The Mechanism Of Hsa＿circ＿0001818 Regulating The Function Of Macrophage In Sepsis

Background:Sepsis is one of the leading causes of death from severe infections worldwide.It is defined as life-threatening organ dysfunction caused by the host’s dysfunctional response to infection.The latest data show that there are about 48.9 million patients with sepsis worldwide,and the mortality rate is as high as 19.7%.Sepsis progresses rapidly and dynamically,with strong individual heterogeneity.Early diagnosis and effective treatment are still facing challenges.Therefore,a thorough understanding of the complex mechanisms of sepsis is critical for the development of new treatment strategies.Macrophage plays an important role in host resistance to pathogens and in regulating the development and progression of sepsis.In sepsis,over-activated macrophage mediates inflammatory cascades,causing cytokine storm to impair body’s immune function.The invasion of pathogen and the reprogramming of macrophage weaken its phagocytic ability,resulting in immunosuppression.In addition,the pathogen induces macrophage death,which prevents them from proliferating properly,making it difficult for the host to resist the invasion of the pathogen.Although many studies have been devoted to exploring the pathogenesis of sepsis in recent years,the exact mechanism by which macrophage function is altered in sepsis remains unclear.Circular RNA（circRNA）is an endogenous non-coding RNA（ncRNA）discovered in recent years,which is involved in the pathogenesis of a variety of diseases by binding to microRNA（miRNA）,binding to proteins,or coding protein.CircRNA is characterized by high stability and strong tissue-specific properties.CircRNA is transferred to the cytoplasm after the formation of nuclear cyclization and is selectively enriched into exosomes,so exosome detection is an effective mean to find differential circRNAin diseases.Our previous study has shown that the expression profile of serum exosomal circRNAis changed between sepsis patient and healthy people.Among them,hsa_circ₀001818（hsa_circRNA₁04670）is up-regulated in serum exosomal of sepsis patients,which has certain value in the diagnosis of sepsis.Bioinformatics analysis revealed that hsa_circ₀001818 may target and bind multiple immune-function related miRNA.Therefore,we speculated that hsa_circ₀001818 may be involved in regulating the function of macrophage in sepsis through competing endogenouse RNA（ceRNA）mechanism.Objective:This study aims to clarify the expression level of hsa_circ₀001818 in the macrophage model of sepsis,and to further explore the regulatory effect of hsa_circ₀001818 on the function of macrophages in sepsis.To clarify that hsa_circ₀001818 regulates macrophage apoptosis through the miR-17-3p/CASP3 axis.Hsa_circ₀001818 regulates phagocytic function of macrophages by targeting miR433-3p and miR-642a-5p.Hsa_circ₀001818 regulates the production of inflammatory cytokines from macrophages by targeting miR-433-3p and miR-642a-5p/ZBTB20/NFκB axis.To provide a theoretical basis for the development of precise therapeutic targets for sepsis.Methods:1.Expression of hsa_circ₀001818 in macrophage model of sepsis（1）Reverse transcription-qPCR（qRT-PCR）was used to detect the expression level of hsa_circ₀001818 in BEAS-2B,HUVEC and THP-1-derived macrophages after LPS stimulation.qRT-PCR was used to detect the expression level of hsa_circ₀001818 in THP-1-derived macrophages after LPS stimulation for different times.（2）Actinomycin D assay was used to detect the stability of hsa_circ₀001818.RNaseR digestion assay was used to identify the ring structure of hsa_circ₀001818.（3）Microscopes were used to observe the morphological changes of macrophages after LPS stimulation.ELISA kits were used to detect the expression level of inflammatory cytokines in the cell culture supernatant after LPS stimulation.Such as Tumor Necrosis Factor-α（TNF-α）,Interleukin-6（IL-6）and Interleukin-1β（IL1β）.Flow cytometry was used to detect the changes in the apoptosis rate of macrophages after LPS stimulation.Fluorescence microsphere phagocytosis assay was used to detect the phagocytic function of macrophages after LPS stimulation.2.Explore the regulatory effect of hsa_circ₀001818 on the function of macrophages in sepsisHsa_circ₀001818 overexpression plasmid and si-hsa_circ₀001818 were transfected to construct hsa_circ₀001818 high and low expression models.qRTPCR was used to detect the transfection efficiency of hsa_circ₀001818.ELISA kit was used to detect the expression level of inflammatory cytokines.Flow cytometry was used to detect the changes in the apoptosis rate of macrophages.Fluorescence microsphere phagocytosis assay was used to detect the phagocytic function of macrophages.3.Explore the molecular mechanism of hsa_circ₀001818 regulating macrophage apoptosis（1）Bioinformatics analysis was used to predict the binding sites between hsa_circ₀001818 and miR-17-3p,and between miR-17-3p and CASP3.Dual luciferase reporter gene assay was used to verify the direct binding between hsa_circ₀001818 and miR-17-3p,and between miR-17-3p and CASP3.