摘要
目的 研究ABCC3基因与膀胱癌细胞增殖、耐药和有氧糖酵解的相关性.方法 利用脂质体2000将ABCC3 siRNA和对照siRNA分别转染人膀胱癌细胞系5637和UMUC-3细胞,并分为siABCC3-1组、siABCC3-2组和对照组.采用Western blot和实时荧光PCR技术检测转染细胞中ABCC3蛋白和mRNA的相对表达水平,通过集落形成实验检测ABCC3对膀胱癌细胞生长的影响,MTT实验检测癌细胞对顺铂的敏感性,并通过检测葡萄糖消耗、乳酸生成以及乳酸脱氢酶A活性了解ABCC3对有氧糖酵解的影响.计量资料用均数±标准差((x)±s)表示,组间比较采用单因素方差分析和LSD方法.结果 siRNA转染后膀胱癌细胞ABCC3和mRNA表达水平都显著下调;与对照组相比,siRNA干扰组集落形成数显著减少;5637细胞siABCC3-1和siABCC3-2干扰后的顺铂IC50值分别为4.02 μg/ml和3.91 μg/ml,UMUC-3细胞siABCC3-1和siABCC3-2干扰后的顺铂IC50值分别为2.54 μg/ml和2.49 μg/ml,与对照组相比,差异均有统计学意义;siRNA转染后膀胱癌细胞乳酸脱氢酶A蛋白表达下调,ABCC3与乳酸脱氢酶A的表达呈正相关(P=0.0362);siABCC3-1和siABCC3-2分别转染5637细胞后,葡萄糖消耗分别减少43.2％和43.7％,乳酸生成分别减少31.3％和29.7％,siABCC3-1和siABCC3-2分别转染UMUC-3细胞后,葡萄糖消耗分别减少33.4％和37.5％,乳酸生成分别减少24.7％和25.2％,与对照组相比,差异具有统计学意义(均P＜0.001).结论 ABCC3是参与膀胱癌细胞增殖、耐药和有氧糖酵解的重要癌蛋白,并有望成为膀胱癌的生物标志物和潜在的治疗靶点.
Objective To study the correlation between ABCC3 gene and bladder cancer cell proliferation,drug resistance and aerobic glycolysis.Methods Lipofectamine 2000 reagent was used for small interfering RNA (siRNA) transfection.Human ABCC3 siRNA and negative control siRNA were transfected into UMUC-3 and 5637 cells separately,and bladder cancer cells were divided into siABCC3-1 group,siABCC3-2 group and control group.Western blot assay was used to evaluate the ABCC3 expression levels.Quantitative real-time polymerase chain reaction (PCR) was performed to determine the ABCC3 mRNA expression levels.The effect of ABCC3 on bladder cancer cell growth was determined by colony formation assay.We also analyzed the sensitivity of cance cells to cisplatin by MTT assay.The effect of ABCC3 on aerobic glycolysis were detected by measuring LDHA protein levels,lactate production and glucose consumption.The measurement data were expressed as ((x) ± s) tandard deviation.The difference between the groups was analyzed by single factor analysis of variance and LSD.Results Both protein and mRNA levels of ABCC3 were significantly decreased after si-ABCC3 transfection.Bladder cancer cells treated with si-ABCC3 exhibited significantly lower colony numbers than that of the control group.5637 cells treated with siABCC3-1 and siABCC3-2 were 4.02 μg/ml and 3.91 μg/ml,respectively.The IC50 values of cisplatin after UMUC-3 cells siABCC3-1 and siABCC3-2 were 2.54 μg/ml and 2.49 μg/ml,respectively.The expression of lactate dehydrogenase A protein in bladder cancer cells was down-regulated and the expression of ABCC3 was positively correlated with the expression of LDHA (P =0.0362).siABCC3-1 and siABCC3-2 were transfected into 5637 cells,respectively.The glucose consumption decreased by 43.2％ and 43.7％ respectively.The lactic acid production was reduced by 31.3％ and 29.7％,respectively.After transfection of UMUC-3 cells with siABCC3-1 and siABCC3-2,glucose consumption decreased by 33.4％ and 37.5％,respectively,and lactic acid production decreased by 24.7％ and 25.2％,respectively,compared with the control group,the difference was statistically significant (P ＜ 0.001).Conclusions ABCC3 is an important oncoprotein involved in cell proliferation,glycolysis and drug resistance.It could be a promising predictive biomarker and potential therapeutic target for bladder cancer.
出处
《国际外科学杂志》
2017年第9期121-128,共6页
International Journal of Surgery
关键词
膀胱肿瘤
细胞增殖
糖酵解
抗药性
肿瘤
ATP结合盒转运蛋白
Urinary bladder neoplasms
Cell proliferation
Glycolysis
Durg resistance,neoplasm
ATP-binding cassette transporter
