| Background and objective:Chlamydia trachomatis is a strictly intracellular gram-negative pathogen.It mainly induces endometritis by persistent infection of the genitourinary tract and can lead to severe complications such as ectopic pregnancy and tubal infertility.So far,the mechanism of Ct persistent infection has not been fully elucidated,and there is no effective vaccine to prevent it.Therefore,a comprehensive and in-depth study of its complex molecular regulation mechanism is the key to effectively preventing Ct persistent infection and blocking the occurrence and development of its serious complications.LncRNA is a non-coding RNA with a length of more than 200 nucleotides.Studies have confirmed that lncRNA can regulate immune response and host cell biological behavior through various mechanisms and promote the proliferation and infection of intracellular pathogens.In this study,based on the differential expression database of lncRNA in Ct persistent infection,the research group planned to screen out key lncRNAs further and analyze the lncRNA-miRNA-mRNA regulatory network.By constructing the cell model of persistent infection and the mouse reproductive tract infection model,the specific mechanisms of key lncRNAs and their targets regulating anti-host cell apoptosis,intracellular survival of Chlamydia,and reproductive tract inflammation in Ct persistent infection were explored.This study will reveal the specific molecular mechanism of persistent infection mediating host cell fate and inflammatory response and provide an essential scientific basis for further searching and developing new Ct prevention and treatment drugs.Methods:1.Hierarchical clustering analysis of differential expression data of lncRNAs and mRNAs after 12 h,24 h,and 40 h of Ct persistent infection;GO and KEGG were used to analyze the function and signaling pathway enrichment of differentially expressed mRNAs in persistent infection.2.q RT-PCR validation of persistent infection lncRNAs and mRNAs chip data,10 up-regulated lncRNAs(LINC01128,MIAT,CYTOR,ZEB1-AS1,FRMD6-AS2,NNT-AS1,LINC00240,EBLN3 P,PANDAR,IRF1),5 down-regulated lncRNAs(LINC00466,SEMA3B-AS1,KRT7-AS,HCG18,LINC00163)and 3 up-regulated mRNAs(NEDD4L,MAP4K4,ZEB1)were randomly selected to detect.3.Bioinformatics software such as Lncbase,ENCORI,miRDB,and Clue Go was used to screen key lncRNAs and mRNAs,and Cytoscape constructed the lncRNA-miRNA-mRNA regulatory network to analyze the enrichment function and signal pathway of the regulatory network.4.q RT-PCR was used to detect the interference effect of lncRNA MIAT,ZEB1-AS1,and IRF1 specific si RNA.FCM and Hoechst staining were used to detect the apoptosis rate of persistently infected cells before and after interference with MIAT,ZEB1-AS1,and IRF1,respectively.JC-1 fluorescent probe was used to detect mitochondrial membrane potential before and after interference with MIAT,ZEB1-AS1,and IRF1.Western blot was used to detect apoptosis-related proteins Bax,Bcl-2,and cleaved caspase 3 before and after MIAT,ZEB1-AS1,and IRF1 interference.IFA witnessed cytochrome c release and Ct infection rate before and after interference with MIAT,ZEB1-AS1,and IRF1.5.Lnc ATLAS analysis and RNA fluorescence in situ hybridization were used to verify the subcellular localization of ZEB1-AS1.RNAfold predicted the minimum free energy secondary structure of ZEB1-AS1.The binding sites of miR-1224-5p to ZEB1-AS1 and MAP4K4 were verified by ENCORI prediction and the double luciferase reporter gene.6.After transfection with siZEB1-AS1,si MAP4K4,miR-1224-5p mimic,and miR-1224-5p inhibitor for 24 h,He La cells were treated with Ct persistent infection.q RT-PCR was used to detect the expressions of ZEB1-AS1,miR-1224-5p,and MAP4K4 before and after transfection.FCM and Hoechst staining were used to detect the apoptosis rate.The mitochondrial membrane potential was detected by JC-1 fluorescent probe.IFA was used to