Screening And Preliminary Study Of LncRNAs Associated With Infection Of Chlamydia Trachomatis

Background and objective:Chlamydia trachomatis(C.trachomatis)is an intracellular bacterium,and is one of the main pathogens that cause sexually transmitted infections worldwide.C.trachomatis usually lead to chronic or asymptomatic diseases and are prone to developing ascending infections.The persistent infection of C.trachomatis can result in serious complications such as epididymitis,prostatitis,endometritis,salpingitis,ectopic pregnancy and infertility.Further research on the pathogenic mechanisms of C.trachomatis and finding more targets for intervention will provide an important experimental and theoretical basis for clinical prevention and treatment of C.trachomatis infection.Due to the characteristics of the intracellular survival of C.trachomatis,it is necessary to evade the immune system and complete intracellular replication by regulating host-related signaling molecules.The only secreted plasmid protein of C.trachomatis discovered by our group has been proved to be essential for C.trachomatis It is secreted into the host cell cytoplasm in the early stage of C.trachomatis infection and participates in the pathogenesis of C.trachomatis.In this study,human cervical cancer epithelial cells HeLa cells were used as model cells to construct a persistent infection model of C.trachomatis serovar E.Based on this model,the differentially expressed lncRNAs,mRNAs and related signals of host IncRNAs during the acute and persistent infection of C.trachomatis were investigated.Subsequently,the transduction pathway associated with differentially expressed lncRNAs and unfolded protein responses were explored to further determine the role of pORF5 in the pathogenesis of C.trachomatis.This study provides an important basis for C.trachomatis to resist host cell apoptosis and maintain cellular homeostasis during infection,and provides new ideas for the study of chlamydial pathogenic mechanism and prediction of potential intervention targets.Methods:1.After treating with human recombinant interferon-gamma(IFN-y)and penicillin G at different concentrations,indirect immunofluorescence assay(IFA)was used to detect changes in the morphology of chlamydial inclusion bodies at different concentrations,and calculate inclusion-forming units(IFUs)of progeny at different concentrations to determine the optimal concentration of persistent inducers.After the removal of persistent inducers,the morphological recovery of C.trachomatis was observed after 16 hours of incubation.At the same time,chlamydial progeny was collected for a new round of infection,and the IFUs of progeny were calculated.2.The formation of ABs at different time points was observed by transmission electron microscopy(TEM).Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the relative transcript levels of genes related to energy metabolism,bacterial division,and membrane structure.Flow cytometry(FCM),Hoechst staining and AO/EB staining were used to detect apoptotic rates of acute and persistent infections after treating tumor necrosis factor alpha(TNF-?)at 12,24,and 40 hours.3.Total RNA from uninfected cells,acute and persistent infected cells at 12,24,and 40 hours were extracted.Differentially expressed lncRNAs and mRNAs were detected by Arraystar v4.0 human lncRNAs expression profile chip.Relative transcript levels of differentially expressed genes(mRNAs:SESN2,GBP3,FZD10,PPP1R3C and DKK-1;lncRNAs:HIF1A-AS1,FGD5-AS1,USP30-AS1 and LINC01556)were validated by qRT-PCR DAVID,KEGG,STRING,Metascape and Cytoscape softwares were utilized to screen enrichment pathways of differentially expressed genes.Co-lncRNA software was used to construct the lncRNA-mRNA co-expression network4.qRT-PCR was used to detect the interference effect of siRNA on FGD5-AS1 expression.After the interference of FGD5-AS1,FCM and Hoechst staining were used to detect the apoptotic rate of infected cells induced by TNF-?.Western blot was used to detect the role of FGD5-AS1 in Wnt signaling pathway after the interference of lncRNA.?