The Mechanism Of MiR-106b-5p Reduces Sepsis-induced Myocardial Injury By Targeting STAT3

Background:The definition of sepsis(sepsis 3.0)describes a syndrome caused by a dysregulated host response to infection,resulting in circulatory dysfunction and organ damage.Sepsis is currently one of the most challenging problems in the medical field and the primary cause of death in critically ill patients with infections.Even in developed countries such as Europe and America,the mortality rate of sepsis remains high,ranging from 30-50%.A study conducted in 2020 with ICU participation from 44 hospitals in China reported that the incidence rate of sepsis in ICU was 20.6%,with a mortality rate of 35.5%.The International Sepsis Campaign updates its sepsis guidelines every four years,with the aim of improving the global diagnosis and treatment of sepsis,and ensuring standardized treatment for more sepsis patients.Despite the efforts of healthcare workers worldwide,there are still many issues that need to be addressed in the prevention,interception,diagnosis,and treatment of sepsis,even though progress has been made in these areas.MicroRNAs(miRNAs)are a class of non-coding small RNAs that are approximately 21 nucleotides in length.They possess gene regulatory functions by binding to specific complementary sequences of target genes,thereby influencing their expression.Differential expression(DE)analysis of genes can identify genes with significant changes in expression levels between disease and healthy states,and has been an important method for studying human disease-causing genes at the molecular level.By analyzing the differential expression of genes between disease and normal physiological conditions,the goal is to identify target genes associated with the disease.This study aims to explore the differential expression of RNA and miRNA between sepsis patients and normal healthy individuals,and to identify target miRNAs,elucidating the relationship between miRNAs and sepsis.This information can be used for the diagnosis or treatment of sepsis.In this study,blood samples will be collected from sepsis patients and healthy volunteers,and gene sequencing will be performed.The aim is to identify target genes through differential gene expression analysis.Further analysis will be conducted to investigate the relationship between the target genes and sepsis,and study their role in sepsis-induced myocardial injury.This will provide new insights into the diagnosis and treatment of sepsis.Part I Sepsis key miRNAs were screened by high-throughput RNA sequencingObjective:To identify key genes and microRNAs(miRNAs)associated with sepsis by high-throughput RNA sequencing.Methods:This study recruited three sepsis patients as the study group and three healthy individuals as the control group.By utilizing high-throughput transcriptome technology,RNA sequencing was performed on the sepsis patient group and the control group.Bioinformatics analysis was utilized to determine the differential expression of mRNA(DEmRNA)and differential expression of miRNAs(DEmiRNAs)between the two groups.Further functional annotation was conducted on DEmRNA targeted by DEmiRNA.A literature search was performed to screen genes potentially involved in sepsis to find differential miRNAs associated with sepsis,and KEGG analysis was performed to derive the pathways potentially involved.Results:In comparison with the normal group,a total of 1199 DEmRNAs and 23 DEmiRNAs were identified in the sepsis patient group.Based on the analysis of DEmiRNA-targeted DEmRNA,hsa-miR-106b-5p(degree=155),hsa-miR-128-3p(degree=128),and hsa-miR-144-3p(degree=79)are the top three DEmiRNAs with the highest coverage in the sepsis patient group compared to the normal control group.According to the analysis by KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway,T cell receptor signaling pathway,cancer pathway,FoxO pathway,and influenza A pathway were significantly enriched pathways in sepsis.Conclusions:We have identified key mRNA and miRNA genes associated with sepsis,with hsa-miR-106b-5p,hsa-miR-128-3p,and hsa-miR-144-3p being potential candidates.Signaling pathways that are significantly enriched in sepsis,including the T-cell receptor signaling pathway,cancer signaling pathway,FoxO signaling pathway,and influenza A signaling pathway,provide new insights into the prevention and treatment of sepsis.Part II The relationship between serum miR-106b-5p expression level and myocardial injury in patients with sepsisObjective:To explore the the correlation between serum miR-106b-5p expression level and myocardial injury in patients with sepsis.Methods:Exploring the correlation between serum expression levels of miR-106b-5p and myocardial injury in sepsis patientsFrom November 1,2018 to