| Objective:Investigate the mechanism and effects of MiR-27a on autophagy after platelet oxidative stress.Method:(1)MEG-01 cells were cultured in vitro.PMA(phorbol-12-myristate-13-acetate)was used to induce MEG-01 cells to differentiate into megakaryocytes which have the function of producing platelets.(2)After MEG-01 cells were differentiated into platelets,the platelets were transfected.Then,the following experiments were carried out,divided into five groups:1 Control group: no intervention into the platelets2 MiR-27a mimic group: MiR-27a mimic was transfected into the platelets3 MiR-27a mimic negative control group: MiR-27a mimic negative control was transfected into the platelets4 MiR-27a inhibitor group: MiR-27a inhibitor was transfected into the platelets5 MiR-27a inhibitor negative control group: MiR-27a inhibitor negative control was transfected into the platelets(3)The five groups of transfected platelets were added to almmol/L concentration of hydrogen peroxide in order to stimulate 2h to produce oxidative stress.The concentration time of hydrogen peroxide was measured by the same subjects.(4)The miRNA levels of MiR-27a in all platelets were measured by real-time PCR;the mRNA levels of the PHB platelets with hydrogen peroxide stimulates were measured by RT-PCR;the protein levels of the PHB/LC3/CD62 P platelets with hydrogen peroxide stimulates were measured by Western blotting.Result:(1)PMA was added to leukemic granulocyte MEG-01 cells.The platelet surface marker CD61 was observed after induction.The flow of CD61 was increased after induction was observed [73.90 + 1.96)vs(7.09 + 0.60)n =3,p<0.05].The CD61 mRNA was also increased after induction was observed [(7.73 + 0.33)vs(1 + 0)n =3,p<0.05].The morphological changes of the MEG-01 cells to the plate type megakaryocytes.MEG-01 cells were successfully differentiated into megakaryocytes by inducing with PMA.(2)The MiR-27a detection results after transfection :Compared with the MiR-27a mimic negative control group,the MiR-27a mimic group had a significantly higher level of MiR-27a [(52.21± 1.87)vs(0.98 ±0.16)n =3,p<0.05].Compared with the MiR-27a inhibitor negative control group,the MiR-27a inhibitor group had a significantly lower level of MiR-27a[(0.054 ±0.022)vs(0.60±0.15)n =3,p<0.05].(3)There was no difference in the PHB mRNA of the platelets following hydrogen peroxide stimulation in the five groups(P > 0.05).The WB detection results are as follows: the MiR-27a mimic compared with the MiR-27a mimic negative control group,PHB protein [(0.3499 ±0.1037)vs(1.081± 0.008366)] and LC3II/I protein [(0.5441 ±0.05774)vs(0.8861 ±0.06064)n =3] and CD62 P protein [(0.2268±0.03362)vs(0.5469 ± 0.05542)n=3] decreased(P < 0.05);the MiR-27a inhibitor group compared with the MiR-27a inhibitor negative control group,PHB protein [(2.927 ± 0.2228)vs(1.045 ± 0.02141)] and LC3II/I protein [(1.571 ± 0.08819)vs(1.021 ± 0)5774)n =3] and CD62 P protein [(1.227 ± 0.09978)vs(0.5349± 0.01527)n =3] increased(P < 0.05).Conclusion:(1)MiR-27a can effectively inhibit PHB protein expression under oxidative stress,which plays an important role in inhibiting platelet autophagy.Thus,MiR-27a can effectively inhibit platelet activation. |
