The Effect Of MiR-34a On CSE Induced Apoptosis Of Human Pulmonary Microvascular Endothelial Cells

Objective:Discuss the effect of CSE intervention on the apoptosis of HPMEC and the expression of miR-34a, and then study the effect of miR-34a on the CSE intervention induced apoptosis of HPMEC.Method:Treat HPMEC with different concentrates (0%、0.5%、1%、2.5%、5%) of CSE for different time(0h、6h、12h、24h). Detect the apoptosis rate by flow cytometrywith double die (Annexin V-FITC/PI). Realtime-PCR were used to assess the expression of miR-34a-5p and miR-34a-3p after different concentrate of CSE treatment for different time. Then analysis the correlation of miR-34a and apoptosis rate of HPMEC. Furthermore, divided cells into four groups:control group (CSE group) with no treatment, miRNA inhibitor negative control transfected group (N+CSE group) transfected with miRNA inhibitor negative control, miR-34a-3p inhibitor transfected group (3p+CSE group) transfected with miR-34a-3p inhibitor, miR-34a-5p inhibitor transfected group (5p+CSE group) transfected with miR-34a-5p inhibitor, treat all groups with1%CSE for12h after giving each group treatments respectively. Measure the apoptosis rate of each group by by flow cytometrywith double die (Annexin-V FITC/PI) and the miR-34a-3p and miR-34a-5p expression by Real-time PCR. Data was presented with Mean±SD, and analysed by using SPSS18.0. P<0.05brings the statistics significance.Result:1.With CSE concentrate increased from0%to1%, the early apoptosis rate of HPMEC was increased(P<0.05). The early apoptosis rate of HPMEC in1%CSE group was (36.23±4.20)%, and there is no statistical difference in early apoptosis rate of HPMEC between1%CSE group and2.5%CSE group. The early apoptosis rate of HPMEC in5%CSE group was less than2.5%CSE group (P<0.05). With1%QSE intervention time prolonged from Oh to12h, the early apoptosis rate of HPMEC was increased (P<0.05). And there is no statistical difference in early apoptosis rate of HPMEC between12h group and24h group.2. With CSE concentrate increased from0%to2.5%, the relative expression of miR-34a-5p and miR-34a-3p were increased (P<0.05). The relative expression of miR-34a-5p and miR-34a-3p in2.5%CSE group were (16.05±0.389)×10-3、(0.75±0.034)×10-3separately,.and there is no statistical difference in early apoptosis rate of HPMEC between2.5%CSE group and5%CSE group. With CSE intervention time prolonged from Oh to12h, the relative expression of miR-34a-5p and miR-34a-3p were increased(P<0.05). And there is no statistical difference in early apoptosis rate of HPMEC between12h group and24h group. 3. Corelation analysis showed that miR-34a-5p expression and HPMEC apoptosis rate were positively correlated (r=0.97,P<0.001), and also miR-34a-3p expression and HPMEC apoptosis rate were positively correlated (r=0.92, P<0.01).4. The early apoptosis rate of N+CSE group,5p+CSE group, and3p+CSE group were (44.52±1.39)%,(34.08±1.29)%,(33.58±0.93)%,. Compared to N+CSE group, the early apoptosis rate of HPMEC in5p+CSE group and3p+CSE group were greatly decreased, and the difference was statistically significant(P<0.05).Conclusion:1. CSE induced increase of apoptosis and miR-34a expression in HPMEC depended on CSE intervention concentrate and time.2. CSE induced increase of apoptosis and miR-34a expression in HPMEC were positively correlated.3. miR-34a inhibitor can reduce CSE induced apoptosis of HPMEC, and miR-34a may play an important role in CSE induced apoptosia of HPMEC.