see cytochrome c release.Western blot was used to detect the expression of apoptosis-related proteins Bax,Bcl-2,and cleaved caspase 3 and further detect the activation of the MAPK signaling pathway.7.Lentivirus vectors LV10N-si NC and LV10N-si MAP4K4 were constructed and then infected RAW 264.7 cells for 48 h.IFA detected the expression of m Cherry red fluorescence,and q RT-PCR detected the transcription level of MAP4K4 to verify the silencing effect of lentivirus transfection.8.IFA detected the effects of si MAP4K4 and ERK inhibitors on Ct and Cm infection rates.Mouse Cytokine Array G2 was used to detect the impact of si MAP4K4 and ERK inhibitors on the expression of 32 cytokines in Cm infection RAW 264.7.Bioinformatics analysis of differentially expressed cytokine enrichment function and signaling pathways.9.BALB/c mouse model of Cm reproductive tract infection was constructed.Mice after infection were treated with LV10N-si NC,LV10N-si MAP4K4,DMSO,and an ERK inhibitor,respectively,and were divided into SPG,Cm,Cm+LV10N-si NC,Cm+LV10N-si MAP4K4,Cm+DMSO,and Cm+PD98059 groups.IFA,q RT-PCR,and IHC were used to verify the effect of LV10N-si NC and LV10N-si MAP4K4.On days 3,6,9,12,15,18,21,24,27,and 30 after infection,the vaginal swab was used to take genital tract secretions of mice,and IFA was used to detect the clearance of Chlamydia in each group.ELISA detected the inflammatory cytokines in serum and reproductive tract secretion of mice.CCK-8 detected spleen cell proliferation.Flow cytometry was used to detect the levels of CD4+,CD8+,and cytokines in spleen cells.10.On the 15 th day after Cm infection,HE staining was used to detect the pathological status of acute inflammation in mouse reproductive tract tissues.IHC detected the expression of inflammatory molecules(MPO,IBA-1,etc.)in reproductive tract slices.On the 60 th day after Cm infection,mice’s reproductive system,liver,spleen,and kidney were isolated,and the degree of hydrosalpinx of mice was observed and scored by naked eyes.The pathological injury of the oviduct,uterine horn,liver,spleen,and kidney was evaluated by HE staining.The differential expression of MAP4K4,MPO,and IBA1 in the fallopian tubes was detected by IHC.Results:1.Hierarchical cluster analysis showed that many lncRNAs and mRNAs with similar expression patterns were clustered at 24 h after persistent infection;GO analysis showed that the differentially expressed mRNAs were enriched in cell fate,innate immune response,and cytokine response.KEGG analysis showed that differentially expressed mRNAs were enriched in the NOD-like receptor signaling pathway,NF-κB signaling pathway,and TNF signaling pathway.2.q RT-PCR results showed that lncRNA MIAT,LINC01128,CYTOR,ZEB1-AS1,FRMD6-AS2,NNT-AS1,LINC00240,EBLN3 P,PANDAR,and IRF1 were up-regulated by persistent infection.LINC00466,SEMA3B-AS1,KRT7-AS,HCG18,and LINC00163 were down-regulated.The expressions of mRNA NEDD4 L,MAP4K4,and ZEB1 were up-regulated by persistent infection,and the results were consistent with the trend of lncRNAs and mRNAs expression profile data.3.Bioinformatics analysis screened out critical lncRNA MIAT,ZEB1-AS1,and IRF1 and constructed the lncRNA-miRNA-mRNA regulatory networks.Clue Go found that the regulatory network is enriched in mitochondrial depolarization response,spliceosomal tri-sn RNP complex assembly,and post-translational protein targeting membrane translocation.4.Compared with the si NC group,q RT-PCR results showed that the corresponding lncRNA transcription levels were significantly down-regulated in si MIAT,si ZEB1-AS1,and si IRF1 groups,respectively(P<0.01).Hoechst staining results showed that apoptosis rates of the Ct-si MIAT group,Ct-si ZEB1-AS1 group,and Ct-si IRF1 group were 17.29%(P<0.01),13.27%(P<0.05),and15.08%(P<0.05),respectively,higher than 9.21% in Ct-si NC control group.The results of flow cytometry were consistent with those of Hoechst staining.JC-1 