（2）qRT-PCR was used to detect the level of miR-17-3p in macrophages after LPS stimulation,and the expression level of miR-17-3p in hsa_circ₀001818 high and low expression models.（3）MiR-17-3p mimic and miR-17-3p inhibitor were transfected to construct miR-173p high and low expression models.qRT-PCR and Western blot were used to detect the expression levels of CASP3 mRNA and protein,and flow cytometry was used to detect the changes in the apoptosis rate of macrophages.（4）qRT-PCR and Western blot were used to detect CASP3 mRNA and protein levels in macrophages after LPS stimulation.（5）Si-hsa_circ₀001818 and miR-17-3p inhibitor were co-transfected in rescue experiment.qRT-PCR and Western blot were used to detect the expression levels of CASP3 mRNA and protein,and flow cytometry was used to detect the changes in the apoptosis rate of macrophages.4.Explore the molecular mechanism of hsa_circ₀001818 regulating macrophage phagocytosis（1）Bioinformatics analysis was used to predict the binding sites of hsa_circ₀001818 with miR-433-3p and miR-642a-5p,and double luciferase reporter assay was used to verify the direct binding of hsa_circ₀001818 with miR-433-3p and miR-642a5p.（2）qRT-PCR was used to detect the levels of miR-433-3p and miR-642a-5p in macrophages after LPS stimulation,and the expression levels of miR-433-3p and miR-642a-5p in hsa_circ₀001818 high and low expression models（3）MiR-433-3p mimic and miR-433-3p inhibitor were transfected to construct miR433-3p high and low expression models.MiR-642a-5p mimic and miR-642a-5p inhibitor were transfected to construct miR-642a-5p high and low expression models.Fluorescence microsphere phagocytosis assay was used to detect the phagocytic function of macrophages.（4）Si-hsa_circ₀001818 and miR-433-3p inhibitor,si-hsa_circ₀001818 and miR642a-5p inhibitor were co-transfected in rescue experiment.Fluorescence microsphere phagocytosis assay was used to detect the phagocytic function of macrophages.5.Explore the molecular mechanism of hsa_circ₀001818 regulating the inflammatory response of macrophages（1）qRT-PCR and Western blot were used to detect the levels of ZBTB20 mRNA,protein and NF-κB pathpathy-related phosphorylated proteins in miR-433-3p high and low expression models,and in miR-642a-5p high and low expression models.（2）ELISA kits were used to detect the levels of inflammatory cytokines in miR-4333p high and low expression models,and in miR-642a-5p high and low expression models.（3）Bioinformatics analysis was used to predict the binding sites of miR-433-3p and miR-642a-5p with ZBTB20,and double luciferase reporter gene assay was used to verify the direct binding of miR-433-3p and miR-642a-5p with ZBTB20.（4）qRT-PCR and Western blot were used to detect the expression levels of ZBTB20 mRNA,protein and NF-κB pathway-related phosphorylated proteins in LPSstimulated macrophages.（5）Si-hsa_circ₀001818 and miR-433-3p inhibitor,si-hsa_circ₀001818 and miR642a-5p inhibitor were co-transfected in rescue experiment.qRT-PCR and Western blot were used to detect the expression levels of ZBTB20 mRNA,protein and phosphorylated protein related to NF-κB pathway,and ELISA kit were used to detect the expression levels of inflammatory cytokines.Results:1.Expression of hsa_circ₀001818 in macrophage model of sepsis（1）After the stimulation of LPS,expression level of hsa_circ₀001818 in THP-1derived macrophages was increased（P<0.05）,while the expression level in BEAS-2B and HUVEC was unchanged（P>0.05）.With extension of LPS stimulation time,expression level of hsa_circ₀001818 in THP-1-derived macrophages increased in a time-dependent manner,and the expression level of hsa_circ₀001818 was initially increased significantly at 48 h after LPS stimulation（P<0.05）.（2）The result of actinomyc in D experiment showed that hsa_circ₀001818 was more stable than the parent gene UBR5（P<0.05）,and the RNase R digestion experiment confirmed the ring structure of hsa_circ₀001818（P<0.05）.