Identification Of Genes And Regulating Pathways Related To High-altitude Adaptation Through Multi-omics Of Myocardial Tissue In Tibetan Pigs

Tibetan pig is a unique type of local pigs on the plateau, has the unique adaptation. The structure and function for hypoxic adaptation of cardiac have an important physiological role. In this study,6 months of age pig for Tibetan pig (TH) raised in Tibet (altitude of about 3000m), Yorkshire pig (YH) moved to Tibet (altitude of about 3000m), Yorkshire pigs (YL) raised in lowlands (altitude of about 100m) and Tibetan pig (TL) moved to Beijing (elevation about 100m) for the study materials, we used the transcriptome RNA-seq, microRNA-seq and iTRAQ proteomic multiple expression genomics for pig heart tissue, identified the key genes, microRNAs(miRNAs) and protein related to hypoxic adaptation and integrated analysis the multi-omics expression in pigs. In addition, the use of dual-luciferase reporter gene system preliminary identified the regulatory role of specific miRNAs to theirs target gene related to high altitude adaptation.By RNA-seq analysis, we obtained 55G clean reads and found the 18585 genes in heart tissue. Among the four groups we had 15701 overlap genes.Through comparative analysis (TH vs. YH,TL vs. YL,TH vs. TL and YH vs.YL) of gene expression, we obtained 480,494,279 and 342 differentially expressed genes (DEGs) (|log2FC|> 1 and P<0.01), respectively. GO and pathways analysis for DEGs results showed enrichment in HIF-1 signaling pathway, and hypertrophic cardiomyopathy (Hypertrophic cardiomyopathy (HCM), cardiovascular development, immunity and other biological functions. In addition, the use of RNA-seq data, we also found 1898 SNVs which including 23 hypoxia-related DEGs for further follow-up study of differential gene laid a foundation.Finally, we obtained 21 DEGs (A2M, AGT, ANXA2, ATF3, CCL2, CCL5, COL3A1, CRYAB, CTGF, DUSP1, EGLN3,FGF1,PFKM,HBB,HBA,NPPA,NPPB,DECR1,TGFB2,PDLIM3 and FOS) related to hypoxia adaptation in Tibetan pig.To further explore the hypoxic adaptation in Tibetan pig, we used the same samples with RNA-seq in miRNA sequencing and identified 220 known miRNAs and 269 new miRNAs. We found 20,29,22 and 26 differentially expressed miRNAs (DE miRNAs) related to through TH vs.YH,TL vs.YL,TH vs.TL and YH vs.YL (|log2FC|> 1 and P<0.05), respectively. Predicting the target genes for DE miRNAs and analysing the GO and pathway of target genes showed that the target genes were all focused on VEGF signaling pathway, MAPK signal pathway, response to hypoxia, vascular regulation, energy metabolism, mitochondria which may be associated with hypoxic term. Comprehensive analysis, we got 12 DE miRNAs (ssc-miR-101, ssc-miR-7136-5p, ssc-miR-10b, ssc-miR-214, ssc-miR-206, ssc-miR-1343,ssc-miR-424-5p,ssc-miR-421-5p,ssc-miR-210,ssc-miR-142-5p,ssc-miR-320 and ssc-miR-4331) related to hypoxia adaptation in Tibetan pig.By iTRAQ protein sequencing, we obtained 2578 proteins. Based on selection criteria FC> 1.2 or <0.83, we got 473,297,394 and 297 differentially expressed proteins (DEPs) through TH vs.YH,TL vs.YL,TH vs.TL and YH vs.YL, respectively. The functional enrichment was showed the DEPs were mainly in HIF signaling pathway, VEGF signaling pathway, MAPK signal pathway, vascular regulation, energy metabolism, mitochondria and other hypoxia-related term. Finally, we got 20 DEPs (MYL7,PTPMT1,PDLIM3,A2M,C6,CRYAB,PDK4,ERK2,ACE,NAPG,CPS1,HSPE1,DECR1,AHSG, ENO3,MRPS26,DCN,COL3A1,COL1 A2 and KRT8) related to hypoxia adaptation in Tibetan pig.By integrating analysis the RNA-seq, miRNA-seq and iTRAQ, we found two DEGs and DEPs (CRYAB、PDLIM3) involved in blood circulation,and corresponding to the two miRNAs (ssc-miR-214、 ssc-miR-320),which suggesting that these miRNAs may be the key miRNAs for hypoxic adaptation. The dual luciferase reporter gene system result found that between miR-320 and DECR1, FGF1 gene had the negative feedback, which proved the miR-320 can regulate the FGF1 and DECRlgene. Eight DEGs and five DE miRNAs were randomly selected to validate the veracity of RNA-seq and miRNA-seq using qRT-PCR, respectively; and also used the Label-free technology validation iTRAQ results in TH vs. YH group.The results showed that the expression corresponds to the trend in sequencing basically. Hence the deep sequencing methods were feasible and efficient.In this study, we used multiple omics technology expanded the number of hypoxic-adaptation-related genes, miRNAs and proteins in Tibetan pig and indicated that the expression patterns of hypoxia-related genes,miRNAs and proteins are significantly altered in the Tibetan pig. We identified the 3 genes in Tibetan pig mainly involved in blood circulation, hypoxic response and heart development, which is conducive to adapt to high altitude Tibetan pig for heart tissue. The study for molecular mechanism of hypoxia adaptation research provided new ideas and ways in Tibetan pig.