Study On The Regulatory Effect Of Non-coding RNA P662 On Brucella Induced Pyroptosis

Objective : This experiment aims to explore the molecular mechanism of LncRNA p662 regulating pyroptosis during the process of Brucella infection of THP1 cells.It provides a certain experimental basis and theoretical basis for further research on the signaling pathways that regulate inflammation and host-bacterial interaction during the process of brucellosis infection;at the same time,it helps to develop targeted drugs for the treatment of brucellosis.Methods:(1)Construct a model of Brucella infecting THP1 cells.qRT-PCR detects the mRNA expression levels of pyroptosis-related factors and LncRNA p662,Western blot detects the protein expression levels of NLRP3 and caspase-1;PI/Hoechst33342 double staining method was used to detect the occurrence of pyroptosis rate;using CPAT and lnc Locator to predict the coding ability of LncRNA p662 and its location in the cell;using nuclear-plasma separation technology combined with qRT-PCR to perform subcellular localization of LncRNA p662.(2)Synthesize si-p662 and transfer it into THP1 cells,detect the expression levels of pyroptosis-related factors and LncRNA p662 mRNA by qRT-PCR,detect the expression levels of NLRP3 and caspase-1protein by Western blot;the incidence of pyroptosis was detected by PI/Hoechst33342 double staining method;using Target Scan and miRDB to predict the binding sites of LncRNA p662 and has-miR-5196-3p;dual luciferase reporter system to detect the targeting relationship between LncRNA p662 and has-miR-5196-3p.(3)Use Target Scan and Mi RDB to predict the binding site of has-miR-5196-3p and NLRP3-3'UTR;dual luciferase reporter system to detect the targeting relationship between has-miR-5196-3p and NLRP3-3'UTR;Brucella infects THP1 cells,qRT-PCR detects the expression level of has-miR-5196-3p mRNA;the has-miR-5196-3p mimics,inhibitor,NC and si-p662+NC,si-p662+ Inhibitor and si-NC+NC were respectively transfected into THP1 cells,qRT-PCR was used to detect the mRNA expression level of pyroptosis factor,Western blot was used to detect the protein expression level of NLRP3 and caspase-1,PI/Hoechst33342 double staining method was used to detect the occurrence of pyroptosis rate.Results:(1)Brucella infected THP1 cells for 4 h,8 h,12 h,and 24 h.The expression levels of LncRNA p662 and NLRP3,caspase-1,IL-1?,and IL-18 mRNA showed an upward trend,and at 24 h The expression was the strongest at the time and the difference was extremely significant(P<0.01);the protein levels of NLRP3 and caspase-1 were consistent with the results of qRT-PCR;Brucella extremely significantly enhanced the incidence of pyroptosis(P<0.01);LncRNA p662 did not coding ability and exists in the cytoplasm.(2)si-p662 significantly inhibited the expression of LncRNA p662 mRNA(P<0.01),and significantly inhibited the mRNA expression of caspase1,GSDMD,and IL-18(P<0.05);the protein levels of NLRP3 and caspase-1 were consistent with qRT-PCR;si-p662 significantly reduced the incidence of pyroptosis(P<0.01);successfully constructed a wild-type and mutant dual-luciferase reporter vector containing LncRNA p662;has-miR-5196-3p mimics significantly reduces the relative luciferase activity of pmir GLO-LncRNA p662-wt about 36%(P<0.01).(3)The wild-type and mutant dual-luciferase reporter vector containing NLRP3-3'UTR was successfully constructed;has-miR-5196-3p mimics significantly reduced the relative luciferase activity of pmir GLO-NLRP3-3'UTR-wt about 26.6%(P<0.01);Brucella had no effect on the expression of has-miR-5196-3p mRNA(P>0.05);compared with the NC group,has-miR-5196-3p inhibitor significantly promoted NLRP3 And ASC mRNA expression(P<0.05),significantly promoted the expression of IL-1 ? and IL-18 mRNA(P<0.01),while has-miR-5196-3p mimics was the opposite;compared with si-NC+NC group,si-p662+NC significantly inhibited NLRP3 mRNA expression(P<0.05),and extremely significantly inhibited the expression of caspase-1,ASC,GSDMD,IL-1 ? and IL-18 mRNA(P<0.01),while si-p662+inhibitor can eliminate the above-mentioned downward trend;The changes of NLRP3 and caspase-1 protein levels and the incidence of pyroptosis are consistent with qRT-PCR.Conclusion:(1)Brucella promotes the expression of LncRNA p662 and the occurrence of pyroptosis during the invasion of THP1 cells,and LncRNA p662 has the potential as a ce RNA.(2)LncRNA p662 promoted the expression of NLRP3 and the occurrence of pyroptosis,and LncRNA p662 and NLRP3-3 ' UTR were targeted to bind to has-miR-5196-3p on the same MRE.(3)LncRNA p662 can promote NLRP3 expression and pyroptosis through the has-miR-5196-3p/NLRP3 axis.It shows that LncRNA p662 can be used as ce RNA to promote inflammation and pyroptosis through the has-miR-5196-3p/NLRP3 axis during the process of Brucella infecting THP1 cells.