differentiation. Regulation of gene expression is the central theme of cell differentiation. Establishing a new cellular phenotype requires suppression of features associated with the prior phenotype or other related phenotypes, which needs switching on and off certain genes. So the results of cell differentiation are characterized by differences between cells in diverse spaces and time.MicroRNAs, a group of newly-discovered non-coding small RNAs involved in organism development by regulating cell proliferation and differentiation, have been found to play a role in committed differentiation and self-renewal of stem cells. Now research on stem cell-related miRNAs is becoming a hot field. Some miRNAs have tissue-specific expression pattern, which hints they may have a close relationship with development and maintenance of that tissue. Results from pristine miRNAs clone and microarray indicate muscle-specific expression pattern of miR-1, miR-133 and miR-206. However, to date, little is known about their functions in muscle development, proliferation and differentiation. Undoubtedly identifing their roles in ASCs proliferation and differentiation will give clues to treatments of muscular injury based on ASCs.In this study, the in vitro model of C2C12 myogenic differentiation was employed to imitate myogenesis process, and expression changes of muscle-specific miRNAs were detected in this model. Then, we evaluated the influence of miR-1 and miR-206 overexpression on the myogenic differentiation, and found their functional targets by bioinformatics and experiments. At the same time, we detected the expression changes of muscle-specific miRNAs during muscle atrophy induced by denervation, and constructed therapeutic vector for further application.Our results provide theoretical support and expremental basis for further research on mechanism of miRNAs regulating skeletal muscle proliferation and committed differentiation.Our study consisted of three parts:1. Detecting expression changes of muscle-specific miRNAs in myogenic differentiation.We established the in vitro model of C2C12 myogenic differentiation and its related TNF-αinhibition model evaluated by morphology,RT-PCR of myogenic differentiation related genes and myosin heavy chain(MHC) immunofluorescence histochemistry. Expressions of miR-1, miR-206 and miR-133 in above mentioned models were detected by Northern blot.2. Studying the roles of miR-1 and miR-206 in the process of myogenic differentiation and exploring their molecular mechanisms.Functional analysis of miR-1 and miR-206 was evaluated by gain-of-function. C2C12 transfected with 200 nm synthesized miRNAs were induced to myogenic differentiation for 48 h and the effects were evaluated by morphology, IF for MHC and western blot for skeletal-α-actin.For analyzing target genes of miR-1 and miR-206, a series of work were done. Firstly, three online softwares for bioinformatics analysis, i.e. PicTar, miRanda and TargetScan4.2, were used for miRNA target prediction. Based on both literatures and biologic function of miR-1/206, we selected several genes as candidate target genes for further study.Secondly, we detected the expression of candidate target genes in the process of myogenic differentiation at protein or mRNA level as well as in the conditions of overexpression miR-1/206. Thus we gave up the genes whose expressions have no correlations with the level of miR-1/206.Finaly, the candidate target genes were verified by luciferace reporter system. To ensure sensitivity and specificity of the system, we construct a positive control vector named pmiR-206- Luc reporter in which the 3′UTR of luciferace gene has a complementary sequence to mature miR-206 sequence. The reconstitute vector, pmiR-206- Luc reporter, the control vector for normalization, pMIR-β-gal and miRNAs were co-transfected to 293 cells. 24 h after transfection,β-Galactosidase and luciferase activities were determined .All transfection data were expressed as luciferase activity normalized byβ-galactosidase activity. To analyze candidate target genes, similar vectors were constructed by cloning the fragments containing presumed target site in 3′UTR of candidate target genes to the 3′UTR of luciferace gene in the vector. The reliability of miRNAs targets was verified by above-mentioned means.3. Exploring expression changes of muscle-specific miRNAs in denervated skeletal muscle and using miR-1 recombinant adenovirus to promot myogenic differentiation in vitro.To explore the expression changes of muscle-specific miRNAs in the process of skeletal muscle atrophy induced by denervation. The mouse model of sciatic nerve resection was prepared. Northern blot was used to examine the levels of muscle-specific miRNAs expression in the gastrocnemius muscle at different time points