MiR-146a-3p As A Potential Therapeutic By Targeting MBD2 To Mediate Th17 Cells Differentiation In Th17 Severe Asthma

Background Asthma is a heterogeneous chronic airway inflammation with different clinical manifestations(phenotype)and different pathological features(endotype).Different endotypes can reflect different pathophysiological processes of the disease,leading to differences in treatment effects.Severe asthma has steroid-resistance,poor treatment effect,high mortality,and bringing a significant economic and medical burden to families and society.T helper 17(Th17)is a subtype of non-T2 asthma,which has airway neutrophil infiltration.It is usually manifested as steroid-resistant and refractory asthma,also known as neutrophilic asthma,and is related to severe asthma.Th17 cells mainly produce cytokines such as interleukin-17(IL-17).IL-17 promotes the activation and chemotaxis of neutrophils to aggravate airway inflammation,which is related to neutrophil infiltration,steroid-resistance,airway hyperresponsiveness(AHR)and airway remodeling.It is an independent risk factor and biomarker of Th17 severe asthma,and is positively correlated with the severity of asthma.Steroids are the main anti-inflammatory treatment for asthma control,and it is generally accepted that Th17-dependent inflammation is less sensitive to steroids.Targeting Th17/IL-17 pathway may be a promising intervention strategy for severe asthma.Previous studies have shown that epigenetics are involved in Th17 severe asthma,but the underlying mechanisms are still unknown.Epigenetic inheritance mainly includes methylation,non-coding RNA and so on.Methyl-Cp G-binding domain2(MBD2),a member of the methylCpg-binding protein family,can recognize DNA methylation marks and regulate their expression,making it a key mediator of many epigenetic processes.Our previous study showed that MBD2 mediates Th17 differentiation by regulating multiple mediators in a mouse model of severe asthma.MBD2 is expected to be a therapeutic target for severe asthma.MicroRNAs(miRNAs),are small non-coding RNAs(18-22nt)that can bind to the 3’-UTR of target genes to negatively regulate gene expression.miR-146a-3p is expressed by leukocytes in peripheral blood,and its function is significantly related to inflammation and innate immunity,with anti-inflammatory effect.Although miR-146a-3p has previously been shown to suppress inflammatory responses in different diseases,tissues,and cells,its underlying mechanisms in severe asthma remain unclear,especially its effect on Th17 cell differentiation.Interestingly,miRNA database bioinformatics analysis showed that MBD2 may be the target gene of miR-146a-3p.We hypothesized that Mi R-146a-3p regulates Th17 differentiation by targeting MBD2 and has a potential therapeutic role in Th17-induced severe asthma.As antiinflammatory therapy for asthma,miR-146a-3p inhibiting Th17/IL17 axis may be an attractive candidate for further preclinical studies,and miR-146a-3p will provide a potential new treatment for Th17-dominated neutrophilic severe asthma.In this study,we aimed to investigate the expression of miR-146a-3p in severe asthma,how miR-146a-3p interacts with MBD2 to regulate Th17 differentiation,and the potential therapeutic role of miR-146a-3p in Th17-associated severe asthma.Based on the previous study,we conducted the following experiments in three steps:(1)eligible healthy volunteers and asthma patients were recruited,Flow cytometry was used to distinguish T2 and Th17 endotypes of severe asthma,the expression of miR-146a-3p in peripheral blood mononuclear cells(PBMCs)was detected by q RT-PCR,the expression of MBD2 in serum was detected by ELISA.(2)A House dust mite(HDM)/lipopolysaccharide(LPS)exposure asthma-like model of bronchial epithelial cells(BECs)was established.The interaction between miR-146a-3p and MBD2 was verified by overexpression and/or knockdown of miR-146a-3p,MBD2 and dual luciferase reporter gene assay,respectively.The effect of miR-146a-3p on the differentiation of Th17 cells in vitro was detected by flow cytometry.