MicroRNA-29a Promotes Apoptosis Of Monocytes By Targeting STAT3 In Sepsis

BackgroundSepsis is the body's infection caused by systemic inflammatory response syndrome(SIRS),sepsis and in the process of development,a variety of tissues and organs of the body are involved.Sepsis is a worldwide leading to ICU patients ranked first death disease,a serious threat to life and health of the patient,but also to society and the family has brought enormous economic burden.In the course of sepsis and development,in addition to the pathogenic microorganisms and toxins invade the body tissues outside the living organism's own immune response also plays an important role in this process.When sepsis occurs,the body's immunity to fight exist and immunosuppression,both in the state of dynamic equilibrium.In addition,anti-inflammatory and pro-inflammatory reactions are also involved in the occurrence of sepsis.Clinical manifestations of sepsis often exhibit diversity,ie without its iconic clinical manifestations,and therefore more and more researchers will move on to look for sepsis-specific biomarkers up,with a view to used in clinical diagnosis of sepsis,treatment and prognosis evaluation.To date,there are a variety of sepsis-associated molecular markers have been found,including blood clotting and inflammation associated with molecular markers,such as C-reactive protein(CRP),procalcitonin,etc.,At present,these molecular markers have been partially used clinically.However,in the above-described molecular markers currently used in the presence of both high sensitivity and specificity is not strong and other shortcomings,so the results are not often used in the diagnosis of sepsis over;scoring system even though it can be used on patients with sepsis prognosis and evaluate the severity of the disease,but it can not be used in the diagnosis of sepsis,but nor can be used in the treatment of sepsis among.So look for new sepsis diagnosis,treatment and prognostic molecular markers in the medical profession has become an urgent problem.miRNA belongs to a class of small non-coding RNA molecules,the length of the mature miRNA is 18 to 25 nucleotides in living organisms can be identified for a specific target mRNA,resulting in post-transcriptional regulation and control of the degradation and translation of mRNA.MiRNA biological organism with a range of life activities related,including cell growth,proliferation,differentiation,apoptosis,tumorigenesis and immune and so on.At present,a large number of studies have confirmed that is closely related to the occurrence and development of miRNA and sepsis.miR-29 is a class of miRNA,in the human lung,kidney,heart and other tissues showed high expression state.miR-29a as an important member of the miR-29 family,which plays an important role in a series of physiological and pathological processes in living organisms,but is not yet miR-29a and sepsis and the development of related research.Signal transducer and activator of transcription(STATs)is a class of protein tyrosine kinase activation of the transcription factor,present in the cytoplasm,with a reactive functional and mediated proliferative signals.JAK/STAT signaling pathway in the major members include JAKs and STATs protein family.Current studies have confirmed,STATs protein family as JAKs substrate,STAT can stimulate the expression of inflammatory mediators and thus influence the development and progression of sepsis.STATs family of proteins regulating physiological processes mediated by a variety of cytokines,which control,including cell growth,differentiation,one of STAT3 as an important member of the family STATs proliferation and apoptosis in living organisms STAT3 signal transduction pathway There is a close link between the various growth factors and signal transduction mediated by cytokines and thus participate in a series of physiological and pathological processes.STAT3 target gene can be carried out by the appropriate regulation to promote cell proliferation,inhibition of apoptosis.and the occurrence and development of malignant cells.Different JAK/STAT signaling pathway tend to have a different role in the development and progression of sepsis in which the JAK2/STAT3 pathway plays in the development of multiple organ dysfunction in sepsis caused by a very important role].By artificial means blocking JAK2/STAT3 signaling pathway can thereby blocking the process of sepsis caused by the occurrence of extreme inflammation,also can occur while suppressing the body's excessive inflammatory response,and ultimately play a protective tissue and organ function.ObjectiveIn the present study,the expression of miR-29a was detected in the peripheral blood leukocytes in patients with sepsis,septic patients to evaluate their prognosis and serum inflammatory factors correlation analysis while changing the expression of miR-29a LI-10-induced human peripheral blood mononuclear THP-1 proliferation,apoptosis and cell cycle of nuclear cells;in addition,we also use bioinformatics software for miR-29a target genes to predict and verify it.This study aims to understand the miR-29a is intended to occur in sepsis and the development process of the mechanism of action,while providing a reliable basis for the clinical diagnosis of sepsis and targeted therapy.MethodWe collected peripheral blood samples of patients with sepsis in August 2015-at the University Affiliated Hospital emergency department XXX Intensive Care Unit(ICU)admitted to August 2016 period,a total of 40 cases;collected over the same period in XXX intensive care unit(ICU)SIRS patients admitted to peripheral blood samples,a total of 35 cases;10 cases over the same period while collecting health checks in XXX hospital outpatient Venous blood samples from healthy volunteers,a total of 10 cases.PCR to detect the expression level of the above-described blood samples mIR-29a,the content detected by ELISA in serum IL-10 and TNF-a in.Analysis of the correlation between miR-29a and IL-10,TNF-a.ELISA was used to detect the expression of IL-10 protein in the different sources of patient monocytes and LPS action with 20 ng/mL of THP-1 cells.Design and synthesis of miR-29a inhibitor,will be transfected into