| Systemic lupus erythematosus(SLE)is an autoimmune disease that can involve multiple organs in the host and has high clinical heterogeneity.Its pathogenesis includes abnormally activated immune cells,abnormal overproduction of antibodies,and various inflammatory factors attacking the host’s autoantigens,leading to a series of inflammatory reactions,including complement activation,deposition of immune complexes and so on.Ultimately,it resulted in the damage to various organs within the host body.T lymphocytes,especially CD4+T lymphocytes,play an important role in assisting B cells in generating antibodies,secreting pro-inflammatory cytokines,and infiltrating inflammatory tissues.In the common involved organs of SLE patients,such as the kidneys and skin,there is a significant infiltration of activated CD4+T lymphocytes,accompanied by tissue damage.CD4+T cells,as helper T cells,have many subsets.In SLE patients,the proportion of CD4+T subsets is unbalanced to varying degrees.However,to date,the specific mechanism of abnormal activation and differentiation of CD4+T cells in SLE has not been fully elucidated.Human endogenous retroviruses(HERVs)are sequences that remain in the host genome after the genetic information of the virus is integrated into the host genome through infection by exogenous retroviruses millions of years ago.Most HERVs have lost the ability to express viral particles following the accumulating mutations,deletions,and recombinations.In pathological conditions,HERVs can be abnormally activated to form viral particles and participate in the occurrence of autoimmune diseases through the molecular mimicry mechanism.To date,little is known about the expression and regulatory role of HERVs in peripheral blood CD4+T cells of SLE patients.Long non-coding RNAs(LncRNAs)are RNA molecules that are longer than 200 nucleotides(nt)and do not encode proteins.Many studies have shown that lncRNAs have vital roles in CD4+T cells of SLE,such as interleukin-21 antisense RNA 1(IL21-AS1)promoting the differentiation of T follicular helper cells,and lncRNA GAS5 inhibiting CD4+T cell activation.Evidence has shown that lncRNAs transcribed from HERVs participating in the regulation of different diseases.There is still a lack of reports on the regulation of lncRNAs derived from HERVs on the activation and differentiation of CD4+T cells in SLE.Our team conducted two high-throughput sequencing,including RNA sequencing(RNA-seq)and whole transcriptome sequencing,on peripheral blood CD4+T cells from SLE patients and healthy volunteers,and screened single-strand RNAs transcribed by HERVs through the J2-double stranded RNAs(dsRNAs)enrichment experiment and the RNase A digestion experiment.This project will reveal the role and mechanism of HERVsderived lncRNAs in the abnormal activation and differentiation of CD4+T cells from SLE patients through the following three parts.Part Ⅰ:detection of HERVs expression in various types of cells,organs in SLE or various autoimmune diseases.Part Ⅱ:exploring the regulatory effect of HERVsderived lncRNAs on the activation and differentiation of CD4+T cells.PartⅢ:exploring the molecular mechanism of HERVs-derived lncRNAs regulating CD4+T cell differentiation.This study revealed that HERVs were in a broad range of transcriptional activation in SLE.HERVs-derived lncRNAs promoted the T-helper 1(Th1)cell differentiation by interacting with protein kinase R(PKR)and regulating the protein expression level of PKR.Part Ⅰ Detection of RNA expression levels of HERVs in SLE and different autoimmune diseasesSection Ⅰ Detection of RNA expression levels of HERVs in different immune cells and tissues of SLE patientsObjective:To investigate the RNA expression levels of HERVs in various immune cells in peripheral blood of SLE patients,including CD4+T cells,CD 19+B cells,CD 14+monocytes,and renal tissue of patients with lupus