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

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 AA-PBMNC 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.