Dynamic Of Gene Expression Profile And Screening Biomarkers In Serum-derived Exosomes LncRNA Of Sepsis Patients

Backgrounds:Sepsis is a Systemic inflammatory response syndrome caused by infection（SIRS）,and its reaction mechanism is not yet clear,pathogenesis is very complex.In recent years,At present,although the study of sepsis has been strengthened,the diagnostic level has been improved,the death rate of sepsis is still increasing year by year.Exosomes are one kind of extracellular vesicles contains a complex RNA and protein,its diameter size in 30nm and 150nm.Exosomes,which carry specific proteins,cytokines and RNA or DNA,Cell communication was carried out by carrying proteins,mRNA,LncRNA,and miRNA in a series of biological processes.At present,the researchers have confirmed that the mRNA,miRNA,LncRNA and protein carried by exosomes will change significantly during the development of the disease.It is of great significance to the prognosis and treatment of the disease.LncRNA,it is a non-coding RNA that have a length greater than 200 nucleotides,It can participate in many important regulatory processes,such as genomic imprinting,x-chromosome silence,transcriptional activation,transcriptional interference,chromatin modification,and nuclear transport.It is found that LncRNA in exosomes is stable,efficient and easy to obtain because of its use as a marker of sepsis,and the application of the disease diagnosis and prognosis treatment has received a lot of attention.Our previous studies found the exosomal LncRNA expression levels are largely changed in sepsis patients’ serum comparing with healthy controls,which suggested that LncRNA of exosomes may be involved in the pathogenesis of sepsis.Therefore,investigating the changes of exosomal LncRNA at different stage of sepsis is important for elucidating the its pathogenesis.Aims:Analysis the trends of exosomal LncRNA expression level in the different stage of sepsis,and exploring the significance of this change.Methods:The serum samples were from the General Hospital of Guangzhou Military Region and Guangzhou No.2 People’s Hospital from October 2014 to December 2017.60 patients with sepsis as the experiment group,and 50 healthy volunteers were matched as control.Extracting and identification the exosomes from serum.using high throughput sequencing and bioinformatics analysis to identify the small RNA,and finding out the differential expressed LncRNA in different stage of sepsis.verification the relative expression of these differential LncRNA by the RTqPCR.The value of a single differential LncRNA expression on the clinical diagnosis of sepsis was measured by receiver operating characteristic curve（ROC curve）analysis.Then,the value of LncRNA expression on the clinical diagnosis of different stage of sepsis was measured by ROC curve analysis.Results:（1）Exosomes were derived from patients’ serum,we found that the exosomes show out typical membraned structure using transmission electron microscope,also we detected CD63,two proteins special expressed in exosomes,by Western blot.（2）LncRNA was identified by High-Throughput Sequencing,and was quantified by Bioinformatics Analysis.Taking |log2（FC）|>=1 and P<0.05 as filter conditions,we finally found out the expression of eight LncRNA were changed.The expression of NR₀73112.1 and NR₀36610.1were significantly increased in the group of sepsis compared with the control group（p<0.05）.the expression of ENST00000548416.was reduced in sepsis shock progression,and there was significant reduced in sepsis shock comparing with the control group,and the expression of ENST00000605833.1 was reduced in the group of sepsis,but was increased in both group of severe sepsis and sepsis shock.Then,we use GO analysis to study the function of targeted genes of LncRNA,and the results show that they play a role in cytoskeleton in cellular component.In molecular function,they play a role in regulate the activity of the small G protein,while in terms of the biological process,they have function on regulate signal transduction mediated Small G protein.（3）The expression levels of NR₀73112.1,NR₀36610.1,ENST00000548416.1 and ENST00000605833.1 were relatively quantified by RT-qPCR,we found that comparing with the control group,NR₀73112.1 and NR₀36610.1was significantly increased in the group of sepsis（P<0.05）,and ENST00000548416.1 was significantly reduced in the group of sepsis（P<0.01）.The expression level of ENST00000605833.1 was reduced in the group of severe sepsis,but was increased in both group of severe sepsis and sepsis shock.All results above were in accordance with Bioinformatics Analysis.（4）The utility in discriminating sepsis from healthy controls of exosomal LncRNA were evaluated by ROC curve analysis.We confirmed that NR₀73112.1have the middle utility for discrimination sepsis patients with healthy control（AUC=0.828,CI95%=（0.634-1.000））.In addition,we also evaluated the utility of discriminating the different stage of sepsis from healthy control,NR₀36610.1 have a high utility for the diagnosis of sepsis,ENST00000605833.1 have a middle level for the diagnosis of sepsis shock（AUC=0.745,CI95%=（0.482-0.995））,and ENST00000548416.1 have a middle level for the diagnosis of sepsis shock（AUC=0.738,CI95%=（0.482-0.995））.Conclusion:In summary,the expression of exosomal LncRNA extracted from serum is closely related to the different stage of sepsis.which could be the reliable diagnostic markers for sepsis.