野生型XPD转染对胆管癌QBC939细胞生物学行为的影响

Transfection of the wild-type xeroderma pigmentosum group D gene alters the biological behavior of human cholangiocarcinoma cell line QBC939

目的:探讨野生型X P D基因对人胆管癌QBC939细胞的生物学影响.方法:用碱裂解法提取空载质粒pEGFP-N2和重组质粒pEGFP-N2-XPD,提取出的质粒以KPNⅠ、BGIⅡ和SPHⅠ酶切鉴定.实验分4组,重组质粒pEGFP-N2-XPD组、空载质粒pEGFP-N2组、脂质体组,并用具有相同遗传背景和代数的QBC939细胞作为空白对照.用脂质体转染法瞬时转染四组细胞.荧光显微镜下观察转染后绿色荧光蛋白报告基因表达情况.提取各组细胞总RNA,合成cDNA,用聚合酶链反应(PCR)检测4组细胞中XPD、p53、cyclin D1、c-myc表达情况.并用四甲基偶氮唑盐(MTT)和流式细胞仪检测细胞增殖及其细胞周期的变化.结果:pEGFP-N2-XPD细胞与pEGFP-N2、脂质体组和空白对照组相比,XPD mRNA表达量明显增加(0.778±0.018vs0.561±0.039,0.544±0.035,0.542±0.034,均P<0.01).pEGFP-N2-XPD细胞中p53mRNA相对表达量与pEGFP-N2、脂质体组和空白对照组比较具有统计学意义(0.421±0.019vs0.256±0.014,0.267±0.015,0.274±0.018,均P<0.01).pEGFP-N2-XPD细胞与其他组相比,cyclin D1mRNA相对表达量明显降低(0.339±0.041vs0.560±0.039,0.558±0.050,0.560±0.041,均P<0.01).pEGFP-N2-XPD细胞与其他组相比,c-myc mRNA相对表达量明显降低(0.355±0.045vs0.570±0.075,0.560±0.041,0.537±0.050,均P<0.01).流式细胞仪检测pEGFP-N2-XPD组细胞周期G1期为81.65%,S期为11.83%,其他组Gl期分别为65.54%、56.61%、63.26%;S期分别为24.10%、29.52%、27.28%,结果具有统计学意义(P<0.05).MTT检测示pEGFP-N2-XPD细胞生长率为0.249±0.02,与其他组相比,细胞增殖力明显减弱(P<0.01).结论:野生型XPD基因可以抑制胆管癌细胞的生长,XPD基因可抑制c-myc、cyclin D1基因的表达,增加p53基因表达.

AIM：To investigate the impact of tranfection of the wild-type xeroderma pigmentosum group D （XPD） gene on the biological behavior of human cholangiocarcinoma cell line QBC939. METHODS：Empty plasmid pEGFP-N2 and recombinant plasmid pECFP-N2-XPD were digested with KPN I,BGI II and SPH I for plasmid identification. Cells were divided into four groups：pEGFP-N2-XPD group,pEGFP-N2 group,Lipofectamine （Lip） group,and blank control group. Cells were transfected with Lipofectamine. The expression of green fluorescent protein （GFP） was observed under a fluorescence microscope. The mRNA expression of wild-type XPD,p53,cyclin D1 and c-myc was detected by reverse transcription-polymerase chain reaction （RT-PCR）. Flow cytometry （FCM） was employed for examining the cell cycle of transfected QBC939 cells. Cell proliferation was detected by methyl thiazolyl tetrazolium （MTT） assay. RESULTS：The relative expression level of XPD mRNA in the pEGFP-N2-XPD group was sig-nificantly higher than those in the pEGFP-N2 group,Lip group and blank control group （0.778 ± 0.018 vs 0.561 ± 0.039,0.544 ± 0.035 and 0.542 ± 0.034,respectively; all P 0.01）. The relative expression level of p53 mRNA in the pEGFP-N2-XPD group was also significantly higher than those in the pEGFP-N2 group,Lip group and blank control group （0.421 ± 0.019 vs 0.256 ± 0.014,0.267 ± 0.015 and 0.274 ± 0.018,respectively; all P 0.01）. The relative expression level of cyclin D1 mRNA in the pEGFP-N2-XPD group was signif i-cantly lower than those in the pEGFP-N2 group,Lip group and blank control group （0.339 ± 0.041 vs 0.560 ± 0.039,0.558 ± 0.050 and 0.560 ± 0.041,respectively; all P 0.01）. The relative expression level of c-myc mRNA in the pEGFP-N2-XPD group was also signifi cantly lower than those in the pEGFP-N2 group,Lip group and blank control group （0.355 ± 0.045 vs 0.570 ± 0.075,0.560 ± 0.041 and 0.537 ± 0.050,respectively; all P 0.01）. FCM results showed that the percentage of cells in G1 phase was signifi cantly higher （81.65% vs 65.54%,56.61% and 63.26%,respectively; all P 0.05） and that in S1 phase was signifi cantly lower （11.83% vs 24.10%,29.52% and 27.28%; all P 0.05） in the pEGFP-N2-XPD group than in the pEGFP-N2 group,Lip group and blank control group. MTT assay revealed that the growth rate of cells in the pEGFP-N2-XPD group was significantly lower than those in the other three groups （all P 0.01）. CONCLUSION：Transfection of the wild-type XPD gene can inhibit the proliferation of human QBC939 cells in vitro,down-regulate the expression of cyclin D1 and c-myc mRNAs,and upregulate the expression of p53 mRNA.