The Function Of IGF2BP1 In LPS Induced Inflammation And Its Mechanism

Background and purposePeriodontitis is associated with chronic inflammation,which affects the supporting tissues of the teeth,even producing the loss of teeth.Recent studies have shown a close relationship between periodontitis and a variety of systemic diseases,thus threatening human health.Therefore,it is of great significance to study the pathogenesis of periodontitis,for maintaining oral health and even general health.RNA binding proteins is involved in the regulation of inflammatory response through various mechanisms.Insulin like growth factor 2 mRNA binding protein 1(IGF2BP1)is a member of the conservative single strand RNA binding protein family,which is related to the development and progression of inflammation.In this paper,the role of IGF2BP1 in LPS induced inflammatory response and its possible mechanism were investigated,providing theoretical basis for finding new targets for imflammatory treatment.Methods1.Real-time quantitative PCR(RT-qPCR)and Western blot were utilized to detect the expression of mRNA and protein of IGF2BP1 in skin squamous cell carcinoma cell line(A431),human THP-1 macrophages,human peripheral blood monocytes(PBMCs)and human skin fibroblasts.The mRNA and protein expression of IGF2BP1 and its dependent genes IGF2,Gli1 in THP-1 macrophages and PBMCs induced by LPS at different concentrations(0,10,100,500,1000ng/mL)were also detected by qPCT and Western blot.2.For THP-1 macrophages,RNAi was utilized to knockdown IGF2BP1,qPCR and Western blot were utilized to detect the mRNA and protein expression of IGF2BP1 and its dependent genes IGF2 and Glil after RNAi knockout and LPS induction.MTT assay and trypan blue staining were utilized to detected Cell survival.qPCR and ELISA were utilized to detect the mRNA and protein expression of TNF-α,IL-1β and IL-6.CRISPR/Cas9 was utilized to knock out IGF2BP1,then after LPS inducing,mRNA and protein expression of IGF2BP1 were detected by qPCR and Western blot,cell survival was detected by MTT,mRNA expression of TNF-αwas detected by qPCR,and protein expression of TNF-α in culture medium was detected by ELISA.LPS was added into the cells after exogenous overexpression of adenovirus wrapped expression vector,protein expression of IGF2BP1 was detected by Western blot,mRNA expression of TNF-α mRNA was detected by qPCR,and protein expression of TNF-α in culture medium was detected by ELISA.3.For LPS-induced THP-1 macrophages,the interaction between IGF2BP1 and(p65,p50)proteins(as NF-κB pathway key factors)was detected by immunoprecipitation.For THP-1 macrophages,protein expression of IGF2BP1,p65 and p50 were detected by Western blot after RNAi knockdown and CRISPR/Cas9 knockout of IGF2BP1 and LPS inducing.Then,DNA binding activity of p65 was detected by ELISA.For PBMCs,the expression of IGF2BP1 was induced by LPS after RNAi knockdown and CRISPR/Cas9 knockout,and the expression of IGF2BP1 was detected by Western blot.DNA binding activity of p65 was detected by ELISA,mRNA expression of TNF-α was detected by qPCR,and protein expression of TNF-αwas detected by ELISA.Results1.IGF2BP1 was highly expressed in A431,significantly expressed in THP-1 macrophages and PBMCS,but not in human skin fibroblasts.IGF2BP1 and its dependent genes IGF2 and Glil were obviously expressed in THP-1 macrophages and PBMCs induced by LPS at different concentrations,and showed concentration dependence.2.In THP-1 macrophages,after targeted RNAi knockdown of IGF2BP1 and LPS induction,the expression of IGF2BP1 and its dependent genes IGF2 and Glil expression were significantly decreased(P<0.05),with no significant change in cell viability(P>0.05),and the expression of TNF-α,IL-1 and IL-6 were significantly decreased(P<0.05).After CRISPR/Cas9 knockout of IGF2BP1 and LPS induction,the expression of IGF2BP1 was significantly decreased(P<0.05),cell viability was not significantly changed(P>0.05),and TNF-α expression was significantly decreased(P<0.05).After exogenous overexpression of 1GF2BP1 and LPS induction,IGF2BP1 expression was significantly increased,and TNF-α expression was significantly increased(P<0.05).3.In THP-1 macrophages,co-immunoprecipitation showed an interaction between IGF2BP1 and NF-κB key factors(p65,p50).After targeted RNAi knockdown and CRISPR/Cas9 knockout of IGF2BP1,LPS was used to induce IGF2BP1,p65 and p50 protein expression were significantly decreased,and DNA binding activity of p65 was significantly decreased(P<0.05).In PBMCs,targeted RNAi knockout and CRISPR/Cas9 knockout of IGF2BP1 and LPS induction,the expression of IGF2BP1 was significantly decreased,the DNA-binding activity of p65 was significantly decreased(P<0.05),and the expression of TNF-α was significantly decreased(P<0.05)..ConclusionIGF2BP1 promotes LPS-induced NF-κB activation and pro-inflammatory cytokines production in human macrophages and monocytes.IGF2BP1 may be one of the potential new therapeutic targets for inflammation.