Identification Of Micrornas From Chinese Qinchuan Bovine Muscle And Adipose Tissues And Functional Analysis Of MiR-10020 During Myogenesis

Micro RNAs（mi RNAs） are a family of 18²4 nucleotide, single-stranded, non-coding RNA molecules, which play an important role in animal growth, development, hormone secretion and disease activities with a post-transcriptional regulation.Currently, culture system on the high yield and quality of beef cattle is the focal point of bovine breeding, high yield producing mainly related to the development of bovine muscle tissue and production of beef in high quality always involved in the development and maintenance of bovine adipose tissue. Therefore, this project focused on Qinchuan cattle as the research object, and construction of sequencing libraries between different developmental stages used bovine muscle and backfat tissues to identify the tissue-specific and temporary-specific mi RNAs. To validate the sequencing data, stem-loop q PCR analysis of mi RNA expression was performed between different tissues, and the luciferase reporter constructs were designed to validate the targets of important mi RNAs. Finally, regulation of bovine skeletal muscle satellite cell proliferation and differentiation was researched using over-expressing and knock-down of the interested mi RNAs. The main work and results were as following:1. Small RNA sequencing analysis of bovine muscle tissues between different stagesFive sequencing libraries were constructed from Qinchuan bovine muscle tissues at two prenatal stages（90 and 200 days） and three postnatal stages（5 days, 12 months postnatal and 24 months postnatal）. The five libraries（hereafter labeled by age of muscle at sampling as FM90, FM200, CM, YM and AM, respectively） were then validated and sequenced with an Illumina Genome Analyzer.Each of the five small RNA libraries generated approximately 15.0 million 40 nt reads, resulting in more than 75.61 million raw reads. After quality control trimming, a total of 74,802,970 were considered to be clean reads downstream analyses. In total, 74.73% of the clean reads belonged to bovine known mi RNAs across five libraries, accounting for 0.90% of the unique reads. Then, 510 unique read types aligned to 537 independent mi RNA loci according to sequence similarity, and a total of 263 mi RNAs were expressed across all tissues. And a total of 125 unique candidate mi RNA were identified from MIREAP prediction of sequence reads not aligned to any known mi RNAs, but could be mapped to a bovine reference genome. Four of these novel mi RNAs were expressed in all five sample types. Thirty-six known bovine mi RNAs were found to be significantly differentially expressed（DE） between prenatal and postnatal stages, and 14 up-regulated and 22 down-regulated DE mi RNAs in bovine postnatal muscle tissues were identified. And novel_mir₉3 was also identified between different muscle stages by q PCR, as well as between different tissues. The results showed that this novel mi RNA was highly expressed in muscle-related tissue or organs（Heart）, and significantly up-regulated in bovine postnatal muscle tissues. The novel mi RNA novel_mir₉3 sequence has been submitted to mi RBase datasets as bta-mir-10020.2. Small RNA sequencing analysis of bovine backfat tissues between fetal and adult stagesTotal RNAs from Chinese Qinchuan bovine backfat at fetal and adult stages were used to construct small RNA libraries for Illumina next-generation sequencing. Sequencing provided a total of 14,071,065 and 14,373,930 raw reads from the fetal and adult backfat tissue libraries. After removing the low quality reads, adaptors, and insufficient tags and sequences, a total of 13,915,411 and 14,244,946 clean reads were obtained, representing 870,506 and 687,753 unique reads.Known bovine mi RNA reads accounted for 59.17% of all sequence reads in the fetal library and 47.89% in the adult library, suggesting that mature mi RNAs were highly enriched in our small RNA libraries. Then, the mi RNA candidates were clustered into 432 and 412 categories corresponding to 457 and 442 independent genomic loci in fetal and adult libraries according to sequence similarity, of which 369 mi RNAs overlapped in both libraries.Based on MIREAP prediction, we identified 36 novel bovine mi RNAs that corresponded to 65 genomic loci. Ten novel mi RNAs were in the fetal bovine library and