关键词: 藏黄牛, 宣汉黄牛, miRNA, 心脏, 高通量测序, 低氧适应性

Abstract:

【Objective】As a kind of non-coding RNA, miRNA is widely involved in various life activities of the organism. This study was aimed to explore the differential expression profiles of miRNA in the heart tissues between Tibetan cattle and Xuanhan cattle, so as to provide the basic data for further study on molecular mechanism of hypoxia adaptation in Tibetan cattle. 【Method】Each three healthy Tibetan and Xuanhan cattle were randomly selected for heart tissue sampling. RNA was extracted from tissues using the Trizol method. An 18 to 30nt fragment was selected by agarose gel electrophoresis, and 3' connector and 5' liner was ligated and then the fragment was enlarged. After gel electrophoresis, three Tibetan cattle and Xuanhan cattle libraries were established, respectively. High-throughput sequencing was performed by using the Illumina HiSeq4000 sequencing platform. The sequence was then filtered and the differentially expressed miRNA of Tibetan cattle and Xuanhan cattle were screened by comparing GenBank and Rfam databases. Functional annotation and signal pathway enrichment analysis of differentially expressed miRNA in Tibetan cattle and Xuanhan cattle. Finally, in order to verify the accuracy of the sequencing data, 8 miRNAs were randomly selected and the expression level of miRNA was detected by RT-qPCR. 【Result】The results showed that Tibetan cattle and Xuanhan cattle had high-quality reads of 17 463 446 and 13 662 812, respectively, while the clean reads were 16 552 296 and 12 055 304, respectively. The highest enrichment of high-quality nucleic acid sequences in Tibetan cattle and Xuanhan cattle were 21 nt, which were 37.5% and 32.1%, respectively. A total of 219 differential expressed miRNAs (48 up-regulated and 171 down-regulated) were obtained. There were 22 terms in the GO function annotation that significantly enriched in the molecular function of differentially expressed miRNAs target genes, such as GO: 0005488 (binding), GO: 0005515 (protein binding) and GO: 0043167 (ion binding). GO: 0005623 (cell), GO: 0044464 (cell component) and GO: 0005622 (cell) were among the 20 terms, which were significantly enriched in the cellular components. While there were 13 terms, which were significantly enriched in biological processes, such as GO: 0035556 (intracellular signal transduction), GO: 0032774 (RNA biosynthesis process) and GO: 0006351 (transcription, DNA templated). Analysis of KEGG signaling pathways revealed that miRNA target genes were significantly enriched to 232 signaling pathways, including the insulin signaling pathway (139 target genes), the mTOR signaling pathway (38 target genes) and the HIF-1 signaling pathway (92 target genes). Among them, 12 miRNA target genes worked together on these three signaling pathways. These results suggested that the differentially expressed miRNAs might participate in the regulation of hypoxia adaptation in Tibetan cattle through these three signaling pathways. Eight miRNAs were randomly selected for RT-qPCR, and the expression profiles were consistent with the sequencing data, indicating that the high-throughput sequencing data was reliable. 【Conclusion】Taken together, the expression profiles of miRNAs in the heart tissues of Tibetan and Xuanhan cattle were obtained in the present study, which laid a foundation for further research on the hypoxia adaptation mechanism of Tibetan cattle.

Key words: Tibetan cattle, Xuanhan cattle, miRNA, heart, high-throughput sequencing, hypoxia adaptability