Expression Analysis Of MiRNA In Siniperca Chuatsi Skeletal Muscle And Study On Its Role In Muscle Regulation

The mandarin fish(Siniperca chuatsi) is carnivorous and naturally lives in the major rivers and lakes of northeastern of China. Because of its high protein content and no fishbone as well as appealing taste, the mandarin fish(Siniperca chuatsi) to be one of the most commercially important and in high demand carnivorous fish species in aquaculture. miRNAs are approximately 22-nt non-coding RNAs that act as negative regulators of gene expression either by inhibiting messenger RNA (mRNA) translation or promoting mRNA degradation through base pairing to the 3'-untranslated region (3'-UTR) of the target mRNAs.In the research of this paper, we extract total RNA from white muscle tissues of the mandarin fish and built up mandarin fish white muscle tissue of small RNA libraries, and then use Solexa high-throughput sequencing technologies for small RNA fragment sequencing in the library. Through a series of bioinformatics software to analyze the small RNA from white muscle tissue, the results identified 166 species of conservative sequence of miRNAs and named after the three new miRNAs,33 kinds of miRNAs in higher abundance in the white muscles.In order to further identification of miRNAs related muscle development in the mandarin fish, the time and space expression analysis of miRNA was performed by RT-qPCR. We choose the miRNAs which with high abundance in white muscle to investigate the expression pattern in different tissues of mandarin fish. To determine whether the expression of these miRNAs changes during fish growth, we compared their expression levels in fast muscles of adult and juvenile Chinese perch. The results showed that miR-la, miR-10c, miR-133a-3p, miR-199-3p, miR-206, miR-214 and miR-222 were specific expressed in white muscle. With the process of the mandarin fish development, miR-10c, miR-143, miR-152, miR-22a, miR-103, miR-107, miR-la, miR-181a-5p, miR-133a, miR-22a, miR-222, miR-152 and miR-140 were expressed with differentia. It can be speculated that these miRNAs might play a crucial role in muscle development.The juveniles were fasted for 1 week, and then fed with a single meal to all individuals to satiation. The miRNAs expression was performed using RT-qPCR at 0 h (before the recovery meal) and at 1,3,6,12,24,48, and 96 h after the single meal feeding. The results showed that the seven miRNAs (miR-133a-3p, miR-10c, miR-107a, miR-140-3p, miR-181a-5p, miR-206 and miR-214) were sharply upregulated or downregulated within 1 h after refeeding. These miRNAs may be the promising candidate miRNAs involved in a fast-response signaling system that regulates fish skeletal muscle growth. We conducted a search on the miRNA and mRNA database of Chinese perch to determine whether the seven differentially expressed miRNAs targeted any of the muscle growth-related genes. We found that miR-10c, miR- 107a, miR-140-3p, and miR-181a-5p were predicted to target the myostatin gene. By dual luciferase reporter assays and target site mutation assays, it was validated that Myostatin and its predicted target site were bona fide target gene and target site of miR-181a-5p, respectively.