December 1,2020,a total of 42 patients with sepsis admitted to EICU were included in the study,with 41 healthy individuals who underwent physical examinations during the same period selected as the control group.Within 24 hours of admission to the ICU,venous blood was collected from patients to assess their APACHE-II score and SOFA score.Collect and record the general clinical and laboratory indicators of the participants.All patients underwent a cardiac ultrasound examination,and if the left ventricular ejection fraction(LVEF)was less than 45%,they were considered to have heart failure.Excluding patients with a history of heart failure.Each participant provided a morning fasting blood sample of 2.5-3ml using a PAXgene? RNA vacuum blood collection tube.The expression level of miR-106b-5p was detected by qRT-PCR.The concentration of cardiac troponin I was measured using the colloidal gold method,C-reactive protein was measured using the scatter turbidimetric method,and procalcitonin(PCT)was measured using the enhanced fluorescence immunochromatography method.We established a ROC curve to evaluate the diagnostic significance of miR-106b-5p in sepsis and sepsis-induced myocardial injury.Performing logistic regression analysis to examine the relationship between miR-106b-5p levels and the occurrence of heart failure.Results:There were no significant differences in age,gender,and body mass index(BMI)between the two groups(P>0.05).However,significant differences were observed between sepsis patients and the healthy control group in terms of blood creatinine(CREA),albumin(ALB),white blood cell count(WBC),C-reactive protein(CRP),and procalcitonin(PCT)levels(P<0.001).Furthermore,the APACHE Ⅱ score and SOFA score of sepsis patients were(13.5±3.42)and(5.57±1.09),respectively.The qRT-PCR results indicate a significant decrease in the level of miR-105b-5p in the serum of septic patients compared to the healthy control group.The Pearson correlation coefficient was used to analyze the correlation between serum miR-106b-5p and related indicators in septic patients.The levels of CRP,PCT and SOFA score were negatively correlated with miR-106b-5p in septic patients.The ROC curve demonstrates an AUC value of 0.722,with a sensitivity of 88.1%and a specificity of 53.7%for diagnosing sepsis.The patients were divided into two groups based on their left ventricular ejection fraction(LVEF)being greater or less than 45%:the group with basic normal heart function(n=19)and the group with heart failure(n=23).Logistic regression analysis showed a negative correlation between miR-106b-5p levels and the occurrence of heart failure,suggesting that miR-105b-5p is a protective factor for the development of heart failure in septic patients.Conclusion:The Serum miRNA-106b-5p is down-regulated in patients with sepsis,suggesting that miRNA-106b-5p is associated with myocardial injury in sepsis.The expression level of miRNA-106b-5p is related to the severity of the disease.Part Ⅲ miR-106b-5p reduces oxidative stress and pyroptosis levels of myocardial injury in septic miceObjective:Exploration of the potential mechanisms by which miR-106b-5p,targeting STAT3,influences the levels of oxidative stress and apoptosis in septic cardiomyopathy.Methods:①Septic model was induced in male C57BL/6 mice aged between 6-8 weeks via performing Cecal Ligation and Puncture(CLP)surgery.After 24 hours of the surgery,cardiac tissue samples were obtained and assessed for the degree of myocardial damage through pathological methods.②The mice were randomly assigned to three groups:control group,model group,and miR-106b-5p group.The control group underwent laparotomy without CLP induction;the model group underwent CLP induction;and the miR-106b-5p group was pre-treated with miR-106b-5p mimics via tail vein injection 1 hour before CLP induction.③The expression levels of creatine kinase-MB(CK-MB),creatine kinase(CK),cardiac troponin Ⅰ(cTn-Ⅰ),interleukin-1β(IL-1β)/IL-18 in serum,as well as the activity of myocardial tissue homogenate superoxide dismutase(SOD)/malondialdehyde(MDA),were measured using the enzyme-linked immunosorbent assay(ELISA).Hematoxylin-eosin staining(HE)was used to evaluate the inflammatory cell infiltration in the cardiac tissues of all groups.TUNEL assay was conducted to detect myocardial cell apoptosis,while immunofluorescence was performed to measure the expression of GSDMD in the cardiac tissues.Furthermore,the protein expression levels of p-STAT3/t-STAT3,p-CamKⅡ/t-CamKⅡ,gp47/gp91,NLRP3,IL-1β/IL-18,GSDMD,ASC,and Caspase-1/4 in the cardiac tissues were detected using the Western blotting method.Results:①Post CLP surgery,the mice exhibited lethargy,disheveled and matted fur,reduced activity,semi-closed eyes with discharge,and a significant decrease in respiratory rate.After 24 hours,myocardial tissue edema and increased tissue gap were observed,accompanied by extensive damage to myocardial cells and significant inflammatory cell infiltration.