| Objective:To investigate the mechanisms of lung cancer caused by radon, BEAS-2B cellswere induced to malignant transformation by radon in vitro to establish a cell model oflung cancer and gene expression of DNMTs, DNA methylation status of tumorsuppressor genes were detected.Methods:The immortalized human bronchial epithelial cells (BEAS-2B) in exponentialgrowth phase were planted onto Transwell membrane with1.5×105per well. Afteradherence, cells were directly exposed to radon with oxygen concentration of21%andtemperature at37℃.Three transwell membrane were exposed to radonsimultaneously.The optimal time and concentration for inducing the malignanttransformation of BEAS-2B cells were determined by CCK-8assay. After the exposure,cells were cultured until to30passages. The exposed cells were trypsinized into dishesfor further growth to40passages. Biological characteristics between the control groupand different passages cells were compared for cell growth, cell morphology, cell cycle,apoptotic rate and mitochondrial membrane potential. The extend of malignanttransformation was detected by soft agar colony formation and nude mice experiment.The expression of DNMT1, DNMT3A, DNMT3B were detected by Q-PCR and themethylation status of the MGMT, ERCC1, XRCC1, RARβ and LINE-1were detectedby methylation-specific PCR, based on modified genome DNA by bisulphate. Then themRNA level of MGMT and RARβ were detected by Q-PCR. Results:(1)The establishment of cell model. The survival rates of BEAS-2B cells wererelated to concentration and time of radon exposure. Appropriate exposure conditionswere20000Bq/m3and30mins. A series of sequential steps emerged among cells whichexposed to radon for10to20passages, including altered cell morphology,heteromorphism and lost of contact inhibition. With increase of passage, morphologicalchanges and growth were similar to cancer cells.The percentage of G1phase in radongroup cells was significantly lower and the percentage of S phase was significantlyhigher compared to control group. The apoptotic rates were significantly lower in cellsafter generation. The MMP was significantly lower in radon group compared to controlgroup.(2)Changes of malignant transformation ability. BEAS-2B cells were malignantlytransformed after exposure to radon for10passages, and capacity of colony formationrate was up to27.27±1.10％in soft ager after exposure for30passages and generationfor40passages(P＜0.05).Rn30-40cells were proved to be totally malignanttransformed by nude mice experiment. Tumor formation rate was30％，significantlyhigher compared to control group(P<0.05). Pathology analysis revealed that tumor cellswith different sizes were distributed diffusely, however, none tumor cells were found incontrol group. The mRNA level of DNMT1gene was higher in the control group thanthe exposure group except for Rn1-20and Rn10-20. The DNMT3A gene expressionwas lower in the control group than the exposure group. DNMT3B was not expressed inneither control nor exposure group.(3)Changes of methylation status. Methylationspecific PCR was used to analyze the DNA methylation of promoter region of MGMT,ERCC1, XRCC1, RARβ and LINE-1gene in control groups (2B,2B-40) and Rn20-40,Rn30-40cells. Methylation of MGMT and RARβ were found in the Rn20-40andRn30-40cells but not in control groups. Methylation of ERCC1and XRCC1were notfound in both the control and radon exposure group. Methylation of LINE-1was foundin control group but not in Rn30-40cells, LINE-1gene was hypomethylation. MGMT,RARβ genes were highly expressed in the control group, and the expression levels of the radon exposure groups were much lower.Conclution:1. A model of malignant transformation of human cells in vitro induced by radonwas established and transplanted tumors were formed in nude mice.2. DNMTs genes expression alteration and hypermethylation of MGMT, RARβgenes and hypomethylation of LINE-1genes may play an important role in malignanttransformation of BEAS-2B cells induced by radon in vitro. |
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