miR-335-5p/ADCY3诱导的再生障碍性贫血患者免疫异常的机制研究

MiR-335-5p-Mediated Dysfunction of T Lymphocytes in Patients with Acquired Aplastic Anemia

目的:探究miR-335-5p/ADCY3失衡对获得性再生障碍性贫血(AA)患者淋巴细胞功能的影响。方法:采集AA患者外周血标本50例,其中重型再生障碍性贫血(SAA)38例,非重型再生障碍性贫血(NSAA)12例,同时采集健康志愿者(HC)外周血标本22例。标本分离单个核细胞(PBMNC)后,用RT-PCR检测miR-335-5p及ADCY3 mRNA的表达量。用RNAimax转染试剂,将阴性对照miR-335-5p及miR-335-5p mimic分别转染至AA患者的PBMNC,流式细胞术检测CD4^+T和CD8^+T细胞增殖、活化及分泌细胞因子的能力。应用双荧光素酶报告基因系统验证miR-335-5p与靶基因的靶向关系。结果:同HC来源的PBMNC(HC-PBMNC)相比,SAA和NSAA来源的PBMNC中miR-335-5p的表达量均明显降低(0.08±0.01 vs 0.74±0.10,P<0.01^**;0.17±0.02 vs 0.74±0.10,P<0.01^**)。而且,SAA来源的PBMNC中miR-335-5p的表达量显著低于NSAA(P<0.01^**)。与对照组相比,体外上调miR-335-5p的表达,AA来源的PBMNC(AA-PBMNC)中,CD4+T和CD8+T细胞增殖能力均明显降低(P<0.05^*和P<0.05^*)。而且上调miR-335-5p的表达可显著抑制AA-PBMNC中CD4^+和CD8^+T细胞的活化(P<0.01^**和P<0.01^**)。此外,上调AA-PBMNC中,miR-335-5p的表达还可明显降低CD4^+TNFα^+T细胞、CD8^+IFNγ^+T细胞和CD8^+TNFα^+T细胞的比例(P<0.01、P<0.05和P<0.05)。靶基因筛查显示,ADCY3在AA-PBMNC中的表达量明显高于HC-PBMNC(1.70±0.15 vs 0.76±0.12,P<0.001)。而且,miR-335-5p可显著抑制ADCY33′UTR野生型质粒的荧光素酶活性(P<0.05)。结论:MiR-335-5p在AA-PBMNC中显著低表达,且与疾病严重程度相关。体外上调AA-PBMNC中miR-335-5p的表达量可纠正患者免疫异常状态。这些改变可能与靶基因ADCY3的抑制增强有关。

Objective:To explore the effect of miR-335-5p/ADCY3 interaction on the lymphocyte function in the patients with aplastic anemia(AA).Methods:Blood samples were collected from 22 healthy volunteers(HC)and 50 AA patients including 38 severe AA(SAA)and 12 non-severe AA(NSAA).Peripheral blood mononuclear cells(PBMNC)were isolated.The expression of miR-335-5p and ADCY3 mRNA was detected by using RT-PCR.Negative control miR-335-5p(NC group)and miR-335-5p mimic(mimic group)were transfected to AA-PBMNC by using RNAimax reagent,respectively.The proliferative ability,activation and cytokines of CD4^+T and CD8^+T cells were measured by flow cytometry.Dual-luciferase reporter assay was used to verify the targeted relationship between miR-335-5p and target gene.Results:The expression of miR-335-5p was significantly downregulated in SAA-PBMNC and NSAA-PBMNC compared with HC-PBMNC(0.08±0.01 vs 0.74±0.10,P<0.01;0.17±0.02 vs 0.74±0.10,P<0.01).Meanwhile,the expression of miR-335-5p in SAA-PBMNC was very statistically significantly lower than that in NSAA-PBMNC(P<0.01).Compared with NC group,upregulation of miR-335-5p in vitro could significantly inhibited the proliferation of CD4^+T and CD8^+T cells in AA-PBMNC(P<0.05 and P<0.05,respectively).And,upregulating miR-335-5p in AA-PBMNC could significantly inhibited the activation of CD4^+and CD8^+T cells(P<0.01 and P<0.01,respectively).The ratio of CD4^+TNFα^+T,CD8^+IFNγ^+T and CD8^+TNFα^+T cell by up-regulating the expression of miR-335-5p from AAPBMNC in vitro was also significantly lower(P<0.01,P<0.05 and P<0.05,respectively).In addition,the expression of ADCY3 was higher in AA-PBMNC than that in HC-PBMNC(1.70±0.15 vs 0.76±0.12,P<0.01).Furthermore,by means of dual-luciferase reporter assay,the luciferase activity of ADCY3′UTR wildtype could be inhibited by miR-335-5p.Conclusions:The expression of miR-335-5p was significantly downregulated in AA,and that correlates with disease severity.Up-regulating miR-335-5p can correct the hyperimmune status in AA patients by targeting ADCY3.These changes may relates with the strengthen of inhibition for targeted gene ADCY3.