nephritis(LN).Methods:Peripheral blood samples were collected from 20 healthy controls(HCs)and 18 SLE patients,and CD4+T cells,CD 19+B cells,and CD 14+monocytes were obtained using immunomagnetic bead sorting technology,respectively;in addition,renal tissue from 10 LN patients and normal adjacent tissues from 10 renal clear cell carcinoma patients were collected.Total RNA was extracted using the TRIzol method and subjected to RT-qPCR experiments.The RNA expression levels of 18 HERVs selected from sequencing results were detected in different immune cells and organs of SLE patients.Results:1.Compared to HCs,the RNA expression levels of 15 HERVs in peripheral blood CD4+T cells of SLE patients was significantly increased,consistent with sequencing results,namely MER21C,ERV316A3-Int,LTR16A,MLT1F1,LTR56,MER4A1,MLT1A0-130,THE1C288,THE1C-376,MLT2B3 and MER65A(P<0.0001),THE1B-367 and MLT1B(P<0.001),LTR40C and THE1B-362(P<0.01).There was no significant difference in the expression of the three HERVs.2.Compared to HCs,the expression of 14 HERVs was significantly increased in CD19+B cells of SLE patients,including LTR40C,MLT1A0,THE1C-288,THE1C-376,MLT2B3,LTR62 and MER65A(P<0.0001),ERV3-16A3-I-Int,MER4A1,MLT1A0-130 and MLT1B(P<0.001),LTR16A,THE1B-367 and THE1C-int(P<0.01).There was no significant difference in the expression of the four HERVs.3.Compared to HCs,the expression of 11 HERVs was significantly increased in CD14+monocytes of SLE patients,including MER4A1,THE1C-288,and MLT2B3(P<0.0001),THE1C-376,and MER65A(P<0.001),MER21C,THE1B-367,MLT1A0,and MLT1B(P<0.01),ERV3-16A3-I Int,and LTR62(P<0.05).There was no significant difference in the expression of the seven HERVs.4.Compared with the normal tissue,the expression of 11 HERVs in LN renal tissue was significantly increased,namely THE1C-288(P<0.0001),THE1B-367,THE1C-376,MLT2B3 and MER65A(P<0.001),MER21C and MLT1A0(P<0.01),LTR16A,MLT1A0-130,MLT1B and THE1C-int(P<0.05).There was no significant difference in the expression of the seven HERVs.Conclusion:HERVs exhibited extensively transcriptional activation in SLE patients.Section Ⅱ Detection of RNA expression levels of HERVs in peripheral blood CD4+T cells of patients with different autoimmune diseasesObjective:To investigate the expression levels of HERVs in peripheral blood CD4+T cells from various autoimmune diseases,including psoriasis,rheumatoid arthritis(RA),Sjogren’s syndrome(SS),dermatomyositis(DM).Methods:Peripheral blood CD4+T cell samples were collected from 16 HCs,20 psoriasis patients,20 RA patients,20 SS patients,and 10 DM patients.Total RNA was extracted by TRIzol method and RT-qPCR was performed to detect the expression of 18 HERVs in peripheral blood CD4+T cells from different autoimmune diseases.Results:1.Compared to HCs,the expression of six HERVs in psoriasis CD4+T cells was significantly increased,namely MER21C,ERV3-16A3-Int and MLT1B(P<0.0001),MLT1F1(P<0.001),LTR56 and MLT1A0-130(P<0.05).The expression of two HERVs was significantly reduced,namely THE1B-367 and LTR40C(P<0.0001),and there was no significant difference in the expression of ten HERVs.2.Compared to HCs,the expression of 8 HERVs in RA CD4+T cells was significantly increased,namely MER21C and MLT1F1(P<0.001),LTR56 and MER4A1(P<0.01),THE1C-288,THE1C-376,MLT2B3,and MER65A(P<0.05).The expression of 2 HERVs was significantly reduced,namely THE1B-367 and LTR40C(P<0.0001),and there was no significant difference in the expression of 8 HERVs.3.Compared to HCs,the expression of six HERVs was significantly upregulated in SS CD4+T cells,namely ERV3-16A3-I Int and MLT2B3(P<0.001),MER21C,THE1C-376,and MLT1B(P<0.01),and THE1C-288(P<0.05).The expression of two HERVs was significantly reduced,namely THE1B-367 and LTR40C(P<0.0001),and there was no significant difference in the expression of ten HERVs.4.Compared to HCs,the expression of 13 HERVs was significantly upregulated in DM CD4+T cells,namely