WWOX基因转染对胆管癌细胞增殖、凋亡及侵袭的影响

WWOX gene transfection inhibits proliferation, accelerates apoptosis, and reduces invasion in human cholangiocarcinoma cell line QBC939

目的:探讨WWOX基因转染胆管癌细胞株QBC939后对其增殖、凋亡与侵袭性的影响.方法:用脂质体转染法将WWOX重组真核表达质粒转染QBC939细胞,建立稳定表达WWOX基因的细胞株.将其分为以下3组:QBC939组,QBC939/con组和QBC939/WWOX组.荧光定量RT-PCR和Westernblot法检测各组WWOXmRNA和蛋白水平的表达;MTT实验检测转染前后各组细胞增殖活性的变化;FCM法检测各组细胞的凋亡;Transwell小室侵袭实验检测各组肿瘤细胞侵袭力的变化.结果:建立了稳定表达WWOX基因的QBC939/WWOX细胞株,WWOXmRNA和蛋白的表达增加[3.71(3.64-3.78)vs1.00(0.98-1.02),1.07(1.02-1.13);0.86±0.03vs0.25±0.01,0.27±0.02,均P<0.05],转染后的QBC939细胞MTT吸光度明显下降(0.63±0.04vs0.90±0.05,0.87±0.04,均P<0.01),FCM显示QBC939/WWOX组的细胞凋亡率明显增高(21.4%±2.35%vs1.24%±0.35%,1.73%±0.48%,均P<0.01),侵袭实验显示转移至下室滤膜的细胞数明显减少(70.00±4.58vs102.33±8.33,107.00±9.00,均P<0.01).结论:WWOX基因能抑制胆管癌细胞株QBC939的增殖,加速肿瘤细胞凋亡并降低其侵袭力,可能作为胆管癌基因治疗的一个新靶点.

AIM： To investigate the effect of transfection with the WW domain-containing oxidoreductase （WWOX） gene on cell proliferation, apoptosis and invasion in human cholangiocarcinoma cell line QBC939. METHODS： A recombinant eukaryotic expression plasmid containing the WWOX gene was introduced into QBC939 cells by liposome- mediated transfection. The mRNA and protein expression of WWOX in QBC939 cells stably transfected with the recombinant plasmid was detected by quantitative RT-PCR and Western blotting, respectively. Cell proliferation was tested by methyl thiazolyl tetrazolium （MTT） assay. Cell apoptosis was assessed by flow cytometry （FCM）. Cell invasion was determined by Tran- swell chamber assay. RESULTS： QBC939 cells stably transfected with the recombinant plasmid were successfully generated. The expression of WWOX mRNA and protein was markedly increased in QBC939 cells transfected with the recombinant plasmid when compared with untransfected QBC939 cells and those transfected with control plasmid [3.71（3.64-3.78） vs 1.00（0.98-1.02）, 1.07（1.02-1.13）; 0.86 ± 0.03 vs 0.25 ±0.01, 0.27 ± 0.02, all P 〈 0.05]. WWOX gene transfection significantly decreased cell proliferation [0.63 ± 0.04 vs 0.90 ± 0.05, 0.87 ± 0.04, both P 〈 0.01] but promoted apoptosis （21.40% ± 2.35% vs 1.24% ± 0.35%, 1.73% ± 0.48%, both P 〈 0.01）. Transwell chamber assay showed that the number of transfected cells that passed the Transwell membrane was significantly less than those of control cells （70.00 ± 4.58 vs 102.33 ± 8.33, 107.00 ± 9.00, both P 〈 0,01）. CONCLUSION： WWOX expression inhibits proliferation, accelerates apoptosis, and reduces invasion in human cholangiocarcinoma cell line QBC939, suggesting that the WWOX gene may be a novel target for gene therapy of cholangiocarcinoma.