Influence Of The Rap2B Gene Expression In Human Bronchial Epithelium Cells Treated By Cigarette Smoke Condensate

Objective To investigate Rap2B gene expression and its promoter methylation in human bronchial epithelium cells(16HBE) treated by cigarette smoke condensate (CSC).1To study the effect of Rap2B gene expression and acute toxicity characteristics of16HBE cells induced by CSC.2To observe the expression of Rap2B gene in16HBE induced by CSC, inferring the role of the Rap2B gene in development process of lung cancer.3To investigate the genome DNA methylation level and Rap2B gene promoter CpG island methylation in16HBE treated by CSC.Methods116HBE cells treated with CSC at different doses (0mg/ml、0.04mg/ml、0.06mg/ml、0.08mg/ml,0.12mg/ml、0.15mg/ml、0.18mg/ml、0.24mg/ml、0.30mg/ml) for24h.Cell counting kit (CCK-8) was used to detect the effects of CSC on proliferation of16HBE cells. Probit analysis was used to calculate the IC50. The16HBE cells were stimulated by CSC at different doses (CTRL, DMSO,1/8IC50,1/4IC5o,1/2IC5o,1IC5o), the cellular morphology were observed by contrast phase microscope at4h,8h,12h,16h,20h and24h. The level of mRNA and protein expressions of Rap2B was detected by both Q-PCR and Western Blot.2During the transformation process, the16HBE cells were treated with CSC (I/8IC50)24h every generation cells. Chromosome aberration analysis, Soft agar cloning experiments and nude mouse transplantation tumor experiments were used to identify the transformed cells. The level of mRNA and protein expression of Rap2B was detected.3The genome DNA methylation level was detected using combined bisulfite restriction analysis(COBRA),the methylation ststus in the promoter regions of Rap2B gene were observed by bisulfite genomic sequencing(BGS).Results1As observed in the Cell Counting Kit-8(CCK-8), exposure to CSC at dosage levels between0and0.3mg/ml decreased cell viability in a dose. IC50of24h exposure was0.08±0.006mg/ml. After24h CSC stimulated, the cells became irregular shape. Compared with controls,20h and24h at0.01mg/ml and0.02mg/ml dose group of mRNA and protein expressions level rise higher(P<0.05), then down at0.08mg/ml(P<0.05).2The P20and P3016HBE cells’chromosome aberration rate increased to12%and18%, there are statistical differences compared with control group(P<0.05). The P30transformed cells could grow on soft agar and there are statistical differences compared with control group(P<0.05). The P30transformed cells could grow into tumors in nude mice. During the transformed process, the level of mRNA and protein expression of Rap2B were increased step by step and there are statistical differences compared with DMSO group(P<0.05).3The methylation of ALU fragment were14.8%(P10),12.8%(P20),13.1%(P30),13.0%(DMSO) and there is no difference between them(P>0/05). The methylation of LINE-1fragment were55.3%(P10),49.8%(P20),49.3%(P30),53.4%(DMSO).Conclusions1Rap2B gene expression increased at low concentrations of group and then decreased at high concentrations of group, it is presume that Rap2B gene was CSC target genes and play an important role in the process of acute poisonous.2The16HBE cells could transformed by CSC, prompting CSC could cause tumour.3The level of mRNA and protein expression of Rap2B were increased step by step during transformed process and Rap2B may play very important role in this process.4The level of genome DNA methylation has changed and there is no obvious change for Rap2B gene promoter regions.