(3)To evaluate the potential therapeutic effect of miR-146a-3p on severe asthma by regulating the differentiation of Th17 cells in vivo.Part Ⅰ The expression of miR-146a-3p in PBMCs of asthmatic patientsObjective Eligible healthy volunteers and asthma patients were recruited,including 30 healthy control group,30 mild/moderate asthma group and30 severe asthma group.The expression of miR-146a-3p in peripheral blood mononuclear cells(PBMCs)was detected by q RT-PCR.Methods(1)Eligible healthy volunteers and asthma patients were enrolled,including 30 healthy control group,30 mild/moderate asthma group and30 severe asthma group.Clinical data were collected.(2)Peripheral blood was extracted,Th2 and Th17 differentiation in peripheral blood was detected by flow cytometry,miR-146a-3p expression in peripheral blood mononuclear cells(PBMCs)was detected by q RT-PCR,and MBD2 expression in serum was detected by enzymelinked adsorption assay(ELISA).Results(1)Severe asthma mainly includes T2 and Th17 asthma;Compared with patients with T2 severe asthma,Th17 severe asthma patients have higher proportion of Th17 cells,lower proportion of Th2 cells,more neutrophils and less eosinophils in peripheral blood.(2)Compared with T2 severe asthma,the expression of miR-146a-3p in Th17 severe asthma was significantly decreased,while the expression of MBD2 was significantly increased.Conclusions(1)The expression of miR-146a-3p in Th17 severe asthma was significantly decreased;(2)miR-146a-3p and MBD2 may be potential biomarker and therapeutic targets for Th17 severe asthma.Part Ⅱ miR-146a-3p mediated Th17 differentiation by targeting MBD2 in vitroObjective To verify the binding site of miR-146a-3p to MBD2,and to explore the interaction between miR-146a-3p and MBD2 and the effect of miR-146a-3p on Th17 cell differentiation in vitro.Methods(1)MBD2 wild-type dual luciferase reporter plasmid(WT MBD2-3’-UTR plasmid)and MBD2 mutant dual luciferase reporter plasmid(MT MBD2-3’-UTR plasmid)were constructed.MT MBD2-3 ’-UTR plasmid,mimic miR-146a-3p,negative control(mimic NC)were transfected into tool cells(293-T cells),and then luciferase was detected by dualluciferase reporter assay.(2)Naive CD4+T cells from BECs and spleen of normal mice were isolated and divided into 14 groups: Control group,HDM/LPS(HL)group,HL+mimic NC group,HL+mimic miR-146a-3p group,HL+mimic miR-146a-3p+si-NC group,HL+mimic miR-146a-3p+si-MBD2 group,and HL+mimic miR-146a-3p+OE-NC group,HL+mimic miR-146a-3p+OE-MBD2 group,HL+inhibitor NC,HL+inhibitor miR-146a-3p group,HL+inhibitor miR-146a-3p+si-NC group,HL+inhibitor miR-146a-3p+si-MBD2 group,HL+inhibitor miR-146a-3p+ OE-NC group,and HL+inhibitor miR-146a-3p+ OE-MBD2 group.HDM/LPS exposure model of bronchial epithelial cells(BECs)was established.BECs were transfected with mimic miR-146a-3p,negative control(mimic NC),inhibitor miR-146a-3p,negative control(inhibitor NC),small interfering RNA targeting MBD2(si-MBD2),negative control(si-NC),MBD2 overexpression plasmid(OE-MBD2),negative control(OE-NC),respectively and co-cultured with naive CD4+T cells.After cocultivation for 24 hours,the cells and cell culture medium were collected.q RT-PCR was used to detect the expression of miR-146a-3p in BECs,Western bloting(WB)was used to detect the expression of MBD2 in BECs and RORγt in CD4+T cells,flow cytometry was used to detect Th17 differentiation of naive CD4+T cells.ELISA was used to detect the expression of IL-17 in the cell culture medium supernatant.Results(1)Compared with mimic miRNA NC,miR-146a-3p effectively reduced WT-MBD2 3’-UTR luciferase activity(p<0.01),but did not change MT MBD2-3’-UTR luciferase activity;(2)Compared with the blank NC group,the relative expression of miR-146a-3p was down-regulated in HDM/LPS(HL)group(p<0.01);Compared with the HL+mimic NC group,the relative expression of miR-146a-3p in the HL+mimic miR-146a-3p group was increased(p<0.01),while the expression of miR-146a-3p did not change in HLimic miR-146a-3p+si-MBD2 grou+mp and HL+mimic miR-146a-3p+OE-MBD2 group.Compared with the HL+ inhibitor NC group,the relative expression of miR-146a-3p in the HL+ inhibitor miR-146a-3p group was decreased(p<0.01),while the expression of miR-146a-3p in HL+inhibitor miR-146a-3p+si-MBD2 group and HL+inhibitor miR-146a-3p+ OE-MBD2 group had no change.