Identification Of Micrornas Related To The Swine Adipose Tissue Development And Primary Research On The Biological Roles Of Mir-103

MicroRNAs (miRNAs) are small ~22-nt regulatory RNAs that regulate the stability and translation of cognate mRNAs at a posttranscriptional level. Growing evidence has demonstrated that miRNAs participate in the regulation of many biological processes related to adipose tissue development. To this day, the full repertoire of miRNAs expressed in developing adipose tissue of swine remains to be determined. Thus, in this study, to gain full insight into the potential role of miRNAs during porcine adipose tissue development, the expression profiles of miRNAs were determined by Solexa sequencing in back subcutaneous adipose tissues samples from an obese phenotype of domestic swine at two stages of development(7day and 240day).Sus scrofa miR-103 (ssc-miR-103) was initially identified by sequence similarity search. There is limited information about the functions of this molecule in pigs. In an effort to uncover miR-103 important in pigs, we first analyzed the expression profile of ssc-miR-103 in normal porcine tissues and calculated GO term enrichment of the targets of miR-103. And then, we examined the integrative tissue expression profile and gene ontology (GO) term enrichment of predicted target genes to determine the global biological functions of miR-103. Final, we examined its biological roles in the differentiation of porcine preadipocytes.There are major results based on investigation above, as follows:1. A total of 12,643,158 and 9,623,166 high quality reads were obtained from the two libraries, one from seven-day-old piglet backfat and the other from 240-day-old adult pig backfat, respectively. The sequence reads were clustered into unique sequences, of which the vast majority were 22 nt in length, consistent with the common size of miRNAs. After removing the reads of low quality and masking adaptor sequences, a total of 21,632,536 clean reads of 18 to 30 nucleotides in length were obtained from the two libraries. The total number of clean sequences in the piglet and adult pig adipose tissue small RNA libraries was 149,957 and 270,913 unique reads, respectively. These unique sequences contained 39,522 common sequences between the adult pig and piglet adipose tissue libraries.2. Combining the data from two libraries identified a total of 227 unique mature miRNAs. Of the 227 miRNAs, 151 of the miRNAs have been deposited in miRBase (release 15.0), 17 have been reported in recent studies and 59 were classified as new porcine miRNAs that have not previously been identified in pig. Sequence analysis results revealed that the majority of identified miRNAs showed length and sequence heterogeneity in porcine adipose tissue, and editing of bases in the seed sequence of the miRNAs were occured frequently. The 214 genomic sequences, encompassing the 227 miRNAs identified above, were predicted to be capable of forming stable hairpin structures characteristic of miRNA precursors.3. According to the criteria for miRNA biogenesis, 78 short RNA-producing loci corresponding to 66 potential novel miRNAs were identified that could be folded into step-loop structures. The absolute sequencing frequencies of these novel miRNAs were much lower in the adult pig and/or piglet adipose tissue samples . In addition, the length of the novel miRNA sequences varied from 18 to 23 nt, with a distribution peak at 21 nt, and their 5' ends comprised most frequently uridine (U).4. The analytic results of expression for 227 miRNAs revealed that 201 miRNAs overlapped between the two libraries, 19 miRNAs were detected only in the piglet adipose tissue library and seven miRNAs were detected only in the adult pig library. Of the 201 coexpression miRNAs, 93 miRNAs were up-regulated, and 33 miRNAs were down-regulated in the adult pig adipose tissue. The expression levels displayed a large range, as reflected by the number of sequence reads, which varied from several counts for rare miRNAs to several million reads for the most abundant miRNAs.5. The 2149 predicted target genes of the 15 most abundant miRNAs in developing swine adipose tissue were analyzed in bioinformatics. The KEGG pathway analysis results showed that significant enriched pathways targeted by miRNA in adipose tissue involved in the three aspect including adipogenesis, cell biological process and nervous system development. The target genes related to lipoid metabolism were used for the GO term enrichment. The results of this analysis showed that the highest enrichment of GO terms was fatty acid biosynthesis process.6. The results of tissue expression profile showed that miR-103 was ubiquitously expressed and detected in all the tissues analyzed, but its expression level varied substantially among the 13 tissues examined. In the case of adult pigs, miR-103 expression is tissue-specific and it was abundantly and specifically expressed in the cerebrum, liver, and spleen.7. The 225 predicted target genes of miR-103 were used for the GO term enrichment. The results of this analysis showed that 5 functional categories of GO terms were significantly overrepresented. These categories included cell process, development process, organisma multicellularis morphogenesis, metabolic process and regulation of biological process. Overrepresentation of the target genes involved in nervous system development was highly specific to miR-1038. The evidences based on expression profile and target genes analysis demonstrated that the miR-103 at least participate in four aspects of biological function including nervous system development, lipid metabolism, hematopoiesis and immunity.9. In normal condition , the level of mature miR-103 was induced about 5-9 fold during peadipocytes differentiation. Antisense inhibition of miR-103 decreased the accumulated a quarter of triglyceride and expression of adipogenic markers such as C/EBPα, PPARαand PPARγ, at early stage of adipogenesis. Meanwhile, the presence of the miR-103 inhibitor resulted in an increase in the RAI14 transcript at both the mRNA and protein levels. These results demonstrate that miR-103 may accelerate the differentiation of porcine preadipocytes by targeting RAI14 mRNA.In a word, our results expand the number of known porcine miRNAs and provide a thorough account of the miRNA transcriptome in porcine adipose tissue. Meanwhile, our data also provide new insights into the global biological role of miR-103.