Screening Of Non-coding RNA Associated With Immune Abnormality In Sepsis By High-throughput Sequencing

Background: Sepsis is an acute and severe infection.It is mainly related to the local inflammatory response of the septic host,and the local inflammatory response of the system is often caused by the infection of various harmful microorganisms.According to clinical experts,the disease is extremely dangerous,and the mortality rate is very high.At present,about 14,000 people worldwide may die from the complications of the disease every day,and they have continued to grow at an average rate of 8%-13% per year in the past 10 years.Although the diagnosis and treatment technology of sepsis has made great achievements and research progress,especially anti-infection technology and organ support technology,the morbidity and mortality of sepsis patients are still high.The current literature and reports have proved that in patients with sepsis in early infection,the body's autoimmune function has appeared to have obvious obstacles,which is difficult to be effectively controlled.Therefore,the studies on the immune functions and underlying mechanisms in sepsis pathogenesis would have great implications in the development of novel ideas and strategies for clinical diagnosis and effective treatment of sepsis.Epigenetics plays a key role in regulating normal cell growth,cell function,carcinogenesis and other diseases.In the human body's immune system,both the maintenance of the stability of the internal immune environment of the body and the protection of the body's cells depend on the participation of immune cells.At present,the results from many different investigators have confirmed that microRNAs(miRNAs)are involved in the critical regulation of both innate immunity and acquired immune.Particularly,miRNAs play a pivotal role in the body's immune regulation mainly through the control of T lymphocytes,B lymphocytes,dendritic cells and various secreted cytokines.However,the expression and function of miRNAs and long-chain non-coding RNAs(lncRNAs)in sepsis are still unknown.Objective: This project aimed to explore the abnormal changes of immune cells and immune factors,miRNAs and lncRNAs in peripheral blood mononuclear cells(PBMC)in patients with sepsis,and screen and analyze the presence of sepsis-specific genes through advanced high-throughput gene sequencing technology.Specific genesmay play an important role in the initiation and development of sepsis,and be used as therapeutic targets and diagnostic markers for sepsis.Methods:1.High-throughput sequencing technique was used in the sequencing of RNA samples purified from PBMCs of sepsis patients and healthy people in order to determine the abnormal expression of non-coding RNAs and specific genes in sepsis pathogenesis.2.PBMC samples were isolated from the peripheral blood samples of normal healthy people and sepsis patients,and used in the analysis of changes of immune cells by flow cytometry in order to detect the changes of specific immune cells such as monocytes in sepsis pathogenesis.3.The plasma sample were prepared from the peripheral blood of patients with sepsis and normal healthy people,and used in the measurement of concentrations of pro-inflammatory factors and anti-inflammatory factors in the plasma by ELISA in order to determine the changes of cytokines in sepsis pathogenesis.4.The relationship between non-coding RNAs,immune cells and inflammatory factors in sepsis pathogenesis was analyzed and determined.Results:1.High-throughput sequencing analysis showed that miRNAs including miR-96-5p,miR-122-5p,miR-122b-3p,miR-146a-5p,miR-182-5p,miR-183-5p,miR-486-5p,miR-582-3p and miR-1298-5p,lncRNAs including RP11-356C4.4,RP5-906C1.1,RP11-802E16.3,RP11-54A4.2,SNHG6,RP11-220I1.1 and SNHG16,and specific genes including ATR,PATZ1,SCRIB and APBA3 were obviously dysregulated in PBMC from patients with sepsis.Among them,mi R-96-5p,miR-182-5p,miR-183-5p,miR-582-3p,RP11-356C4.4,RP5-906C1.1,RP11-802E16.3and RP11-54A4.2 were significantly up-regulated,but miR-122-5p,mi R-122b-3p,miR-146a-5p,miR-1298-5p,SNHG6,RP11-220I1.1,SNHG16,ATR,PATZ1,SCRIB and APBA3 were down-regulated significantly in PBMC from sepsis patients.The results suggested that the abnormally-expressed miRNAs,lncRNAs and specific genes may play an important role in sepsis pathogenesis.2.ELISA was used in the detection and analysis of plasma cytokine concentrationsin patients with sepsis and healthy controls.As compared with normal healthy people,the levels of pro-inflammatory factors IL-1,IL-6,TNF-?,IL-17 and GM-CSF increased significantly in sepsis patients;however,the levels of transforming growth factor-?(TGF-?),interferon-gamma(IFN-?)and IL-4 levels were significantly decreased.Flow cytometry analysis discovered that the percentages of T cells and B cells decreased significantly,but the percentage of monocytes and MDSCs increased significantly in the PBMC from sepsis patients as compared with normal people.3.mi R-182-5p,miR-486-5p,RP11-356C4.4,RP5-906C1.1,RP11-802E16.3 and RP11-54A4.2,which were up-regulated in sepsis patients,had a significant negative correlation with CD3+ T cells,CD3+CD4+ double positive cells,CD19+ cells and CD4+Th2 cells,but had a significant positive correlation with CD33+CD11b+ cells.The down-regulated mi R-146a-5p,SNHG6,RP11-220I1.1,SNHG16,ATR,PATZ1,SCRIB and APBA3 in sepsis patients cells showed a significant positive correlation with CD3+ T cells,CD3+CD4+ double positive cells,CD19+ cells and CD4+Th2 cells,but showed a significant negative correlation with CD33+CD11b+ cells.In addition,miR-182-5p,miR-486-5p,RP11-356C4.4,RP5-906C1.1,RP11-802E16.3 and RP11-54A4.2,which were up-regulated in sepsis patients,showed a significant negative correlation with the low levels of IL-4 and TGF-?,but showed a positive correlation with the high levels of IL-1,IL-6,IL-17,TNF-?,GM-CSF and IL-10.The down-regulated mi R-146a-5p,SNHG6,RP11-220I1.1,SNHG16,ATR,PATZ1,SCRIB and APBA3 in sepsis patients were significantly positively correlated with low levels of IL-4 and TGF-?,but significantly negatively correlated with the high levels of IL-1,IL-6,IL-17,TNF-?,GM-CSF and IL-10.Conclusion: As compared with healthy people,sepsis patients have abnormal immune regulation.Specifically,pro-inflammatory cytokines are significantly increased,and lymphocytes are suppressed,suggesting that lymphocyte apoptosis could occur.Non-coding RNAs including miR-96-5p,miR-182-5p,miR-183-5p,miR-582-3p,RP11-356C4.4,RP5-906C1.1,RP11-802E16.3 and RP11-54A4.2 were up-regulated in patients with sepsis;while non-coding RNAs including mi R-122-5p,miR-122b-3p,miR-1298-5p,NHG6,RP11-220I1.1,SNHG16 and specific genes such as ATR,PATZ1,SCRIB and APBA3 were down-regulated.These abnormallyexpressed non-coding RNAs are involved in the regulation of abnormal immune functions in sepsis and may be used as potential therapeutic targets for sepsis;however,the underlying mechanisms of non-coding RNA functions in sepsis still needs further investigation.