results showed that MMP in Ct-si MIAT and Ct-si ZEB1-AS1 group was significantly lower than that in the Ct-si NC group(P<0.01).Western blot results showed that compared with the Ct-si NC group,the ratio of Bcl-2/Bax was down-regulated,and cleaved caspase 3 was elevated in the Ct-si MIAT and Ct-si ZEB1-AS1 groups(P<0.05).IFA results showed that compared with the Ct-si NC group,the release of cytochrome c in Ct-si MIAT and Ct-si ZEB1-AS1 groups was significantly increased.IFUs of the Ct-si MIAT group was significantly higher than that of the Ct-si NC group(P<0.01),and the mean diameter of the inclusion body in the Ct-si MIAT group was 1.5 times that in the Ct-si NC group(P<0.01).5.Lnc ATLAS prediction results showed that 53.2% of ZEB1-AS1 was localized in the cytoplasm of He La cells,and RNA-FISH results showed that ZEB1-AS1 was mainly distributed in the cytoplasm.RNAfold predicted that the most stable structure of ZEB1-AS1 included the stem region,the inner ring,and the hairpin ring region.ENCORI prediction and the double luciferase reporter gene results showed that miR-1224-5p had interaction sites with ZEB1-AS1 and miR-1224-5p with MAP4K4.6.q RT-PCR and Western blot results showed that ZEB1-AS1 could indirectly regulate the expression of MAP4K4 through the ce RNA mechanism of miR-1224-5p adsorption.Hoechst results showed that miR-1224-5p overexpression increased the apoptosis rate of He La cells,while Ct infection reduced the apoptosis rate induced by miR-1224-5p overexpression(P<0.01).Ct-si MAP4K4 significantly promoted the apoptosis of Ct persistently infected cells(P<0.01).Flow cytometry results were consistent with Hoechst staining results.JC-1 results showed that compared with the negative group,miR-1224-5p mimic and si MAP4K4 could decrease MMP(P<0.01)and significantly increase cytochrome c release(P<0.05).However,Ct persistent infection could alleviate the effects of miR-1224-5p overexpression on MMP and cytochrome c release(P<0.05).Western blot results showed that compared with the negative control group,miR-1224-5p mimic and Ct-si MAP4K4 group decreased Bcl-2/Bax ratio and increased cleaved caspase 3 expression(P<0.05).Compared with the IFN-γ control group,the level of p/t-ERK in the persistent infection group was increased(P<0.05),and the level of p/t-ERK in the Ct-si ZEB1-AS1 or Ct-si MAP4K4 group was significantly decreased(P<0.05).7.IFA results showed that the expression of red fluorescence was observed in LV10N-si NC or LV10N-si MAP4K4 group.q RT-PCR results showed that LV10N-si MAP4K4 significantly down-regulated MAP4K4 compared with the LV10N-si NC group(P<0.05).8.IFA results showed that siMAP4K4 and PD98059(20 μM)significantly inhibited the infection rate of Ct in He La cells compared with the control group(P<0.05).Si MAP4K4 and PD98059 could significantly inhibit the infection rate of Cm in RAW 264.7 cells(P<0.01).Mouse Cytokine Array results show that compared with the control group,G-CSF,IL-12,and IL-6 were up-regulated after si MAP4K4 treatment,CTACK,CCL11,IL-2,IL-10,IL-17,TNF-α,and THPO were down-regulated after PD98059 treatment.After si MAP4K4+PD98059 co-treatment,CTACK,CCL11,IL-2,IL-10,IL-12,IL-17,KC,MIP-2,s TNFR1,TNF-α,and THPO all showed down-regulation trend(P<0.05).GO,and KEGG analysis showed that the differentially expressed cytokines and chemokines were enriched in the inflammatory response,T cell proliferation regulation,JAK-STAT signaling pathway regulation,leukocyte mediated immune regulation,etc.9.Lentivirus can infect mouse spleen cells successfully.q RT-PCR results showed that compared with the control group,MAP4K4 expression in the spleen,liver,kidney,and uterus of the Cm-LV10N-si MAP4K4 group was significantly down-regulated(P<0.05),and IHC results showed that compared with the control group,the expression of MAP4K4 in the uterus of Cm-LV10N-si MAP4K4 group was down-regulated.IFA results showed that LV10N-si