Primary Studies On Distribution Of MiR-206 And Its Expression And Biological Function During Muscle Differentiation

Wounds such as road accidents, gunshot injuries, fire burns and some internal medicine diseases like diabetes, tumors and palsies always accompany with muscle injuries. Muscle regeneration is relatively difficult on the conditions of serious and large area of muscle injury or loss. Finally the raw surface will be replaced by fibrous connective tissue and form scar, which may disenable the movement of body and badly influence nerves and blood-vascular system, and eventually develop into obduracy wound. For this reason, studies on the process and regulatory mechanism of myogenesis and how to promote the early period reparation have become into popular topics in wound recovery science.MicroRNAs (miRNA) are a class of 21~23nt small noncoding single strand RNAs highly conserved among eukaryotic cells and are processed in cytoplasm by Dicer from ~70-90nt precursors with characteristic stem-loop secondary structures. MiRNAs are implicated in various many biology events, such as growth and development, proliferation and differentiation, tumor genesis, viral infection, et al. In the past, researches mainly focused on three traditional RNAs which are related with the protein generation including mRNA, tRNA and rRNA. Ever since the discovery of small RNAs: miRNAs and siRNAs, hundreds of miRNA have been identified from human and nematode. Studies on plant genome as Arabidopsis thaliana indicate that most of the miRNAs can regulate transcription factors related with the development processes, especially the processes of the phenotype formation and cell differentiation. MiR-206 was first cloned and identified from muscle tissue of human and mouse with a length of 22 nucleotides processed from 73nt precursor with stem-loop structure. The coding gene of miR-206 localizes in chromosome 6 cluster with miR-133 and the core sequence of miR-206 is same with miR-1. According to the analysis by oligonucleotide microarray we noticed that expression of miR-206 during skeletal muscle differentiation took in some kind of tissue-specific and period-specific manner. All of the above evidences suggest that miR-206 may play a similar role in promote muscle differentiation like miR-133 and miR-1. Therefore, we detected the expression of miR-206 among various organs and tissues in C57 mice firstly in the present study. Then C2C12 cells were used as a model to imitate myogenesis process in vitro, and miR-206 expression was detected in this model. At the same time we constructed the adenoviral expression vector of miR-206 and synthesized its 2'-O-methyl antisense oligonucleotides in order to get over expression of and low expression of miR-206 in C2C12 cells in vitro to see the influence of miR-206 expression changes in myoblast differentiation. The results are shown as the following:1.MiR-206 expresses strongly in skeletal muscle and cardiac muscle, while its expression in small intestine, spleen and lung is relatively poor, and in brain, kidney and liver is none, which indicate the distribution of miR-206 is tissue-specific.2.C2C12 cells were induced to differentiation into muscle cells in vitro and the myogenesis model was detected by inverted microscope, RT-PCR, western-blot, cell immunofluorescence histochemistry, flow cytometry and confirmed to be successful. On the foundation of differentiation model, miR-206 was detected by northern hybridization and the result showed that the expression level increasing gradually during muscle differentiation with a character of phase specificity.3.MiR-206 gene was successfully cloned and constructed into adenovirus vector. Genomic sequence of miR-206 was amplified by PCR and firstly constructed into sequencing vector pUCm-T, and then shuttle vector pAdTrack-CMV. Subsequently miR-206 was constructed into backbone plasmid pAdEasy-1 by homologous recombination in competence E coli BJ5183. The positive recombinant pAdEasy-1 containing miR-206 gene was linearized by Pac I and transfected into 293 cells to package recombinant adenovirus vector. The resultant adenovirus vector was named Adv/miR-206 which was confirmed to be correct by PCR and northern-blot.4.Primary investigation on biological function of miR-206. The C2C12 cells were transfected with Adv/miR-206 and then incubated in growth medium. We noticed that the C2C12 cells still differentiated into muscle cells without differentiation condition and formed enlarged polynucleation myotubes. Cell immunofluorescence histochemistry by MHC staining suggested that MHC expression in cytoplasm was positive. While we didn't see the same changes in Adv/GFP transfected control group. The results suggested that raising the expression of miR-206 can promote myogenesis of C2C12 cells even without the differentiation condition. At the same time we used antisensenucleic acids technique to inhibit miR-206 expression in C2C12 cells, then added differentiation medium and noticed that the myoblast differentiation of C2C12 cells were delayed obviously. After 48h of differentiation culture, we counted the number of cells that were elongated twofold longer than normal proliferating C2C12 and the total number of cells in every vision-field under the inverted microscope to calculate the proportion of differentiation. The proportion of antisense strands transfected group was 35%, while the normal differentiation induction group reached 87%, indicated that inhibiting the expression of miR-206 could depress the myogenesis ability of C2C12 cells even in the condition of differentiation. Therefore, miR-206 was presumed to be an important regulatory factor participating in myogenesis process and maintaining the differentiation station.5.Cell cycle of the Adv/miR-206 transfected cells was analyzed by flow cytometry, and we noticed that the percentage of S-period cells was obviously lower than normal C2C12 cells. The Adv/miR-206 transfected C2C12 cells existed from division cycle to make preparation for differentiation. While the transfection of Adv/GFP had very tiny influence on cell cycle. We also transfected the recombined adenovirus vector Adv/miR-206 into marrow mesenchymal stem cells and observed that the transfected MSC changed in cellular morphology as elongating in cell body and increasing in proportion of tart cell, while Adv/GFP transfected control group didn't have the similar alteration. Upon all of the experimental results above, we presumed the rising of miR-206 expression might simulate some development signals to promote myoblast differentiation process by urging cell to exist from division cycle through cyclin.