-catenin protein expression and nuclear translocation were observed by western blot and IFA,respectively5.pORF5 lentiviral vector was constructed.The expression of pORF5 protein was verified by qRT-PCR,western blot,and IFA.The total RNA of pORF5-transfected cells and control cells were extracted for Arraystar v4.0 human lncRNAs expression profile chip detect differentially expressed lncRNAs induced by pORF5.qRT-PCR was used to verify expressions of differentially expressed genes(lncRNAs:GAS5,RP11-42015.3,H19,ZFAS1,BASP-AS1,FGD5-AS1,CRHR1-IT1,and CALML3-AS1;mRNAs MAP3APK3,DDIT3,SESN2,FZD10,MAP3K13,NKRF,CASP2,and DKK-1).Multiple softwares were utilized to screen enrichment pathways differentially expressed genes6.The interference effect of siRNA on the expression of ZFAS1 was used by qRT-PCR.Western blot was used to detect the expressions of apoptosis-related proteins Bax and Bcl-2 before and after interference.FCM and Hoechst staining were used to detect the apoptotic rate of infected cells.Then,the progeny of C.trachomatis after the interference of ZFAS1 was measured.7.After pretreatment with ERK inhibitor(PD98059),p38 inhibitor(SB202190),and JNK inhibitor(SP600125),TNF-? was added to induce the apoptosis of pORF5-transfected cells and control cells.Western blot detected the expression of apoptosis-related proteins.Subsequently,the phosphorylated levels of MAPK/ERK and MAPK/p38 induced by TNF-? were detected by western blot after the interference of ZFAS 1.8.HeLa cells were exogenously stimulated with 5 ?g/mL or 20 ?g/mL of endotoxin-free pORF5 protein,respectively.Expression levels of autophagy-related molecules LC3,SQSTM1/p62 and Beclin-1,and UPR-related molecules BiP,p-PERK,PERK,p-eIF2?,eIF2?,p-IRE1?,IRE1?,ATF6,ATF4 and CHOP in C.trachomatis-infected cells,pORF5-transfected cells,and HeLa cells stimulated with exogenous proteins at 12,24,40 h were detected by western blot.At the same time,IFA detected the aggregation of LC3 and nuclear translocation of ATF4,and agarose gel electrophoresis was used to observe the splicing of XBP1 mRNA after qRT-PCR amplification.9.After autophagy was inhibited with 3-MA,western blot was used to detect the expression levels of UPR-related molecules in C.trachomatis-infected cells,pORF5-transfected cells,and HeLa cells stimulated with exogenous proteins at 12,24,40 h.IFA detected LC3 aggregation and ATF4 nuclear translocation,and agarose gel electrophoresis was used to detect the splicing of XBP1 mRNA after qRT-PCR amplification.10.PERK inhibitor(GSK2606414)and IRE1 inhibitor(4?8C)was added to inhibit the UPR downstream pathway,followed by western blot to detect autophagy-and UPR-related molecules in C.trachomatis-infected cells,pORF5-transfected cells,and HeLa cells stimulated with exogenous proteins at 12,24,40 h.IFA detected the aggregation of LC3 and nuclear translocation of ATF4,and agarose gel electrophoresis detected the splicing of XBP1 mRNA after qRT-PCR amplification11.Western blot was used to measure the phosphorylation of MAPK/ERK in C.trachomatis-infected cells,pORF5-transfected cells,and HeLa cells stimulated with exogenous proteins at 12,24,40 h.Subsequently,PERK inhibitor and IRE1 inhibitor were used to detect the phosphorylation levels of ERK in C.trachomatis-infected cells,pORF5-transfected cells,and HeLa cells stimulated with exogenous proteins12.ERK inhibitor was applied(PD98059)to monitor the expression levels of autophagy-and UPR-related molecules in C.trachomatis-infected cells,pORF5-transfected cells,and HeLa cells stimulated with exogenous proteins.IFA detected the aggregation of LC3 and the nuclear translocation of ATF4.The splicing of XBP1 mRNA after