(3)Compared with the blank NC group,the expression of MBD2 protein was increased in HDM/LPS(HL)group(p<0.01);Compared with the HL+inhibitor NC group,the expression of MBD2 protein in the HL+inhibitor miR-146a-3p group was increased(p<0.01);Compared with the HL+inhibitor miR-146a-3p group,the expression of MBD2 protein in the HL+inhibitor miR-146a-3p+si-MBD2 group was decreased(p<0.01),while the expression of MBD2 protein in the HL+inhibitor miR-146a-3p+OE-MBD2 group was significantly increased(p<0.01);Compared with the HL+mimic NC group,the expression of MBD2 protein in the HL+mimic miR-146a-3p group was decreased(p<0.01);Compared with HL+mimic miR-146a-3p,the expression of MBD2 protein in HL+mimic miR-146a-3p+si-MBD2 group was further decreased(p<0.01),while the expression of MBD2 protein in HL+mimic miR-146a-3p+ OE-MBD2 group was significantly increased(p<0.01);(4)Compared with blank control group,the positive rate of Th17 in HL group was increased(p<0.01),and further increased after transfection with inhibitor miR-146a-3p(p<0.05).Compared with HL+inhibitor miR-146a-3p group,positive rate of Th17 decreased after transfection with inhibitor miR-146a-3p+si-MBD2(p<0.01),while positive rate of Th17 increased after transfection with inhibitor miR-146a-3p+OE-MBD2(p<0.01).Compared with HL group,the percentage of Th17 positive was significantly decreased in HL+mimic miR-146a-3p(p<0.01),and further decreased after transfecting miR-146a-3p+si-MBD2 group(p <0.05),while positive rate of Th17 was increased after transfecting mimic miR-146a-3p+OE-MBD2 simultaneously(p<0.01).The protein expression trend of RORγt(the key transcription factor of Th17)in CD4+T cells detected by WB was consistent with the trend of Th17 positive rate.In addition,the concentration of IL17 in the supernatant of cell culture medium detected by ELISA was also similar to the trend of Th17 positive rate.Conclusions(1)miR-146a-3p directly binds to MBD2 3’-UTR to inhibit the expression of MBD2;(2)miR-146a-3p inhibits Th17 cell differentiation by targeting MBD2.Part Ⅲ miR-146a-3p regulates Th17 cell differentiation and its potential therapeutic effect in a mouse model of severe Th17 asthma in vivoObjective A Th17-dominated neutrophilic asthma mouse model was constructed to evaluate the regulation of miR-146a-3p on Th17 cell differentiation and the potential therapeutic effect of miR-146a-3p in severe asthma in vivo.Methods(1)Wild type C57BL/6 mice were divided into 4 groups: blank control group,severe asthma group,severe asthma +NC Agomir group,and severe asthma +miR-146a-3p Agomir group,with 6 mice in each group.Normal saline,NC Agomir and miR-146a-3p Agomir were inhaled by atomization,respectively.Then,the intervention effect of miR-146a-3p Agomir on severe asthma was evaluated by observing the behavioral changes,airway hyperresponsiveness,total and differential cell counts of alveolar lavage fluid(BALF),and pathological changes of lung tissue.(2)lung tissue and CD4+T cells from spleen of mice in each group were obtained.Immunohistochemistry was used to detect MBD2,Ly6 g and ECP,Western blot was used to detect the expressions of MBD2 and RORγt,q RT-PCR was used to detect the expression of miR-146a-3p in lung tissue of mice in each group,flow cytometry was used to detect the positive rate of Th17,and ELISA was used to measure the concentration of IL-17 in BALF.Results(1)Compared with the blank control group,the mice in the severe asthma group and the severe asthma +NC Agomir group showed humanlike asthma symptoms after sensitization and challenge,while the mice in the severe asthma +miR-146a-3p Agomir group showed no obvious asthma symptoms after challenge.