after denervation.To construct the miR-1 adenovirus vector, genomic fragment containing miR-1-1 was amplified by PCR and firstly constructed into pAdTrack-CMV. Subsequently miR-1 was constructed into backbone plasmid pAdEasy-1 by homologous recombination in competence BJ5183. The positive recombinant pAdEasy-1 containing miR-1 gene was linearized by PacⅠand transfected into 293 cells to pachage recombinant adenovirus. The production of mature miR-1 was confirmed by northern blot. The influence of myogenic differentiation by miR-1 recombinant adenovirus was evaluated in C2C12 cells infected by the adenovirus by morphology, IF for MHC and Western blot for skeletal-α-actin.By analysis the three researches above, the following results can be obtained:1. Establishment of the in vitro model of C2C12 myogenic differentiation and its related TNF-αinhibition model. C2C12 cells were induced to myogenic differentiation by DMEM containing 2% horse serum for 3-5 d. The enlarged polynucleation myotubes formed accompanied with strong positive signal of MHC IF in cytoplasm and upregulation of myoD, myoG as well as skeletal-α-actin detected by RT-PCR. These results indicated the in vitro model of C2C12 myogenic differentiation was successful. In the TNF-αinhibition model, the above-mentioned index were all inhibited to different degree, which indicate the TNF-αinhibition model was successful too.2. Identifing expression changes of muscle-specific miRNAs in above-mentioned models. In the model of C2C12 myogenic differentiation,all three miRNAs upregulate during muscle differentiation. While the expression level of all three were inhibited with the existence of TNF-αin the differentiation medium.3. miR-1 and miR-206 can promot myogenic differentiation. Cells were continuously cultured in growth medium for 24 h after transfection and then transferred to differentiation medium for 48 h before immunostaining for MHC and Western blot for skeletal-α-actin. The results from MHC and skeletal-α-actin indicate synthesized miR-1 and miR-206 promot the differentiation.4. CCND1 and FOXP1 are target genes of miR-1/206. Three online softwares for bioinformatics analysis, i.e. PicTar, miRanda and TargetScan 4.2, were used for predicting targets of miR-1 and miR-206. We selected four genes including CCND1, FOXP1, PFTK1 and EDN1 as potential targets of miR-1/206. The further detection of their expression changes in the process of myogenic differentiation and miR-1/206 overexpression eliminate PFTK1 and EDN1 out of the list. Then we construct vectors named pmiR-Luc -CCND1-3′UTR,pmiR-Luc-FOXP1-3′UTR-1 and pmiR-Luc-FOXP1-3′UTR-2, which have the potential target sites of miR-1/206 from 3′UTR of CCND1 or FOXP1. The results of experiments demonstrated that: the pmiR-Luc-CCND1-3′UTR luciferase activity for miR-1 and miR-206 decreased 47.4% and 49.3%, respectively; the pmiR- Luc-FOXP1-3′UTR-2 luciferase activity for miR-1 and miR-206 decreased 46.9% and 40.7%, respectively. However, the pmiR-Luc-FOXP1-3′UTR-1 luciferase activity was not affected by miR-1 and miR-206. The results of target verification experiments demonstrated that CCND1 and FOXP1 are targets of miR-1/206.5. Identifying the expression changes of muscle-specific miRNAs in denervated skeletal muscle. The mouse model of sciatic nerve resection was successfully established. The expression of miR-206 was obviously up-regulated with the time denervation, however, those miR-1 and miR-133 was decreased firstly and then gradually recovered with the time elapse.6. Construction of miR-1 recombinant adenovirus and observation of its effect on promoting myogenic differentiation. After evaluation and sequencing, we constructed the miR-1-1 recombinant adenovirus successfully which could produce high level mature miR-1 in infected 293 cells. C2C12 infected miR-1 recombinant adenovirus were induced for myogenic differentiation for 72 h, and the index of morphology, immunostaining for MHC, western blot for skeletal-α-actin indicate the miR-1 recombinant adenovirus could promot myogenic differentiation.Conclusion:1. The in vitro model of C2C12 myogenic differentiation and its related TNF-αinhibition model were established successfully.In addition, we found the expression features of the muscle-specific miRNAs in the two models.2. miR-1 and miR-206 are positive regulators of myogenic differentiation, and CCND1 and FOXP1 are two target genes.3. The expression changes of muscle-specific miRNAs were revealed in denervated skeletal muscle, which offered new view to explore the mechanisms of skeletal muscle atrophy in the level of miRNAs.4. The miR-1 recombinant adenovirus was constructed successfully and proved to promot myogenic differentiation in vitro in primary experiment, providing basis for repairing muscular injuries with miRNAs.