| BackgroundSystemic lupus erythematosus(SLE)is a chronic,multisystem autoimmune disease with a striking heterogeneity of clinical presentations that can involve multiple systems and cause multi-organ damage.The pathogenesis of SLE is complex and has not yet been fully elucidated.N6-methyladenosine(m6A)modification is one of the most important epigenetic modifications in eukaryotes and dynamically and reversibly regulates the transcription,processing,splicing,translation and degradation of RNA without changing the DNA sequence.m6A modification is known to play a vital role in the occurrence and development of autoimmune diseases.Long noncoding RNA(lnc RNA)are a group of transcripts that are longer than 200 nucleotides and lack protein-coding ability.They can directly influence the development,activation and differentiation of immune cells and regulate innate and adaptive immune responses.Substantial evidence has shown that some of the many lnc RNA implicated in SLE have emerged as therapeutic targets and viable biomarkers.Previous studies have revealed the critical roles of the m6A modification of lnc RNA in some diseases,but the association of m6A and lnc RNA in SLE remains unclear.ObjectiveThis study therefore aims to discern m6A-related lnc RNA in SLE,evaluate their diagnostic value in patients with SLE,and explore their possible mechanisms in the pathogenesis of SLE,providing evidence for disease etiology and prevention.MethodsCombined analysis of methylated RNA immunoprecipitation sequencing(Me RIP-seq)and transcriptomic RNA sequencing(RNA-seq)in PBMCs of SLE patients and healthy controls,identified 6 lnc RNA with significant differences in m6A peaks and expression levels.Quantitative reverse transcription-polymerase chain reaction(q RT-PCR)was used to validate potential m6A-related lnc RNA.Then,we explored the association between the expression levels of the differentially expressed lnc RNA and the clinical characteristics of SLE patients usingc~2 test and Spearman’s rank correlation test and utilized receiver operating characteristic(ROC)curves to investigate their potential value as biomarkers for SLE.In addition,the m RNA and protein levels of m6A methyltransferases were measured by q RT-PCR and Western blot in SLE patients and healthy controls,and Spearman’s rank correlation was used to analyse its correlation with the aberrantly expressed lnc RNA.After knockdown of METTL3 in Jurkat cells,the expression level of LINC02035 was measured by q RT-PCR.We also conducted Me RIP-q PCR to identify whether LINC02035 was a target of METTL3.Nuclear-cytoplasmic fractionation was conducted to determine the localization of LINC02035 in Jurkat cells.Bioinformatics tools were used to predict the mi RNAs that can bind to LINC02035 and the m RNAs that can bind to mi R-107,which were further confirmed by luciferase reporter assay,western blot and q RT-PCR.After sh-LINC02035 or mi R-107 mimics or si-PTEN transfection,Western blot and RT-PCR were used to detect the expression of mi R-107 and/or PTEN,and cell counting kit-8 assay and flow cytometry analysis were performed to explore the effect on the proliferation of Jurkat cells.Finally,the sh RNA targeting LINC02035 in parallel with mi R-107 inhibitor were co-transfected into Jurkat cells,followed by a series of functional experiments.ResultsPhase 1:Screening lnc RNA with significant differences in m6A peaks and expression levels in PBMCs of SLE patientsBased on the results of Me RIP-seq and RNA-seq in PBMCs of 3 SLE patients and 3healthy controls,a total of six lnc RNA with significant differences in m6A peaks and expression levels were selected,namely,CYTOR,LINC02035,LINC00847,LINC01001,LINC02446 and ERVK13-1.Phase 2:Verification of m6A-related lnc RNA and m6A methyltransferases in PBMCs of SLE patients(1)The expression levels of six potential m6A-related lnc RNA were determined in PBMCs of 102 SLE patients and 107 healthy controls.Decreased expression of LINC02035,LINC00847 and ERVK13-1 were observed in SLE patients(LINC02035:Z=-4.029,P<0.001;LINC00847:Z=-3.631,P<0.001;ERVK13-1:Z=-4.762,P<0.001).We further explored the association between the expression levels of three differentially expressed lnc RNA(LINC02035,LINC00847 and ERVK13-1)and the clinical characteristics of 102 patients with SLE.The expression level of LINC02035was associated with pericarditis,pyuria and serum creatinine(all P<0.05).The expression level of LINC00847 in PBMCs was associated with anti-ds DNA antibodies and haemoglobin(all P<0.05).The expression level of ERVK13-1 was associated with pericarditis,alopecia,and anti-centromere protein B(CENPB)antibodies(all P<0.05).The areas under the ROC curves of LINC02035 and ERVK13-1 were over 0.700,indicating certain diagnostic value.