| Part 1Objective: Detecting the expression levels of Bin1 in NSCLC cells and in the embryo lung cell line 2BS,then establishing high expression Bin1 NSCLC cell line with Bin1 lowest expressed NSCLC cells by gene transfection to study the effect of Bin1 overexpression on cell proliferation,invasion and migration.Methods:1 Quantificational real-time polymerase chain reaction(q RT-PCR)and Western blot assays were used to evaluate Bin1 expression levels in four NSCLC cell lines of H1975,H1650,A549,H460 and the embryo lung cell line 2BS.2 H1974 cells which express lowest level of Bin1 were transfected with Bin1 lentiviral vector(Bin1+)or empty vector plasmid(pCDH).In addition,H1975 cells was performed as the Con group.Flow cytometry(FCM)was used to obtain the stable transfected Bin1 cell line.qRT-PCR and Western blot assays were used to detect the effect of Bin1 transfection.3 MTT and clone formation assays were used to detect the effect of cell proliferation ability on H1975 cells of Bin1 overexpression.4 FCM assay was performed to evaluate the effect of Bin1 on cell cycle in H1975 cells.5 Scratch test and transwell chamber experiment were used to evaluated the effect of Bin1 on cell migration and cell invasion in H1975 cells.Results:1 qRT-PCR and Western blot analysis showed the lowest level expression of Bin1 in H1975 cells compared to H1650,A549,H460 and 2BS.2 The expression of Bin1 mRNA and protein in Bin1+ H1975 cells increased significantly(P<0.05),compared with pCDH and Control groups,which confirmed that Bin1 transfected into H1975 cells successfully.FCMwas used to selected for stability Bin1+ and pCDH cells.3 The growth rate of Bin1+ cells at 24 h,48h,72 h,96h,120 h were0.0543±0.021,0.1249±0.034,0.2348±0.027,0.3594±0.019,0.4923±0.031,respectively.The prolifertion rate of pCDH cells at 24 h,48h,72 h,96h,120 h were 0.0634±0.024,0.3549±0.037,0.4988±0.047,0.9857±0.019,1.524±0.053,respectively.The growth rate of Con cells at 24 h,48h,72 h,96h,120 h were 0.0619±0.029,0.3624±0.042,0.4874±0.039,0.9957±0.023,1.5097±0.046,respectively.The result demonstrated that Bin1 could inhibit the growth of H1975 cells(P<0.05).Colony formation assay demonstrated that the colony ability of Bin1+ cells was decreased compared to pCDH and Con cells(P<0.05).These results indicated that Bin1 could inhibit the abilities of proliferation in H1975 cells.4 The ratio of G0/G1 phase of Bin1+ group,p CDH group and Con group were(67.12±1.74)%,(55.81±1.48)%,(56.41±2.03)%,respectively.Compared with p CDH group and Con group,the ratio of G0/G1 phase of Bin1+ group significantly increased(P<0.05),and the difference was not significant between pCDH group and Con group(P>0.05).These results showed that the DNA synthesis in Bin1+ cells was limited,and the cells were prevented from entering the S phase from G0/G1 phase and then induced cell cycle arrest to occur in H1975 cells.5 The migration distance of Bin1+ cells,pCDH and Con groups at 12 h were(25.3±2.6)?m,(58±5.2)?m and(60.21±4.2)?m,respectively.The migration distance of Bin1+ cells,pCDH and Con groups at 24 h were(58.7±3.8)?m,(92.3±4.3)?m and(89.7±5.8)?m,respectively.Compared with pCDH and Con groups,the migration distance was shorter in Bin1+ cells(P<0.05).There were no significant difference between pCDH group and Con group(P>0.05).Transwell experiments showed that the number of transmembrane cells in the Bin1+ group,pCDH group and Con group were50.50±3.15,124.00±4.25,130±4.37,respectively.Compared with pCDH and Con groups,the number of transmembrane cells in the Bin1+ group significantly decreased(P<0.05),the difference was not statisticallysignificant between pCDH group and Con group(P>0.05),indicating that Bin1 could inhibit the invasive ability of H1975 cells(P <0.05).Part 2Objective: Identifying the effect of Bin1 on the expression of PD-L1 of NSCLC by gene transfection and siRNA methods,and exploring the associated mechanisms.Methods:1 qRT-PCR and Western blot assays were used to evaluate PD-L1 expression levels in four NSCLC cell lines of H1975,H1650,A549,H460 and the embryo lung cell line 2BS and the effect of Bin1 on PD-L1 expression in H1975 cells.2 Bin1-si RNA and NC-siRNA plasmid were transfected into H460 cells.qRT-PCR and Western blot assays were used to detect the effects of RNA silencing of Bin1 and the effect of Bin1 low expression on PD-L1 expression.3 c-MYC-siRNA and NC-si RNA were transfected into H1975 cells to confirme whether c-MYC was involved in the mechanisms of PD-L1 regulation.Results:1 q RT-PCR and Western blot analysis showed the highest level expression of PD-L1 in H1975 cells compared to H1650,A549,H460 and2 BS.An analysis of PD-L1 expression in Bin1-overexpressed H1975 cells revealed significantly decreased expression of PD-L1 with qRT-PCR and Western blot when compared to pCDH group and Con group(P<0.05);2 Knock-down of Bin1 caused a significant increase in expression of PD-L1 compared to the Con group(P<0.05).3 siRNA knockdown of c-MYC caused a reduction in the level of PD-L1 mRNA and protein compared to the Con group(P<0.05).These results indicated that Bin1 could regulate the expression of PD-L1 in c-MYC-dependent pathways.Conclusions:1 The expression level of Bin1 was lowest in H1975 cells,Bin1 stableexpressed H1975 cells was constructed successfully.2 Bin1 could inhibit the abilities of proliferation,migration and invasion in H1975 cells.3 The expression level of PD-L1 was highest in H1975 cells and Bin1 could reverse the PD-L1-mediated immune escape via inactivating c-MYC. |
PDF Full Text Request