THP-1 cells to inhibit the expression of miR-29a cells.PCR to detect the expression of transfected cells of miR-29a.With 20 ng/mL LPS acting on transfected THP-1 cells,MTT assay proliferation,apoptosis rate Hoechst staining cells.LPS(20 ng/ml)and LPS + IL-10(10 ng/mL)is treated separately for THP-1 cells,MTT cell viability assay,flow cytometry apoptosis rate;western blot method STAT3 and expression of the cells was detected p-STAT3 protein.Bioinformatics software for miR-29a target genes to predict.Were constructed STAT3 3'UTR WT(wild type)and STAT3 3'UTR Mut(mutant)plasmid vector and the miR-29a mimic,respectively,were co-transfected expression HTP-1 cells,the cells was detected luciferase reporter gene.The miR-29a mimic transfection were human peripheral blood from healthy volunteers and the mononuclear cells cultured monocytic THP-1,using PCR and western blot method of expression in these cells STAT3 mRNA and protein were detected.The miR-29a and/or STAT3 lentiviral vector transfected THP-1 cells,followed by 20 ng/ml LPS acting on the cell apoptosis rate,MTT assay cell survival after transfection by flow cytometry rate.ResultsFluorescence quantitative PCR results showed that the plasma of patients in the control group relative expression of miR-29a was 0.235 ± 0.110,sepsis patients was 0.733 ± 0.126.SIRS patients was 0.419 ± 0.093.Compared with the control group,the plasma of patients with sepsis group of miR-29a expression levels were significantly higher(t = 3.122,P=0.035<0.05);compared with the control group,plasma levels in patients with SIRS group,the expression of miR-29a significantly higher(t = 2.091,P = 0.044<0.05);compared with SIRS patients,plasma of patients with sepsis group of miR-29a expression levels were significantly higher(t = 2.322,P?0.046<0.05).ELISA showed that the expression level of serum IL-10 in the control group was(233.175 ± 25.624)pg/mL,for the patients with sepsis)549.367 ± 30.098)pg/mL,SIRS patients was(331.119 ± 29.033)pg/mL.Compared with the control group,sepsis patients serum IL-10 expression levels were significantly higher(t ?4.355,P = 0.029<0.05);compared with the control group,SIRS patients serum expression levels of IL-10 significantly higher(t = 3.761,P = 0.041<0.05);compared with patients SIRS,sepsis patients serum levels of IL-10 expression was significantly higher(t = 2.716,P = 0.043<0.05).Serum TNF-a expression in the control group of patients was(189.233 ± 19.259)pg/mL,sepsis patients was(411.189 ± 24.106)pg/mL,SIRS patients was(290.611 ± 22.735)pg/mL.Compared with the control group,the serum of patients with sepsis group,the expression level of TNF-a increased significantly(t = 6.811,P = 0.023<0.05);compared with the control group,the serum of patients with SIRS group,the expression levels of TNF-? significantly higher(t =4.655,P = 0.038<0.05);compared with SIRS patients,patients with sepsis group serum TNF-? expression level was significantly higher(t = 4.316,P = 0.044<0.05).miR-29a and IL-10 was positively correlated(r = 0.407,P = 0.041<0.05);on the correlation between the expression of TNF-? was content in the peripheral blood of patients with sepsis between miR-29a,the results It showed a positive correlation with miR-29a was TNF-?(r = 0.576,P = 0.038<0.05).ELISA showed that,compared with the control group,the patients with sepsis mononuclear cells of IL-10 expression levels were significantly higher(P<0.05).By ELISA after using 20 ng/mL LPS role of THP-1 cells at 0,4,8,12 h when the expression of IL-10 were detected,the results show that over time,LPS effect descendants monocytes the expression levels of IL-10 THP-1 gradually increased.Fluorescence quantitative PCR results showed that compared with the control group,miR-29a inhibitor cells in miR-29a expression levels were significantly lower(P<0.05),showed that the constructed expression of miR-29a inhibition of THP-1 cell line was successfully constructed.MTT assay test results showed that the transfected miR-29a inhibitor of THP-1 cells at 48 h,72 h,96 h proliferation rate when compared with the control group was significantly higher(P<0.05)in.Hoechst staining test results show,miR-29a inhibitor cell apoptosis rate was significantly lower than the control group group(P<0.01).With time,the control group and the role of cells in the survival rate of cells IL-10 were increased gradually;wherein compared to the control group,the survival rate of cells treated with IL-10 at each time point was significantly higher(P<0.05).Compared with the control group,IL-10 effect of cell apoptosis was significantly lower(P<0.05).Over time,the expression level of STAT3 and p-STAT3 protein gradually increased.Bioinformatics prediction results show that,STAT3,as a target gene of miR-29a.Compared with the control group,miR-29a mimic STAT3 3'UTR WT co-transfected with luciferase expression of transfected group was significantly lower(P<0.05);and miR-29a mimic and STAT3 3'UTR Mut co-transfected cells the enzyme luciferase expression level is compared with the control group no significant difference(P>0.05).In healthy volunteers isolated from peripheral blood mononuclear cells after miR-29a expression regulation could inhibit the expression of STAT3 mRN cells and protein(P<0.05);In cultured human monocytic THP-1,after the upregulation of miR-29a can significantly inhibit the expression of STAT3 mRN cells and protein(P<0.05).Compared with the miR-control group,miR-29a group apoptosis rate was significantly higher(P<0.05);and miR-control + STAT3 group apoptosis rate was significantly lower(P<0.05);and miR-29a + no STAT3 group apoptosis rate and miR-control group compared significant difference(P>0.05).miR-29a group in 1,3,4,5 days cell viability was significantly lower than that miR-control group(P<0.05);miR-control + STAT3 cell survival group was significantly higher than miR-control group(P<0.05);and miR-29a + non-STAT3 group and miR-control group compared significant difference(P>0.05).ConclusionmiR-29a expression was high status in the peripheral blood of patients with sepsis,it is possible to suppress the expression of STAT3,weakening the role of IL-10 protection monocytes,thereby promoting apoptosis and inhibit cell proliferation.