qRT-PCR amplification was visualized by agarose gel electrophoresis.Results:1.It was confirmed that 75 U/mL of IFN-y and 75 U/mL of penicillin G could significantly reduce the infection ability of C.trachomatis(P<0.01)and the volume of inclusion bodies(P<0.05).Under the TEM,the structure of the persistently infected inclusions appeared loose.The abnormally enlarged aberrant body(AB)were confirmed the successful construction of a persistent infection model.Compared with the acute infection group(32.400±1.800)× 106,the IFUs after removing IFN-y was(12.600 ±0.520)× 106,and the IFUs after removing penicillin G was(14.400 ± 1.375)×106,the IFUs without removing IFN-y was(3.600±0.520)× 106,and the IFUs without removing penicillin G group was(3.900 ±0.300)× 106.The chlamydial infectivity was partially restored after removing persistent inducers(P<0.05).After inducing apoptosis,compared with HeLa cells(33.63%),the apoptosis rate of C.trachomatis acute infection and persistent infection was 13.12%and 14.77%(P<0.05)at 12hpi,was 21.76%and 18.71%(P<0.05)at 24 hpi,and was 25.49%(P>0.05)and 26.32%(P>0.05)at 40 hpi.C.trachomatis showed an anti-apoptotic effect at 12 and 24 hpi.The anti-apoptotic ability of C.trachomatis decreased with the infected time.2.The results of qRT-PCR showed that expressions of screened differentially expressed IncRNAs HIF1A-AS1,FGD5-AS1,USP30-AS1,and LINC01556 were up-regulated at the corresponding infected time point,and expressions of differentially expressed mRNAs GBP3 were up-regulated at 12 h in acute infection and 40h in persistent infection,expressions of SESN2,FZD10,PPP1R3C,and DKK-1 were down-regulated at the corresponding infected time point.Alternations of these differentially expressed genes were consistent with the results of the chip.The lncRNA-mRNA co-expression network of candidate lncRNA molecules was constructed,and their enrichment pathways including "Spliceosome","Ubiquitin Mediated Proteolysis","Oocyte Meiosis" and "Wnt Signaling Pathway".3.After successfully interfering with the expression of FGD5-AS1(P<0.01),the expression level of ?-catenin protein in the interference group was lower than that in the non-interfered group(P<0.01),the nuclear translocation phenomenon was partially suppressed.FCM results showed that the apoptosis rate of the C.trachomatis-infected cells in the interfered group(15.05%)was higher than the non-interfered group(11.08%)(P<0.05)after TNF-? induced apoptosis.Hoechst staining results showed that the apoptotic rate in the interfered group(17.70%)was higher than that in the non-interfered group(11.12%)(P<0.05)after TNF-? induced apoptosis.The anti-apoptotic ability of C.trachomatis-infected cells was weakened4.The pORF5-transfected cells were successfully constructed.qRT-PCR results showed that expressions of differentially expressed lncRNAs GAS5,RP11-42015.3,H19 and ZFAS1 were up-regulated,expressions of lncRNAs BASP1-AS1,FGD5-AS1,CRHR1-IT1,and CALML3-AS1 were down-regulated;expressions of differentially expressed mRNAs MAPKAPK3,DDIT3,SESN2,and FZD10 expression were up-regulated,and expressions of MAP3K13,NKRF,CASP2,and DKK-1 were down-regulated.Alternations of these differentially expressed lncRNAs and mRNAs were consistent with the results of the chip The candidate lncRNA molecule ZFAS1 was screened.After interference with its expression(P<0.01),western blot results indicated that the Bcl-2/Bax ratio was down-regulated(P<0.01)in the ZFAS1 interfered group.Hoechst staining results showed that after TNF? induced apoptosis,the apoptosis rate in the ZFAS1 interfered group was 14.49%,higher than the non-interfered group with an apoptosis rate of 10.61%(P<0.05).FCM results showed that the apoptosis rate of the ZFAS1 interfered group(16.02%)was higher than that of the non-interfered group(10.90%)(P<0.05)The progeny IFUs of the ZFAS1 interfered group was(2.28 ±0.2265)× 