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[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.