MiRNA Biomarker Screening Of Serum And Myocardium In Rat Traumatic Hemorrhagic Shock Model, And Mechanism Research

Objective:Traumatic hemorrhagic shock (THS) as one of clinical common disease with the characteristics of the pathogenesis of complex, long duration and high mortality, has been widely paid attention to by people, and variety of inflammatory cytokine involved in regulating of it, and however the detailed mechanism was still entirely illuminate, especially the mechanism of miRNA involving of THS need to further eluciate. To address it, the rat THS model was established based on acute mechanical injury intially, and then confirmed that optium testing time of serum and myocardium based on the detection of serum cytokines and myocardium macrophage maker proteins, using high throughput sequencing and bioinformatics to obtain the differential expression miRNAs in it with the verification of miRNA biological function to elucidate the machanism, and further provided a noval tools and potentially target for the prevention and treatment of THS from miRNA.Methods:Firstly, rat THS model was established using acute mechanical injury method, and serum and myocardium samples were collected after Oh, 1h,2h,4h,8h,16h,24h, and 48h modeling, and serum cytonines espression level, including Tumor necrosis factor-alpha(TNF-α), Interleukin-2(IL-2), Interleukin-6(IL-6), Interleukin-10(IL-10), etc, was detected using enzyme linked immunosorbent assay (ELISA) to confirm the optium testing time of serum miRNA assay. Similarly, the myocardium macrophage maker proteins, including CD68, inducible nitric oxide synthase(iNOS), and Arginase-1(ARG1), was detected by Immunohistochemistry(IHC) to confirm the optium testing time of myocardium miRNA assay.Secondly, small RNA(sRNA) deep sequencing of rat serum was performed using Ⅲumina HiSeq4000, and analysis by bioinformatics to obtain the differential expression miRNAs and clustar, and the target genes of miRNA regulated were enriched by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enriching. After then, the 5 signigicant up-regulated and 5 significant down-regulated miRNAs were screened, and amplified by qRT-PCR followed with correlation analysis of confirmed miRNAs to cytokines.Finally, sRNA deep sequencing of rat myocardium was performed using Ⅲumina HiSeq4000, and analysis by bioinformatics to obtain the differential expression miRNAs and clustar, and the target genes of miRNA regulated were enriched by GO and KEGG enriching. After then,5 miRNAs were screened of which was associated with toll-like receptors-4(TLR-4) signal pathway, and confirmed by qRT-PCR. Then, the regulation of miR-155 to TLR4 was directly confirmed by Dual-Luciferase reporter assay system, and miR-155 mimic was synthesis in vitro with intraperitoneal injection, and the myocardium cytokines, including TNF-a, transforming growth factor-βTGF-β), IL-6, IL-10, was assay, and myocardium pathology and macrophage maker proteins was determined by Hematoxylin-eosin(HE) and IHC, and also assay of macrophage maker proteins by Western blot.Results:1) Rat THS model was correctly established using acute mechanical injury method, and the expression level of inflammatory factors, including TNF-a and IL-6, was significantly decreased with modeling time prolonging, and reached to the valley value after 4 h modeling, and the the expression level of anti-inflammatory factors, including IL-2 and IL-10, as significantly increased with modeling time prolonging, and reached to the peak value after 4 h modeling. These indicated that 4 h modeling was an optium testing time of serum. Similarly, after 16 h modeling, the expression of macrophage maker proteins, including CD68, iNOS, ARG1, was significantly changed, and indicated that the 16 h modeling was an optium testing time of myocardium.2) After high throughput sequencing of sreum, a total of 86 significant differential expression miRNAs was identified, including 68 known miRNAs and 18 noval miRNAs. Wherein, the expression level of miR-92a-1-3p was consistent with the results of high throughput sequencing of sreum, and was negative correlation to IL-6 and CRP, and positive correlation to IL-10.3) After high throughput sequencing of myocardium, a total of 744 significant differential expression miRNAs was identified, including 729 known miRNAs and 15 noval miRNAs. Wherein, the expression level of miR-155 was consistent with the results of high throughput sequencing of sreum, and could regulate 3’-UTR of TLR4. Significantly, the synthesis miR-155 mimic could significantly inhibited the expression of type-M1 macrophage maker proteins iNOS and its secretive cytokines, including TNF-α and IL-6, and facilitated the expression of type-M2 macrophage maker proteins ARG1 and its secretive cytokines, including IL-10 and MCL1.Conclusion:This study obtained several significantly differetial expression miRNA in serum and myocardium after THS using high throughput sequencing, bioinformatics and molecular biological technique, and provided a novel idea and target of THS associated disease treatment. Meanwhile, it also provieded a signigicant reference of the mechanism studying of THS induced myocardium injury from miRNAs, and exhibited a centain applicaton value of it.