nine were in the adult bovine library; 17 of these overlapped in both libraries. DE analysis demonstrated that 173 mi RNAs consisted of 87 up-expressed mi RNAs and 86 down-expressed mi RNAs in fetal sequencing libraries.Based on stem-loop q PCR, 15 specific mi RNAs were detected, and the results showed that mi RNAn25 and mi RNAn26 were highly expressed in backfat tissue, suggesting these small RNAs play a role in the development and maintenance of bovine subcutaneous fat tissue.3. Character analysis of bovine sequencing librariesBecause of specificity of the cleavage site, mi RNA has some preference on bases at different positions. For example, the first base from the 5’ end has a strong preference of U, but resistant to G; bases from position 2 to 8（seed sequence） on the 5’ end are usually resistant to U; bases from position10（this position is the cleavage site when mi RNA regulates m RNA） has a strong preference of A. In five muscle tissues and two backfat tissues, a total of 534 unique known mi RNAs were identified, and lots of them contained isomi Rs. Between bovine fetal and adult backfat tissues, only bta-mi R-204 was significantly DE in bovine adipose tissues, and only bta-mi R-133 a in bovine muscle tissues was significantly up-regulated. Our analyses of the entire mi RNAomes identified in bovine muscle and backfat tissues produced 12 clusters of highly correlated mi RNAs, with each including 25²33 units. And cluster gray was reserved for unassigned units. Significantly up-regulated mi R-204 in backfat was contained in blue cluster, and highly corrected with mi R-143; significantly up-regulated mi R-133 a in muscle was contained in red cluster, and highly corrected with mi R-1.4. Prediction of mi RNA targets and mi RNA annotationWe used mi Randa, PITA and RNAhybrid to predict potential target genes that regulated by 36 DE mi RNAs between prenatal and postnatal stages.In total, target analysis comparison yielded 3,069 unique genes potentially regulated by DE mi RNAs. This resulted in 4,305 mi RNA-target interactions; 2,306 of these were targeted by up-regulated mi RNAs and 2,269 were targeted by down-regulated mi RNAs. Diseases and biological function analyses and the KEGG pathway analysis revealed that these targets were statistically enriched in functionality for muscle growth and disease. Putative targets for mi RNAn25 and mi RNAn26 were predicted, and a total of 2,416 putative target sites for mi RNAn25 and 672 target sites for mi RNAn26 were identified in the cattle’s backfat. Further analyses revealed that 126 mi RNAn25 targets and 44 mi RNAn26 targets relating to lipid metabolism.In total, 1359 mi R-10020 targets were identified, and 27 targets were annotated in muscle growth and development. RT-q PCR analysis between different tissues showed that MYLPF, MYL3 and ANGPT1 genes were muscle tissue-specific genes, and KLF4 and CAPN3 genes were muscle tissue-enriched genes.5. Identification of bta-mi R-10020In order to determine whether bta-mir-10020 can directly target MYLPF, MYL3, ANGPT1, KLF4 and CAPN3, we designed luciferase reporter constructs that included either the wild or mutant-type 3’UTR of the above five genes. Two types of constructs were then transfected into HEK293 cells respectively, and no convincingly reduced activity was observed between wild and mutant luciferase reporters. For cotransfection with bta-mir-10020 mimics, over-expression of the bta-mir-10020 significantly reduced the luciferase activity of the wild-type ANGPT1 reporter compared to the mutant-type. To validate the results of the dual luciferase reporter assay, we also constructed wild and mutant-type expression cassettes of the target gene ANGPT1 3’UTR using the p Ds Red-Express-N1 vector, which encoded the Disco-soma red fluorescent protein（Ds Red）. These wild or mutant-type cassettes were then cotransfected with bta-mir-10020 mimics into HEK293 cells, and appreciable Ds Red fluorescence was identified to be reduced in those cotransfected with the wild-type cassette and mi RNA mimics. These results demonstrated that the bta-mir-10020 directly targeted the bovine ANGPT1 3’UTR.In bovine skeletal muscle satellite cell, over-expressing of mi R-10020 significantly decreased Pax7 expression, and further inhibiton of myogenic differentiation; no significant effect in myogenic proliferation and differentiation by inhibiting mi R-10020 expression.