②Compared to the control group,the levels of CK/CK-MB/cTn-Ⅰ in the serum were significantly increased in the model group,while in the miR-106b-5p group,the levels of CK/CK-MB/cTn-Ⅰ were between those of the control and model groups.Compared to the control group,the SOD levels were significantly decreased,and MDA levels were significantly increased in the model group.In the miR-106b-5p group,the levels of SOD and MDA were between those of the control and model groups.Compared with the control group,the expression levels of cell pyroptosis-related factors IL-1β and IL-18 were significantly increased in the model group,while the expression levels in the miR-106b-5p group were intermediate between those of the control and model groups.③The HE staining revealed that the myocardial tissue of the control group mice was normal,without any significant edema or infiltration of inflammatory cells.The model group exhibited evident myocardial tissue edema,widened tissue spaces,a large number of myocardial cell granular degenerations,and significant infiltration of inflammatory cells.On the other hand,the miR-106b-5p group exhibited a lighter degree of myocardial tissue edema,with only a few instances of myocardial cell granular degeneration and a minor infiltration of inflammatory cells.④TUNEL assay revealed that compared with the control group,the model group showed a significant increase in myocardial cell apoptosis,while the miR-106b-5p group showed a level of myocardial cell apoptosis that was intermediate between the control and model groups.⑤Immunofluorescence analysis showed that the expression of GSDMD in the myocardial tissue of the model group was significantly higher than that in the control group,while the expression of GSDMD in the miR-106b-5p group was intermediate between the control and model groups.⑥The results of Western blot detection showed that the expression levels of p-STAT3,p-CamKⅡ/t-CamKⅡ,gp47/gp91,NLRP3,IL-1β/IL-18,GSDMD,ASC,and Caspase-1/4 in the model group were significantly higher than those in the control group.However,in the miR-106b-5p group,the expression levels of these indicators were intermediate between the control group and the model group,and only the expression level of t-STAT3 showed no difference among the groups.Conclusions:MiR-106b-5p acts through targeting STAT3 to reduce oxidative stress in septic cardiomyopathy induced by CLP,and blocks the Caspase-1 pathway,reducing the level of myocardial cell pyroptosis,thus alleviating sepsis-induced myocardial injury.Part Ⅳ miR-106b-5p reduces oxidative stress and pyroptosis levels in myocardial injury by target STAT3Objective:To explore the mechanism by which miR-106b-5p regulates oxidative stress and cell apoptosis in sepsis-induced myocardial injury through targeting STAT3.Methods:AC 16 cells(human cardiac myocyte cell line)were treated with lipopolysaccharide(LPS)and then transfected with miR-106b-5p mimics NC/mimics/inhibitor NC/inhibitor and STAT3-shRNA plasmid using Lipofectamine 3000.The cells were divided into six groups:mimics NC,miR-106b-5p mimics,inhibitor NC,miR-106b-5p inhibitor,inhibitor NC+STAT3-shRNA,and miR-106b-5p inhibitor+STAT3-shRNA group.The qRT-PCR method was used to detect the transfection efficiency of miR-106b-5p,and the dual luciferase reporter assay was used to validate the interaction between miR-106b-5p and STAT3.Western blotting was used to detect the protein expression levels of p-STAT3/t-STAT3,p-CamK Ⅱ/t-CamKⅡ,gp47/gp91,NLRP3,GSDMD,ASC,and Caspase-1.TUNEL assay was performed to detect myocardial cell apoptosis,and immunofluorescence assay was used to detect the expression of GSDMD in myocardial cells.Results:The qRT-PCR results indicate that Lipofectamine 3000 can efficiently transfect miR-106b-5p mimics/inhibitor into AC 16 cells.The dual-luciferase reporter assay results indicate that miR-106b-5p can effectively bind to the STAT3 plasmid.The results of Western Blot analysis showed a significant decrease in the expression levels of p-STAT3,p-CamK Ⅱ/t-CamK Ⅱ,gp47/gp91,NLRP3,GSDMD,ASC,and Caspase-1 in the LPS+mimics group compared to the mimics NC group.Compared to the inhibitor NC group,the expression levels of all indicators were significantly increased in the inhibitor group.However,after co-transfection with STAT3-shRNA plasmid,there was no significant difference in the expression levels of each indicator between the inhibitor NC+STAT3-shRNA group and the inhibitor+STAT3-shRNA group.Conclusions:At the cellular level,miR-106b-5p reduces oxidative stress and cell pyroptosis in myocardial cells by targeting STAT3.