Effect Of Epsin2 On BBB Function And Expression Of Related Factors In Brain Metastasis Of Non-small Cell Lung Cancer

[Objective]:The expression of Epsin2 and related factors VEGFR2,VE-cadherin,Occludin and ZO-1 in blood-brain barrier brain microvascular endothelial cells(b.End.3)during brain metastasis of non-small cell lung cancer(NSCLC)were studied by establishing an in vitro blood-brain barrier(BBB)model.Using chemically synthesized siRNA(small interfering RNA)to interfere with the expression of Epsin2 in b.End3,the effect of Epsin2 on the expression of blood-brain barrier function and related factors in vitro was studied,which provided a new theoretical basis and potential therapeutic direction for NSCLC brain metastasis..[Methods]:1.A model of in vitro blood-brain barrier(BBBM)was established using a single layer of mouse brain microvascular endothelial cells;a non-contact co-culture blood tumor barrier model(BTBM)was established using a single layer of mouse brain microvascular endothelial cells and human non-small cell lung cancer cells.2.After successful modeling of BBBM and BTBM in vitro,the transmembrane endothelial resistance(TEER)of the above two models was measured by transmembrane endothelial resistance meter and the change trend was recorded.The BBBM and b.End3 cells in BBBM and BTBM were extracted after 5 days of culture.Total RNA was extracted by spin column method,and the expression levels of protein and mRNA of Epsin2,VEGFR2,VE-cadherin,ZO-1 and Occludin were detected by immunofluorescence(IF)and qPCR,respectively.3.The chemically synthesized siRNA was used to interfere with the expression of Epsin2 in b.End3 cells,and then the changes in expression with proteins and mRNA of Epsin2,VEGFR2,VE-cadherin,ZO-1,Occludin in normal b.End3 and Epsin2 siRNA b.End3 were detected by IF,WB and qPCR.4.In vitro BBBM was established as an experimental group using Epsin2 siRNA b.End3 cells,and a normal group were established using normal b.End3 cells.After successful modeling,co-culture was continued for 5 days to detect changes in TEER and A549 cell invasion and migration in normal and experimental groups.[Results]:1.Detection of BBBM and BTBM in vitro;1.1 The test of transmembrane endothelial cell resistanceThe TEER values of BBBM and BTBM increased slowly in the first 2 days.There was no significant difference between the two groups on the first day(P>0.05).On the second day,the TEER of BBBM increased more than BTBM,the difference was statistically significant(P<0.05)..On the third day,the TEER in the BBBM group continued to rise,but the BTBM began to decline,and the difference was statistically significant.On the 4th and 5th day,TEER decreased in both groups,and the difference was statistically significant(p<0.05).1.2 The result of IFThe results of IF showed that the expression of Epsin2,VEGFR2 and VE-cadherin in b.End3 in BTBM was higher than that in BBBM group,and the expression of ZO-1 and Occludin was decreased.The difference was statistically significant(p<0.05).1.3 The result of qPCRCompared with b.End3 in BBBM group,the mRNA expression levels of Epsin2,VEGFR2 and VE-cadherin in b.End3 in BTBM were increased,and the expression levels of ZO-1 and Occludin were decreased,the difference was statistically significant(p<0.05).2.The detection of Epsin2 after b.End3 transfection chemical synthesis of Epsin2 siRNA2.1 The result of IFThe results of IF showed that the expression of Epsin2,VEGFR2 and VE-cadherin in Epsin2 siRNA b.End3 was decreased compared with normal b.End3,and the expression of ZO-1 and Occludin was increased.The difference was statistically significant(p<0.05).2.2 The result of qPCRCompared with normal b.End3,the mRNA expression levels of Epsin2,VEGFR2 and VE-cadherin in Epsin2 siRNA b.End3 decreased,and the expression levels of ZO-1 and Occludin increased,the difference was statistically significant(p<0.05).2.3 The result of WBCompared with normal b.End3,the protein expression of Epsin2,VEGFR2 and VE-cadherin was decreased in Epsin2 siRNA b.End3,and the expression of ZO-1 and Occludin was increased,and the difference was statistically significant(p<0.05).3.The TEER of the normal group BBBM was slightly lower than that of the experimental group on the first day,but the difference was not statistically significant(P>0.05).The BBBM of the experimental group was higher than that of the normal group(P<0.05).4.The result of a549 cell invasion test:the average number of transmembrane cells in the experimental group was significantly reduced in the normal group,the difference was statistically significant(P<0.05).[Conclusion]:1.In vitro BBBM and BTBM can be successfully constructed using a single layer of b.End3 and A549 cells.2.After non-contact co-culture of A549 and b.End3,the expression levels of Epsin2,VEGFR2 and VE-cadherin increased,the expression levels of tight junction proteins ZO-1 and Occludin decreased,and BBB function was destroyed.This indicates that the increase in the expression of Epsin2 and its downstream factors,VEGFR2 and VE-cadherin,and the decrease in the expression of tight junction proteins ZO-l and Occludin may lead to an increase in permeability of brain microvascular endothelial cells,thereby destroying the blood-brain barrier.Integrity,in turn,facilitates the occurrence of NSCLC brain metastases.3.After interfering with the expression of Epsin2 in b.End3 cells,the expression of VEGFR2 and VE-cadherin was also decreased,and the expression of ZO-1 and Occludin was increased,which proved that there is an upstream and downstream regulation relationship between Epsin2-VEGFR2-VE Cadherin.Epsin2 may also regulate the expression of ZO-1 by the action of VEGFR2.4.After successfully using siRNA to interfere with the expression of Epsin2 in b.End3,the expression levels of VEGFR2 and VE-cadherin also decreased,BBB function was reduced,and the expression of tight junction proteins ZO-1 and Occludin increased.The TEER of BBBM constructed by Epsin2 siRNA b.End3 was higher than that of normal endothelium,indicating that the former had higher BBBM integrity than the latter.Moreover,the invasive ability of A549 to BBB in the BBBM composed of endothelial cells after transfection was also significantly decreased,suggesting that the deletion of Epsin2 in b.End3 may inhibit the invasive ability of a549 cells to the blood-brain barrier in vitro.