Mechanism Of M6A Methylation Involved In LPS-induced Changes In Endothelial Cell Permeability

Objective(1)To investigate the effect of LPS on m6 A methylation level and permeability of endothelial cells;(2)reveal the molecular biological mechanism of m6 A methylation involved in regulating endothelial cell permeability.Methods:(1)HUVEs cells were treated with different concentrations of LPS for 24 hours,and real-time PCR was used to detect the expression of METTL3 and ALKBH5 m RNA.(2)500 ng/m L LPS intervention HUVEs cells for 24 h,real-time PCR and Western blot detection Claudin-5,Occludin and VE-caherin m RNA and protein expression;At the same time,the methylation detection kit was used to detect the change in the methylation level of m6 A,and the cell permeability related experiment was used to detect the changes in cell permeability and related indicators.(3)the application will Lipofectamine3000 METTL3(NM＿019852)recombinant transfection HUVECs cells,application of concentration is 2.5 mu g/m L puro screening for 4 weeks,build METTL3 had stable expression cell lines,detecting METTL3 expression effect on cell permeability.(4)Three interfering si RNA sequences of ALKBH5(NM＿017758)were transfected into HUVECs cells,and the transfection efficiency was detected by real-time PCR and Western blot.ALKBH5 knockdown stably transfected cells were screened with2.5μg/m L puromycin for 4 weeks.The effect of ALKBH5 knockdown on endothelial cell permeability was detected.Results:(1)After HUVEs cells were treated with different concentrations of LPS for 24 h,real-time PCR confirmed that LPS at 125,250,500 and 1000ng/m L significantly promoted the expression of ALKBH5 m RNA.Compared with the control group,LPS of50,125,250,500,1000,2000ng/m L significantly inhibited the expression of METTL3 m RNA.(2)The results of real-time PCR and Western blot showed that HUVECs cells were treated with 500ng/m L LPS for 24 hours.Compared with the control group,LPS significantly inhibited the m RNA and protein expression of Claudin-5,Occludin and VE-caherin.(3)The results of cell permeability test and analysis showed that the OD488 value of the normal group(N)was(0.2561±0.068),and that of the LPS intervention group was(0.437±0.048).Compared with the N group,the fluorescence intensity of LPS intervention group was significantly enhanced,and the permeability coefficient of monolayer cells was significantly increased,which was(1.814±0.447)times of the normal group,and the difference was statistically significant.(4)The knockdown efficiency of ALKBH5 m RNA was(66.3±11.1)%,(48.2±8.7)%and(65.4±8.6)%,respectively.The interference efficiency of proteins were(64.3±15.3)%,(46.2±22.7)% and(55.7±8.5)%,respectively.(5)Compared with the normal group,the expression of METTL3 m RNA and protein in the GV657/METTL3 group was significantly increased,and the expression levels were(5.368±0.469)and(2.241±0.509),respectively.(6)The results showed that ALKBH5 m6 A methylation level in sh RNA group was significantly higher than that in N group,which was(180.628+35.283)%.The results of cell permeability experiment showed that compared with LPS intervention group,the cell permeability of LPS+sh RNA group decreased by(16.330±2.539)%,and the difference was statistically significant.(7)Compared with the N group,the methylation level of m6 A in GV657/METTL3 group was significantly enhanced,which was(156.764±22.754)% compared with the N group,and the difference was statistically significant.The results of cell permeability experiment showed that compared with the LPS intervention group,the cell permeability of the LPS+GV657/METTL3 group decreased by(14.632±10.376)%,and the difference was statistically significant.Conclusion:(1)LPS could promote the expression of m6 A methyltransferase ALKBH5 and inhibit the expression of demethylase METTL3.(2)the m6 A methylation involved in regulating the cell permeability of LPS intervention.