摘要
目的探讨人膜联蛋白A4(AnnexinA4,ANXA4)对肝癌细胞株MHCC97H黏附能力的影响及其相关机制。方法化学合成针对ANXA4序列特异性小干扰RNA,用脂质体LipofectamineTM2000转染人肝癌细胞株MHCC97H;RT-PCR和western blot检测RNA干扰后ANXA4 mRNA和蛋白质的表达水平;黏附实验检测ANXA4表达下降对细胞黏附能力的影响;荧光定量RT-PCR检测RNA干扰后黏附相关基因表达的变化。结果荧光定量RT-PCR及western blot结果表明,经siRNA干扰后,ANXA4 mRNA和蛋白质表达水平明显降低;细胞黏附实验结果显示,与空白对照组的0.293±0.019和Mock组的0.305±0.018相比,实验组为0.234±0.013,差异有统计学意义(P<0.05)。荧光定量RT-PCR结果显示,与阴性对照组相比,实验组ITGA5、ITGB1、E-cadherin mRNA表达水平下调,ICAM1、CD44、OPN mRNA表达水平上调,差异均有统计学意义(P均<0.05)。结论 ANXA4可能通过调节黏附相关基因的表达水平影响细胞的黏附能力,参与肝癌的发生和发展进程。
Objective To investigate the effects of ANXA4 on cellular adhesion and and underlying mechanism during hepatocarcinogenesis.Methods Two pairs of small interfering RNA（siRNA） targeting to ANXA4 were chemically synthesized and transfected into MHCC97H cells by using lipofectamineTM2000.The levels of mRNA and protein of ANXA4 were determined by reverse transcription PCR and western blot respectively.The effects of decreased expression of ANXA4 on adhesion ability of MHCC97H cells were detected by adhesion test,and the expressions of adhesion-related genes were observed by real-time fluorescent RT-PCR.Results Both the expressions of mRNA and protein of ANXA4 in MHCC97H cells were effectively inhibited by three groups of ANXA4 siRNA（siRNA-1,siRNA-2 and the mixture of siRNA-1and siRNA-2） following interference of 24,48 and 72 h at the concentration of 100 nmol / L.Compared with blank control group（0.293 ± 0.019） and Mock group（0.305 ± 0.018）,the absorbance of experimental group was（0.234 ±0.013）（P 0.05）.The results of real-time fluorescent PCR showed that the mRNA expressions of adhesion-related genes ITGA5,ITGB1 and E-cadherin were repressed,while ICAM1,CD44 and OPN were induced.The differences were statistically significant compared with the negative control group（P 0.05）.Conclusion ANXA4 may promote cell adhesion of hepatocytes by regulating certain adhesion-related genes expressions and involve in the progress of hepatocarcinogenesis.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2013年第8期588-591,共4页
Chinese Journal of Clinical Laboratory Science
基金
国家自然科学基金(81060199)
广西自然科学基金(2010GXNSFA013151)
