The Expression And Function Of MiRNAs In Chicken Liver And Skeletal Muscle

Liver metabolism and skeletal muscle development are two important biological processes in chicken. microRNAs (miRNAs) are a class of small non-coding RNAs about22nuleotides in length, which regulate gene expression by interacting with the3’untranslated region (UTR) of target mRNA. miRNAs play important roles in liver metabolism and skeletal muscle development. In the current study, a series of expression and function studies about the miRNAs in chicken liver and skeletal muscle were conducted. The main results are as follows:(1) miR-122expression in various tissues was detected using real-time quantitative RT-PCR (qRT-PCR). The result showed miR-122was highly expressed in liver. Using miRNA target prediction software,364potential target genes of miR-122were predicted, of which the genes P4HA1, PKM2, TGF-β3, FABP5and ARCN1related with liver metabolism were selected for further investigation. By qRT-PCR it was found that miR-122was up-regulated during the liver development and down-regulated during the isolation and cultivation of hepatocytes, the expression of the5genes were not affected during the liver development, P4HA1, TGF-β3and ARCN1were down-regulated during the isolation of hepatocytes, and FABP5was down-regulated and up-regulated during the isolation and cultivation of hepatocytes, respectively. By dual luciferase reporter assays and target site mutation assays it was validated that the5genes and their predicted target sites were bona fide target genes and target sites of miR-122, respectively. Through the overexpression of miR-122in chicken cell line, it was shown that miR-122regulates TGF-(33in mRNA level, whereas it does not regulate P4HA1, PKM2and ARCN1in mRNA level. These results indicated that miR-122plays roles in liver metabolism by regulating the5genes in chicken.(2) After the inhibition of miR-122in primary chicken hepatocytes, the genes were analyzed by trancriptome sequencing. The result showed123genes were differentially expressed, of which64were up-regulated and59were down-regulated. Gene ontology (GO) analysis showed21genes were involved in liver metabolism, indicating that miR-122may regulate liver metabolism by affecting these genes. Through comparison of sequencing and qRT-PCR, it was found that after the inhibition of miR-122P4HA1, PKM2and TGF-β3were up-regulated, FABP5was down-regulated, and ARCN1was not affected. The Western Blotting analysis showed the P4HA1protein was up-regulated by the inhibition of miR-122. These results further validated that miR-122regulate these target genes.(3) After the RNA interference of TGF-β3in primary chicken hepatocytes, it was found that3genes downstream the TGF-β3signaling pathway were differentially expressed, of which ACTA2was up-regulated, and LEF1and CCNB2were down-regulated, indicating that miR-122may affect these genes to regulate liver metabolism by targeting TGF-P3.(4) miR-126expression in various tissues was detected using qRT-PCR. The result showed miR-126was expressed in liver very low. Using miRNA target prediction software,54potential target genes of miR-126were predicted, of which the gene Spredl related with liver metabolism was selected for further investigation. By qRT-PCR it was found that miR-126was up-regulated during the liver development and down-regulated during the isolation and cultivation of hepatocytes, the expression of Spredl was not affected. By dual luciferase reporter assays and target site mutation assays it was validated that Spredl and its predicted target site were bona fide target gene and target site of miR-126, respectively. Through the overexpression of miR-126in chicken cell line, it was shown that miR-126does not regulate Spred1in mRNA level. These results indicated that miR-126plays roles in liver metabolism by regulating Spredl in chicken.(5) After the cGH treatment of primary chicken hepatocytes, the miRNAs were analyzed using deep sequencing. The result showed216chicken miRNAs were identified. Among the identified chicken miRNAs56were affected by cGH, of which29were up-regulated and27were down-regulated, indicating these miRNAs may take part in liver metabolism.(6) miR-1a expression in various tissues was detected using qRT-PCR. The result showed miR-la was highly expressed in skeletal muscle. Using miRNA target prediction software,247potential target genes of miR-la were predicted, of which the genes ACVR1and Spredl related with muscle development were selected for further investigation. Another further investigated gene was CNBP, which was previously predicted as miR-133a target gene and validated as miR-la target gene later. By qRT-PCR it was found that miR-la was wavily expressed during the thigh muscle development and it was highly expressed at4week-old stage, the expression of ACVR1and Spredl was not affected, and CNBP was at first down-regulated and then up-regulated. By dual luciferase reporter assays it was validated that the3genes were bona fide target genes of miR-la, and by target site mutation assays it was validated that the predicted target sites in ACVR1and Spredl were bona fide target sites of miR-la. Through the overexpression of miR-la in chicken cell line, it was shown that miR-la regulates Spredl in mRNA level, whereas it does not regulate ACVR1and CNBP in mRNA level. These results indicated that miR-la plays roles in skeletal muscle development by regulating the3genes in chicken.(7) miR-133a expression in various tissues was detected using qRT-PCR. The result showed miR-133a was highly expressed in skeletal muscle. Using miRNA target prediction software,287potential target genes of miR-133a were predicted, of which the gene BIRC5related with development was selected for further investigation. By qRT-PCR it was found that miR-133a was down-regulated firstly and up-regulated later during the thigh muscle development, the expression of BIRC5was not affected. By dual luciferase reporter assays and target site mutation assays it was validated that BIRC5and its predicted target site were bona fide target gene and target site of miR-133a, respectively. These results indicated that miR-133a plays roles in skeletal muscle development by regulating BIRC5in chicken.