107,which was lower than that of the non-interfered group(5.88 ± 0.2893)× 107,and the chlamydial infectivity was significantly reduced(P<0.01).5.After TNF-? induced apoptosis,compared with the phosphorylation levels of ERK,JNK,and p38 in control cells,phosphorylation levels of ERK and p38 were increased by 1.77 times(P<0.05)and 1.34 times(P<0.05)in pORF 5-transfected cells,respectively.There was no significant difference in the phosphorylation level of JNK(P>0.05).The ratio of Bcl-2/Bax of control cells in the ERK inhibition group was(0.454 ± 0.024),and the ratio of pORF5-transfected cells was(0.513 ± 0.019),there was no significant difference between the two cell lines(P>0.05);The ratio of Bcl-2/Bax of control cells in the JNK inhibition group was(0.418 ± 0.057),and the ratio of pORF5-transfected cells was(0.513 ± 0.019),which is higher than the ratio in control cells(P<0.05);The ratio of Bcl-2/Bax of control cells in the p38 inhibition group was(0.427 ± 0.110),and the ratio of pORF5-transfected cells was(0.380 ± 0.072),there was no significant difference between the two cell lines(P>0.05).After inducing apoptosis,compared with the phosphorylation levels of ERK and p38 in control cells,there was no significant difference in the phosphorylation level of ERK in ZFAS1-interfered group(P>0.05),and phosphorylation level of p38 decreased by 0.56 times(P<0.01).6.C.trachomatis infection and endogenous and exogenous stimulation of pORF5 protein can up-regulate the phosphorylation levels of PERK,eIF2?,IRE1,and the expression levels of BiP,ATF4 and CHOP proteins.At the same time,the transformation of LC3 was induced,p62 expression was decreased,and Beclin-1 expression was increased.ATF6 degradation,nuclear translocation of ATF4,and splicing of XBP1 mRNA were promoted7.After inhibited autophagy by 3-MA,C.trachomatis and pORF5 stimulation still can increase the phosphorylation levels of PERK,eIF2?,and IRE1,and the expression levels of BiP,ATF4,and CHOP.ATF6 degradation,nuclear translocation of ATF4,and splicing of XBP1 mRNA were promoted.After the addition of PERK inhibitor,autophagy induced by C.trachomatis and pORF5 stimulation was suppressed.The transformation of LC3 was repressed,the expression of p62 and Beclin-1 had no alternation After the inhibition of IRE 1,autophagy induced by C.trachomatis and pORF5 was partially repressed,the transformation of LC3 was smothered,the expression of Beclin-1 and p62 were changed slightly.8.Both C.trachomatis infection and pORF5 can activate MAPK/ERK pathway.After inhibiting PERK,the phosphorylation levels of ERK activated by C.trachomatis and pORF5 were lower than that of the control group(P<0.05),while IRE1 inhibition did not change the phosphorylation levels of ERK9.After inhibiting the MAPK/ERK,autophagy induced by C.trachomatis and pORF5 was repressed.The transformation of LC3 was decreased;the expression of Beclin-1 and p62 was slightly changed.However,UPR was still activated by C.trachomatis and pORF5,the phosphorylation levels of PERK,eIF2?,and IRE1 were up-regulated,and the expression levels of BiP,ATF4,and CHOP were increased.ATF6 degradation,nuclear translocation of ATF4,and splicing of XBP1 mRNA were promoted.Conclusions:1.An in vitro model of C.trachomatis serovar E persistent infection induced by IFN-y was successfully constructed2.Acute and persistent infection of C.trachomatis can cause differential expressions of lncRNAs and mRNAs in host cells Differentially expressed lncRNA FGD5-AS1 participates in the anti-apoptotic process of C.trachomatis by regulating the Wnt signaling pathway.3.The C.trachomatis plasmid protein pORF5 up-regulates lncRNA ZFAS1 to activate the MAPK/p38 pathway to resist host cell apoptosis and affect the infection rate of progeny4.C.trachomatis and pORF5 can activate the UPR pathway;pORF5 activates the MAPK/ERK pathway through UPR to involve in autophagy.