LTR56 and MLT2B3(P<0.0001),MER21C,LTR16A,MLT1F1,THE1C-288,THE1C-376,LTR62 and MER65A(P<0.001),ERV3-16A3-Int,MER4A1 and MLT1A0(P<0.01),MLT1A0-130(P<0.05).The expression of 2 HERVs was significantly reduced,namely LTR40C(P<0.0001)and THE1B-367(P<0.01),There was no significant difference in the expression of three HERVs.Conclusion:CD4+T cells in different autoimmune diseases exhibited varying degrees of HERVs activation.Section Ⅲ Detection of RNA expression levels of HERVs in human peripheral blood CD4+T cells at different activation time pointsObjective:To investigate whether the expression of HERVs is timedependent in activated CD4+T cells.Methods:Peripheral blood samples from 3 normal individuals were collected and CD4+T cells obtained from sterile sorting were activated with anti-CD3 and anti-CD28 antibodies at different time points,including 0 h(unactivated),6 h,12 h,24 h,48 h,and 72 h.Cells were collected for the RT-qPCR experiment to detect the expression of 18 HERVs at different activation time points.Results:Compared to the inactive resting state,the expression levels of 7 HERVs were significantly upregulated to varying degrees at 6 and 12 hours of activation.However,after 12 hours,the expression levels decreased to or below the resting state,namely LTR16A,THE1B-367,MER4A1,MLT1A0,THE1C-288,THE1C-376,and MLT1B.The expression of 8 HERVs also showed an upward trend within 12 hours of activation,and after 12 hours,the expression decreased to or below the resting state,namely MER21C,LTR40C,LTR56,MLT1A0-130,MLT2B3,THE1B-362,MER65A,and THE1C-int.The expression of two HERVs showed a downregulation trend at 6 hours of activation,an upregulation trend at 12 hours of activation,and a decrease in expression to or below the resting state after 12 hours,namely MLT1F1 and LTR62;Only one HERVs,ERV3-16A3-Int,showed an upregulation trend at 6 hours of activation,and was downregulated to 24 hours after 6 hours,with significant upregulation after 24 hours.Conclusion:The expression changes of HERVs in CD4+T cells with different activation states were in a time-dependent fashion.Part Ⅱ Characteristics and biological functions of lncRNA MER21CObjective:To investigate the attribute characteristics of lncRNA MER21C transcribed by HERVs and its regulatory function on the activation and differentiation of CD4+T cells.Methods:1.The Spearman correlation analysis was used to study the correlation between MER21C in CD4+T cells from SLE patients and the Systemic Lupus Erythematosus Disease Activity Score(SLEDAI).2.The full length of MER21C was identified by rapid amplification of cDNA ends(RACE)and the sequence length of MER21C was verified by northern blot.3.Querying the genomic location and species conservation of MER21C in the human genome(Hg19),as well as predicting the secondary structure of lncRNA using the online website.4.ZV302-MER21C-3 ×flag expression plasmid was transfected into HEK293T cells,and the protein encoding ability of MER21C was detected by western blot.5.Fluorescence in situ hybridization(FISH)was used to the subcellular localization of MER21C in the unactivated and activated CD4+T cells,naive CD4+T cells,and Thl cells,respectively.6.In human peripheral blood CD4+T cells,after overexpression of MER21C by the lentivirus infection,total RNA was extracted for RNAseq.After overexpression of MER21C by lentivirus infection,the ratio of activated CD4+T cells was detected by flow cytometry.7.After the specific antisense oligonucleotide(ASO)interference with MER21C,RT-qPCR was to detect IFNG mRNA levels,flow cytometry was performed to identify the proportion of Thl cell differentiation,and the chemotaxis assay was to detect the chemotactic function of Thl cells.Results:1.The expression level of MER21C in CD4+T cells of SLE patients was significantly positively correlated with SLEDAI score(R=0.7082,P<0.001).2.The full length of lncRNA MER21C is 