The Expression Profiles And Function Analysis Of MiR-1 And MiR-133 In Goat Skeletal Muscle Tissue And Cell

MicroRNAs (miRNAs) are length of 19-22nt non-coding small RNA molecules, which exist in viruses, plants and animals widely. MiRNAs are new members controlling gene expression at the post-transcriptionai level which promote mRNA degradation or inhibit translation by complementary binding to the 3’untranslated regions (UTRs) of special target mRNAs. Emerging evidence has demonstrated that miRNAs play a critical role in cell proliferation and differentiation, cell death, gene regulation and tumorigenesis. At present, study on miRNAs is still at a preliminary stage. The researchers focused on discovering the model of miRNAs and some miRNAs related with the function of growth, development and disease, while study on miRNAs about important livestock梘oat is relatively little. The study aims to analyze the expression rules of miR-1, miR-133 and their target genes (HDAC4 and SRF) during goat skeletal muscle developmental process and skeletal muscle satellite cells and functional research, in order to understand its important role in the goat muscle development.1. The spatio-temporal distribution of miR-1, miR-133 and their target genes (HDAC4 and SRF) in goat. Two important miRNAs (miR-1 and miR-133) were selected as the research object in numerous miRNAs that could afftect the goat growth and development. The prediction of the target genes for miR-1 and miR-133 was performed using TargerScan and miRBD. To further understand the biological function of miRNAs in goat, the expression profiles of miR-1, miR-133 and their target gene s (HDAC4 and SRF) were detected in ten goat tissues (heart, liver, spleen, lung, kidney, small intestine, fat, longissimus dorsi muscles, pectoral muscles and crural muscles) by the real-time qRT-PCR. The results showed that both of the two miRNAs appeared to be tissue specific and miR-1 and miR-133 were specifically expressed in heart and skeletal muscle (longissimus dorsi muscles), while there was the least expression in other tissues. In this study, miR-1 was induced during goat muscle cell differentiation and its expression levels was increased from newborn to three months old, and decreased with the individual maturing. That indicated miR-1 was necessary during the muscle rapid growth, which could promote bulk copy of fibroblasts. The expression level of miR-1 was relatively reduced when the process of myoblasts differentiation was weakened. However, the expression level of miR-133 showed the increasing trend in goat growth process, which indicated miR-133 played a significant role in the adult muscle cells. Since miR-1 and miR-133 regulated important processes during skeletal muscle development, such information would be very important for understanding the mechanism of muscle formation during neonate development.In addition, the expression levels miR-1 and miR-133 in skeletal muscle of Anhui white goats and Boer goats were found to be significantly different (P<0.05).Both of HDAC4 and SRF also appeared to be tissue specific and HDAC4 and SRF showed the least expression in heart and skeletal muscle (longissimus dorsi muscles). MiR-1 and miR-133 was the most abundance in goat skeletal muscles. And the target genes of miR-1 and miR-133, HDAC4 and SRF predicated to be muscle transcription factors which involved in muscle proliferation and differentiation were the least abundant transcript in skeletal muscles. Therefore, our study suggested that the increase of miR-1 and miR-133 expression may lead to decrease of HDAC4 and SRF expression. And more studies will need to be done to determine the regulation patterns of miR-1 and miR-133 and their predicated target genes.2. The expression rules of miR-1, miR-133 and their target genes (HDAC4 and SRF) in goat skeletal muscle satelliate cells (SSC). The expression levels of miR-1 decreased in F6, F9 and F12 goat SSCs along with cell algebraic increasing, while the expression levels of miR-133 increased, which showed the expression trend was similar with goat tissues. In addition, the expression trend of HDAC4 and SRF in goat SSCs was similar with miR-1 and miR-133.3. Preliminary validation of the potential targets of miR-1 and miR-133. To provide the direct evidence to validate the HDAC4 and SRF genes are the targets of miR-1 and miR-133, miR-1 and miR-133 mimics were transfected into goat SSCs. Then, we detected the expression of HDAC4 and SRF mRNA and protein expression by real-time PCR and Western blotting. Firstly, for explorating transfection conditions, miRNA negative control mimics (red light) were transfected into goat SSCs. We found that the transfection concentration was 100nM; the transfection efficiency was the highest. The Western blotting results showed that HDAC4 and SRF protein expression in miR-1 and miR-133-transfected expressed at a lower level compared with Mock. However, we didn’t found that the change of HDAC4 and SRF mRNA level after the miR-1 and miR-133 mimics transfection. Therefore, we preliminary suggested that miR-1 and miR-133 regualted HDAC4 and SRF gene expression by inhibiting HDAC4 and SRF protein translation rather than promoting HDAC4 and SRF mRNA degradation, which indicated miR-1 and miR-133 regualted HDAC4 and SRF gene expression at the post-transcriptional level.In summary, the expression rules of miR-1, miR-133 and their target genes (HDAC4 and SRF) in goat tissues and SSCs and miR-1 and miR-133 regualted HDAC4 and SRF gene expression at the post-transcriptional level, which provided a foundation for further clarify the molecule mechanism of miR-1 and miR-133 in goat skeletal muscle growth and development.