（3）After the stimulation of LPS,the secretion of inflammatory cytokines（TNF-α,IL6 and IL-1β）was increased（P<0.05）,the apoptosis rate of macrophages was increased（P<0.05）,and the phagocytic function of macrophages was decreased（P<0.05）.2.Over-expression and inhibition of hsa_circ₀001818 affected the function of macrophages in sepsisOver-expression of hsa_circ₀001818 increased the levels of inflammatory cytokines（TNF-α,IL-6 and IL-1β）secreted by macrophages（P<0.05）,increased the apoptosis rate and decreased the phagocytic function（P<0.05）.Inhibition of hsa_circ₀001818 reduced the levels of inflammatory cytokines（TNF-α,IL-6 and IL-1β）secreted by macrophages（P<0.05）,reduced apoptosis rate and enhanced phagocytic function（P<0.05）.3.Hsa_circ₀001818 increased the apoptosis rate of macrophages by targeting miR17-3p/CASP3（1）Hsa_circ₀001818 and miR-17-3p,and miR-17-3p and CASP3 can be directly targeted.（2）Level of miR-17-3p in macrophages was decreased after LPS stimulation（P<0.05）,and the expression level of miR-17-3p was decreased in the hsa_circ₀001818 high expression model（P<0.05）,and the expression level of miR-17-3p was increased in the hsa_circ₀001818 low expression model（P<0.05）.（3）Over-expression of miR-17-3p decreased the apoptosis rate of macrophages and the expression levels of CASP3 mRNA and protein（P<0.05）.Inhibition of miR17-3p increased the apoptosis rate of macrophages and the expression levels of CASP3 mRNA and protein（P<0.05）.（4）The levels of CASP3 mRNA and protein in macrophages increased after LPS stimulation（P<0.05）.（5）In the rescue experiment,co-transfection of si-hsa_circ₀001818 and miR-17-3p inhibitor could partially alleviate the decrease of CASP3 mRNA and protein expression levels and apoptosis rate mediated by si-hsa_circ₀001818（P<0.05）.4.Hsa_circ₀001818 inhibits macrophage phagocytic function by targeting miR433-3p and miR-642a-5p（1）Hsa_circ₀001818 could directly target miR-433-3p and miR-642a-5p.（2）The expression levels of miR-433-3p and miR-642a-5p in macrophages were decreased after LPS stimulation（P<0.05）,and the expression levels of miR-4333p and miR-642a-5p were decreased in the hsa_circ₀001818 high expression model（P<0.05）.The expression levels of miR-433-3p and miR-642a-5p were increased in the hsa_circ₀001818 low expression model（P<0.05）.（3）Phagocytic function of macrophages was enhanced after overexpression of miR433-3p and miR-642a-5p（P<0.05）.Phagocytosis function of macrophages decreased after silencing miR-433-3p and miR-642a-5p（P<0.05）.（4）In the rescue experiment,co-transfected of si-hsa_circ₀001818 and miR-43 3-3 p inhibitor,si-hsa_circ₀001818 and miR-642a-5p inhibitor could partially alleviate the enhanced phagocytosis of macrophages mediated by si-hsa_circ₀001818（P<0.05）.5.Hsa_circ₀001818 enhances the level of inflammatory cytokines producted by macrophages by regulating ZBTB20/NF-κB axis through miR-433-3p and miR642a-5p（1）After the over-expression of miR-433-3p and miR-642a-5p,the levels of inflammatory cytokines（TNF-α,IL-6 and IL-1β）secreted by macrophages were decreased（P<0.05）,and the levels of ZBTB20 mRNA,protein and NF-κB pathpathy-related phosphorylated protein were decreased（P<0.05）.After silencing miR-433-3p and miR-642a-5p,the levels of inflammatory cytokines（TNF-α,IL-6 and IL-1β）secreted by macrophages were increased（P<0.05）,and the levels of ZBTB20 mRNA,protein and NF-κB pathpathy-related phosphorylated proteins were increased（P<0.05）.（2）miR-433-3p and miR-642a-5p can be directly targeted to ZBTB20.（3）The expression levels of ZBTB20 mRNA,protein and NF-κB pathway-related phosphorylated protein in macrophages were increased after LPS stimulation（P<0.05）.（4）In the rescue experiment,co-transfected of si-hsa_circ₀001818 and miR-43 3-3 p inhibitor,si-hsa_circ₀001818 and miR-642a-5p inhibitor could partially alleviate the decreased levels of ZBTB20 mRNA,protein and phosphorylated proteins related to the NF-κB pathway and the decreased secretion of inflammatory cytokines（TNF-α,IL-6 and IL-1β）mediated by si-hsa_circ₀001818（P<0.05）.Conclusions:1.Hsa_circ₀001818 is highly expressed in macrophage model of sepsis and is involved in the regulation of macrophage function.2.Hsa_circ₀001818 increases the apoptosis rate of macrophages by targeting miR17-3p/CASP3.3.Hsa_circ₀001818 inhibits phagocytic function of macrophages by targeting miR433-3p and miR-642a-5p.4.Hsa_circ₀001818 enhances the level of inflammatory cytokines producted by macrophages by regulating ZBTB20/NF-κB axis through miR-433-3p and miR642a-5p.