(2)With the increase of methacholine concentration(0,0.39,0.78,1.56,3.12 mg·m L-1),airway resistance gradually increased in each group.Compared with the blank control group,the airway resistance of the severe asthma group and the severe asthma +NC Agomir group was significantly increased(p<0.01);Compared with the severe asthma +NC Agomir group,the airway resistance of the severe asthma +miR-146a-3p Agomir group was significantly decreased(p<0.01);(3)Compared with blank control group,the total number of cells,neutrophils and eosinophils in BALF of mice in the severe asthma group and the severe asthma +NC Agomir group were increased,and the neutrophils were increased more significantly(p<0.01).Compared with the severe asthma +NC Agomir group,the total number of cells,neutrophils and eosinophils in BALF in the severe asthma +miR-146a-3p Agomir group were significantly decreased(p<0.01);(4)Compared with those in blank control group,H&E staining of lung tissue of mice in severe asthma group and severe asthma +NC Agomir group showed partial alveolar and lung tissue structure destruction,airway contraction and swelling,epithelial cell shedding and mucus embolism in the airway,and inflammatory cell infiltration such as neutrophils,eosinophils and lymphocytes around the airway were obvious,while most of them were neutrophils.However,the degree of lung tissue destruction,bronchial constriction and swelling and inflammatory cell infiltration around the airway were significantly improved in the severe asthma +miR-146a-3p Agomir group.(5)Compared with the blank control group,immunohistochemical staining of lung tissues of mice in the severe asthma group and the severe asthma +NC Agomir group showed that MBD2 was more expressed in lung tissues(p<0.01),and mainly distributed in bronchial epithelial cells.Ly6g(neutrophil-specific antibody)and ECP(eosinophilic cationic protein)were increased(p<0.01),but Ly6 g was more significantly increased.The levels of MBD2,Ly6 g and ECP in lung tissue of mice in severe asthma+miR-146a-3p Agomir group were significantly decreased(p<0.01);(6)Compared with the blank control group,the results of q RT-PCR showed that the expression of miR-146a-3p in the lung tissues of mice in the severe asthma group and the severe asthma +NC Agomir group decreased(p <0.01).The expression of miR-146a-3p in lung tissue of mice in severe asthma +miR-146a-3p Agomir group was significantly increased(p<0.01).Western Blot results showed that the expression level of MBD2 in the lung tissues of mice in the severe asthma group and the severe asthma +NC Agomir group was significantly increased(p<0.01),while the expression level of MBD2 in the lung tissues of mice in the severe asthma +miR-146a-3p Agomir group was significantly decreased(p<0.01);(7)Compared with blank control group,flow cytometry results showed that the positive rate of Th17 was significantly higher in severe asthma group and severe asthma +NC Agomir group(p<0.01),but significantly lower in severe asthma +miR-146a-3p Agomir group(p<0.01).The expression of RORγt protein in spleen tissues of each group was consistent with the positive rate of Th17 detected by flow cytometry(p<0.01).In addition,ELISA detection of IL17 cytokine concentration in BALF of each group also showed the same trend as Th17 positive rate(p<0.01).Conclusions(1)The mouse model of Th17 severe asthma was established successfully;(2)the miR-146a-3p inhibit Th17 cells differentiation in vivo,significantly reduce airway hyperresponsiveness,airway inflammation and airway mucus secretion,relieve severe asthma.37 Figures,3Tables,117 References...