(2)The expression levels of four m6A methyltransferases were determined in PBMCs of 74 SLE patients and 74 healthy controls.Decreased expression levels of METTL3,METTL14,WTAP,and FTO were observed in SLE patients(METTL3:Z=-5.999,P<0.001;METTL14:Z=-4.313,P<0.001;WTAP:Z=-3.349,P=0.001;FTO:Z=-5.999,P=0.017),among which the expression level of METTL3 was the lowest.Additionally,the m6A level in PBMCs of SLE patients was significantly lower than that in controls(t=-3.663,P=0.003).Therefore,METTL3 was selected as the m6A methylase in the current study.Consistent with the expression level,the protein level of METTL3 was also significantly decreased in PBMCs of patients with SLE compared with that in healthy controls(t=4.936,P=0.008).Phase 3:Verification of differentially expressed lnc RNA and METTL3 in T cells of SLE patients(1)Three differentially expressed lnc RNA in PBMCs were further verified in T cells from 102 SLE patients and 102 healthy controls.The results showed that the expression levels of LINC02035,LINC00847 and ERVK13-1 in T cells of SLE patients were significantly downregulated compared with those in healthy controls(LINC02035:Z=-4.120,P<0.001;LINC00847:Z=-2.203,P=0.028;ERVK13-1:Z=-4.604,P<0.001).We further explored the relationship between the expression levels of three differentially expressed lnc RNA and the clinical characteristics of 102 patients with SLE.The expression level of LINC02035 was associated with Raynaud’s phenomenon,cylindruria,anti-C1q antibodies,platelets,and the SLEDAI score(all P<0.05).The expression level of LINC00847 was associated with fever,rash,taking glucocorticoids,anti-C1q antibodies and the SLEDAI score(all P<0.05).The expression level of ERVK13-1 was associated with fever,cylindruria,taking glucocorticoids,anti-C1q antibodies,the SLEDAI score and high-density lipoprotein cholesterol(all P<0.05).We found that the AUCs of LINC02035 and ERVK13-1 were over 0.700,indicating certain diagnostic value.(2)To further verify the expression level of METTL3 in T cells,46 SLE patients and46 healthy controls were recruited,and the results showed that the m RNA levels of METTL3 were decreased in T cells of patients with SLE(Z=-3.514,P<0.001).Consistent with the expression level,the protein level of METTL3 was also lower in T cells of SLE patients(t=3.116,P=0.036).Phase 4:METTL3-mediated m6A is associated with the upregulation of LINC02035(1)q RT-PCR results indicated that METTL3 expression levels were positively correlated with the levels of LINC02035 in PBMCs and T cells of SLE patients(PBMCs:r_s=0.811,P<0.001;T cells:r_s=0.844,P<0.001).(2)We found decreased expression of LINC02035 in METTL3 knockdown Jurkat cells(t=5.911,P<0.001).Moreover,LINC02035 was a target of METTL3(t=-4.993,P=0.038).Phase 5:Functional study of m6A modifications of LINC02035(1)Silencing LINC02035 significantly increased the proliferative capability of Jurkat cells(all P<0.05).(2)LINC02035 was mostly located in the cytoplasm of Jurkat cells,which indicated that LINC02035 may sponge mi RNA.By utilizing a bioinformatics tool,we found that mi R-107 contains a complementary binding site within LINC02035,which was further confirmed by a luciferase reporter assay.Jurkat cells with LINC02035 knockdown exhibited increased expression of mi R-107(t=-10.248,P<0.001).In addition,the expression of mi R-107 was significantly elevated in the T cells of SLE patients(Z=-4.808,P<0.001),and the expression of LINC02035 was negatively correlated with mi R107 expression(r_s=-0.428,P=0.023).(3)Jurkat cells transfected with mi R-107 mimic presented a markedly increased proliferation ability(all P<0.05).(4)By utilizing a bioinformatics tool,we found that PTEN contains the complementary binding site within mi R-107,which was further confirmed by a luciferase reporter assay.Decreased m RNA and protein expression of PTEN were observed in mi R-107-overexpressed Jurkat cells(both P<0.05).In addition,the m RNA and protein expression of PTEN were significantly reduced in T cells of SLE patients(both P<0.05),and the expression of PTEN was negatively correlated with mi R-107expression(r_s=-0.538,P=0.001).(5)Silencing PTEN significantly increased the proliferative capability of Jurkat cells(all P<0.05).(6)Jurkat cells with LINC02035 overexpression showed decreased PTEN m RNA and protein levels(both P<0.05).In addition,the expression of PTEN was positively correlated with LINC02035 expression in T cells of SLE patients(r_s=0.596,P=0.001).(7)Rescue experiments showed that co-transfection with sh-LINC02035 and mi R-107 mimic not only increased the m RNA and protein expression of PTEN(both P<0.05)but also induced the protein expression of p-AKT(t=5.677,P=0.005).Furthermore,Jurkat cells co-transfected with sh-LINC02035 and mi R-107 mimic showed increased proliferative ability compared to the expression in Jurkat cells not transfected with mi R-107 mimic.Conclusions(1)Three m6A methylation-related lnc RNA were found to be associated with the pathogenesis of SLE,namely,LINC02035,LINC00847 and ERVK13-1.(2)The lnc RNA(LINC02035 and ERVK13-1)in PBMCs and T cells may serve as potential novel biomarkers for SLE.(3)LINC02035 was regulated by m6A modification and promoted the expression of PTEN by sponging of mi R-107,inhibiting the activation of the AKT pathway and eventually regulating the proliferation of T cells. |
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