MAP4K4 significantly reduced the bacterial load in the vagina of mice on the 6th day after infection,and PD98059 significantly reduced the bacterial load in the vagina of mice on the 6th day,9th day,12 th day,and 15 th day after infection.CCK-8 test results showed that the stimulation indexes of spleen cells in SPG,Cm,Cm+si NC,Cm+si MAP4K4,Cm+DMSO,Cm+PD98059,and Con A groups were 0.971±0.01,1.83±0.08,1.73±0.08,2.78±0.16,1.52±0.15,2.26±0.03,3.16±0.09,respectively,infection group was significantly higher than SPG control group.The spleen cell stimulation index of the Cm+si MAP4K4 group was higher than that of the Cm+si NC group;the Cm+PD98059 group was higher than the Cm+DMSO group(P<0.01).Flow cytometry results showed that on the 7th day after infection,the proportion of CD4+,CD8+ cells in the infected group was higher than that in the SPG control group,and the ratio of CD8+ cells in the Cm+si MAP4K4 group was significantly higher than that in Cm+si NC group(P<0.05).On the 15 th day after infection,the ratio of CD4+/CD8+ in SPG,Cm,Cm+si NC,Cm+si MAP4K4,Cm+DMSO,and Cm+PD98059 group spleen cells was 1.64±0.20,1.84±0.04,1.80±0.07,2.26±0.01,1.96±0.16,1.92±0.03,respectively,infection group was significantly higher than SPG control group,Cm+si MAP4K4 ratio was higher than Cm+si NC group(P<0.05),compared with SPG control group,CD8+/IFN-γ,CD8+/TNF-α,CD4+/IFN-γ,and CD4+/TNF-α levels in the infected group were significantly increased,while CD8+/IL-4 and CD4+/IL-4levels were not significantly changed(P<0.05);CD8+/IFN-γ,CD8+/TNF-α,and CD4+/TNF-α levels in Cm+si MAP4K4 group compared with Cm+si NC control group were significantly increased(P<0.05),while CD8+/IL-4 and CD4+/IL-4 levels were not significantly changed.There were no significant differences in IFN-γ,TNF-α,and IL-4 levels between Cm+DMSO and Cm+PD98059groups.10.On the 15 th day after infection,total uterine horn enlargement was observed in the Cm,Cm+si NC,Cm+si MAP4K4,and Cm+DMSO groups,while no uterine enlargement was observed in the Cm+PD98059 group.HE staining results showed that the infiltration of inflammatory cells in the Cm+si MAP4K4 group was more severe than that in the Cm+si NC group.At the same time,there were only a few inflammatory cells in the Cm+PD98059 group compared with the Cm+DMSO group.On day 60 after infection,the incidence of hydrosalpinx observed by the naked eye was 100% in other infection groups,except for Cm+si NC and Cm+si MAP4K4 group,which was75%(n=4).The hydrosalpinx score was 0.00±0.00(SPG),3.50±0.58(Cm),3.00±2.16(Cm+si NC),1.50±1.29(Cm+si MAP4K4),3.75±1.71(Cm+DMSO),and 1.25±0.50(Cm+PD98059),respectively,PD98059 could significantly reduce the hydrosalpinx score caused by Cm,while si MAP4K4 showed no significant difference.HE staining results showed that tubal lumen dilated and inflammatory cells infiltrated mainly by monocytes occurred in all infection groups compared with the SPG control group.The score of tubal dilation degree in the Cm+si MAP4K4 and Cm+PD98059 groups was significantly lower than that in the control group,and the score of inflammatory cell infiltration in the Cm+PD98059 group was markedly lower than that in the control group.Conclusions:1.Ct inhibited mitochondrial apoptosis pathway and Chlamydia growth by upregulating lncRNA MIAT.2.Ct regulates miR-1224-5p /MAP4K4 molecular axis by upregulation of lncRNA ZEB1-AS1 and activates MAPK/ERK signaling pathway to resist apoptosis of host cells and influence Chlamydia infection rate.3.Ct inhibited T cell immunity and IFN-γ secretion by upregulation of MAP4K4 and promoted Chlamydia survival and chronic inflammatory injury of mouse reproductive tract.4.MAPK/ERK signaling pathway is closely related to the clearance of Chlamydia in the mouse reproductive tract.It aggravates tubal edema caused by Chlamydia infection by promoting the secretion of pro-inflammatory factors. |
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