1976nt,and northern blot shows a specific band of approximately 1821 bp.3.LncRNA MER21C is located on the anti-sense chain of the coding gene DDX60L on the chromosome 4,and has high conservatism only in primates,with multiple secondary structures present.4.The full length sequence of MER21C does not have the function of encoding protein.5.MER21C is localized in both the cytoplasm and nucleus of unactivated CD4+T cells,while in activated CD4+T cells,MER21C is mainly localized in the nucleus.MER21C is localized in both the cytoplasm and nucleus of naive CD4+T cells,while mainly distributed in the nucleus in Thl cells.6.RNA-seq showed that compared with the negative control group(MER21C-NC),there were 907 differentially expressed genes in CD4+T T cells overexpressing MER21C,of which 458 genes were up-regulated and 449 genes were down regulated.The GO pathway enrichment analysis suggested that most of the differentially expressed genes were enriched in cell migration,chemotaxis,metabolic pathways and inflammatory mediator secretion pathways.7.Compared with the negative control group(MER21C-NC),there was no significant difference in the activation proportion of CD4+T cells after overexpression of MER21C,the mRNA expression levels of C-C chemokine receptor type 5(CCR5)were significantly increased(P<0.05),and there was no significant difference in the proportion of CD4+CCR5+cells detected by flow cytometry.The mRNA expression levels of C-X-C motif chemokine receptor 3(CXCR3)showed an upward trend,and the proportion of CD4+CXCR3+cells detected by flow cytometry was significantly increased(P<0.05).8.Compared with the negative control group(ASO-NC),the expression of IFNG mRNA after interfering with ASO-MER21C was significantly reduced(P<0.05)and the percentage of Thl cells was significantly reduced.Under the chemotaxis of CXCL9 and CXCL10,compared with ASO-NC,there was no significant change in the migration quantity of Th1 cells after interfering with MER21C.Conclusion:1.HERV MER21C is an antisense lncRNA with a total length of 1976nt and does not have the ability to encode protein.2.The expression of MER21C is elevated in Th1 cells,promoting the differentiation of Th1 cells.Part Ⅲ Molecular mechanism of lncRNA MER21C regulating Th1 differentiationObjective:To explore the specific mechanism of lncRNA MER21C regulating Thl cell differentiationMethods:1.RNA pull-down and mass spectrometry were used to analyze and screen the proteins that bind to MER21C in Th1 cells.2.RNA binding protein immunoprecipitation(RIP)-qPCR was used to reversely verify the binding protein to MER21C.3.Western blot was performed to detect the expression of the target protein in naive CD4+T cells and Thl cells.The target protein inhibitor specifically inhibits the target protein and induces differentiation of naive CD4+T cells into Thl cells in vitro.The effect of the target protein inhibitor on Th1 cell differentiation was detected by flow cytometry.4.After ASO specific interference with MER21C,western blot was used to detect changes in the protein level of the target protein in Th1 cells.Results:1.RNA pull-down combined with mass spectrometry analysis screened 323 proteins that bind to lncRNA MER21C.2.The RIP-qPCR experiment confirmed the interaction between MER21C and protein PKR(P<0.05).3.Compared to naive CD4+T cells,the protein expression of PKR was increased in Th1 cells.PKR inhibitor(PKR-IN-C16)at a concentration of 5 μM significantly inhibited the expression of PKR protein.The flow cytometry results showed that a decrease in PKR expression could significantly inhibit the differentiation of Th1 cells(P<0.05).4.After interfering with MER21C,the protein expression of PKR in Th1 cells decreased.Conclusion:LncRNA MER21 C may promote Th1 cell differentiation by binding to the protein PKR and increasing its expression. |
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