Identification, Characterization And Function Analysis Of LincRNA In The Fetal Skeletal Muscle Of Porcine

The fetal skeletal muscle development is an important determinant of muscle growth and meatquality in porcine. At present, most research focused on the protein-coding genes and small RNAinvolved in muscle development but not the long non-coding RNA. Previous studies showed that longnon-coding RNA also has significant role in the development and differentiation of muscles. Here, usingRNA sequencing technology, we first systematically identified lincRNA in porcine fetal skeletal muscleand characterized its molecular features on the first time. In addition, we investigated the function of aninter-species conserved lincRNA. The main results of this study are as following:Section1: The reads produced by RNA sequencing were mapped to the genome and thenassembled by using tophat and cufflinks, respectively, which resulted in a total of54,550transcripts.Finally, we received570lincRNAs after using our pipeline filter, which corresponds to476gene loci.These lincRNA averaged1043nt and spanning an average of2.5exons. The ratio of lincRNA that hadonly two exons accounted for65.7%of the total.The average FPKM value of lincRNA is twelve,91.2percent of them are below fifteen. LincRNAs are distributed in the different chromosomes except for Ychromosome. Using BLASTN,28and6porcine lincRNA were found to have orthologs in the humanand mouse lincRNA (E value <10-5) respectively, which account for5%and1.05%of the totallincRNA.35%of lincRNA have no repetitive elements, while1.76%of the total lincRNA haverepetitive elements more than80%of its length. Based on the GO analysis, we found that neighboringprotein-coding gene of lincRNA are rich involved in the process such as muscle development andtranscriptional regulation. Among the ten lincRNAs randomly selected, two lincRNA were highlyexpressed in fetal muscles compared to adult muscles. These results indicated that lincRNA have shorterlength, less exons, lower expression and less conservative among the species, and a proportion of thelincRNA are alternative spliced. Most lincRNA were not generated from the repetitive sequence. A fewproportion of porcine lincRNA may have an important regulatory role in muscle development andtranscriptional regulation.Section2: The alignment results by BLASTN revealed that an unknown lincRNA was conservativeamong porcine, mouse and human. For porcine, this conservative lincRNA gene had two transcripts thatshowed a quite different expression between fetal and adulthood of muscles. For mouse, thisconservative lincRNA gene had seven transcripts in ensemble database, three of which were detectedand were differential expressed during differentiation of C2C12cell line.3’RACE and RT-PCR showedthat iso1has six transcripts and iso4has two transcripts, which showed a different structure within its3’terminal. These transcripts were differential expressed between fetal and adults of muscles, in whichiso1-1showed more different expression. RNA interference experimental results revealed that a slightdecline of iso1and iso4transcripts lead nearly2.5fold (P <0.01) decrease of two differentiation markergenes, myog and MHC, which showed that the expression of iso1and iso4may promote thedifferentiation of C2C12cells. Iso1and iso4mainly expressed in the cell nucleus, with a moresignificantly combination ability with PRC2protein complex when compared with the IgG antibodies(P <0.01). Section3: Three CpG island were found to be existed in the upstream promoter of iso1and iso4.These three CpG islands CPG1-1, CPG2and CpG3had no methylation sites and showed nomethylation difference between0days and5days differentiation of cells. The expression of iso1andiso4are more significant increased (P <0.01) than negative control after dealing with0daydifferentiation of cells using5-Aza-dc. The results showed that DNA methylation may be involved inthe regulating expression of iso1and iso4. In addition, the expression of iso1was significantlyincreased using low dose (2uM) of5-Aza-dc, while the similar scenarios for iso4needed high dose (5uM) of5-Aza-dc. The results showed that iso1was more sentive to5-Aza-dc compared to iso4and thelevel methylation of CpG1may have a different effect on the expression of iso1and iso4.