| microRNAs (miRNAs) is a class of 22-nucleotide-long noncoding RNAs, which can combine with target gene’3’-UTR region to form dimers and inhibit the translation of target genes or directly degrade it. In this way, microRNAs have achieved the function of regulating the expression of target genes. It has been estimated that miRNAs can regulate the expression of more about 30% of the human genes, which involved in a variety of biological processes, such as cell proliferation, differentiation and apoptosis. Mitochondria is the energy factories in cell. The amount of of mitochondria seriously decides the level of energy metabolism status in cell. There has been many differents about mitochondrial content in various cells, it is caused by the spicial energy demand. However, the heterogeneity of muscle fibers is closely related with different energy metabolism in the different muscle fiber types. The slow muscle fiber has great mitochondrial content, which cause the energy metabolism mainly operated by aerobic respiration. Mitochondria in the fast muscle fibers has a low content, which cause the energy metabolism mainly operated by anaerobic respiration. In the meat quality research, slow-twitch muscle hold a low glycogen and a high mitochondria content, it is induced slow-twitch muscle fibers has a lower glycolytic potential. Therefore, the slow muscle fiber has contributed to a great meat quality.This study uesd C2C12 cells as a model, and intended to explore the function of miR-23a and miR-27b affects mitochondrial copy number in cell. Moreover, through studying mitochondrial copy number variations revealed the influence on energy metabolism status and muscle fiber types composition in myoblast. The results are as follows:1. This study has found C2C12 has reached the highest transfection efficiency (about 43%) when Lipofectamine 2000 concentrations was 1.56μl/cm2 and mimic (or inhibitor) concentrations was 150 nm.2. It was verified that the Mef2c gene was a direct target gene of miR-23a and the Foxj3 gene was a direct target gene of miR-27b by dual luciferase report system. We also have identified the Mef2c gene’s 3’UTR had two binding site with miR-23a. Through highly expressing of miR-23a and miR-27b, we had found the expression levels of Meflc gene significantly declined 7.14 times (p<0.01). Moreover, highly expressing miR-27b has casused Foxj3 gene significantly decreased to 9.09 times (p<0.01).3. Through highly expressing miR-23a and miR-27b in C2C12, it was significantly inhibited the synthesis capacity of mitochondrial copy number (p<0.05).4. miR-23a and miR-27b with reducing mitochondrial copy number could significantly reduced the level of energy metabolism in cells (p<0.05), such as significantly reduced mitochondrial F0F1-ATP synthetase genes’s expresion (such as ATP6, ATP8, ATP12, TMEM70) expression (p<0.01), and energy metabolism marker genes AMPK expression level.5. Reduced mitochondrial copy number in cells could significantly inhibited C2C12 cell’s differentiative capacity, and frustrated forming normal myotube, it was brought about myotube losted the direction of the law.6. It was proved that miR-23a and miR-27b could inhibited the synthesis capacity of the four muscle fiber types, which could reduced slow fiber type’s expression level upto 3.04 to 4.37 times (p<0.01), however, the expression of fast fiber only decreased with 1.00 to 1.52 times (p<0.05). Which has caused the composition of slow fiber type in the myotube significantly reduced (p<0.01).The above results has showed that the mitochondrial copy number could changes by miRNA regulation, which also might influence muscle differentiative capacity and the composition of muscle fiber types through changed energy metabolism level in cells. This has provided the basis data for the study of epigenetic regulation of muscle fiber types, and has opened up a new research direction in the research of meat quality. |
