微小RNA-520b靶向调节肝脏磷酸果糖激酶抑制胰腺癌细胞侵袭并促进其凋亡  

MicroRNA-520b inhibits pancreatic cancer cell invasion and promotes apoptosis by targeting phosphofructokinase liver type

作　　者：刘玉华[1] 毛爱红 王佳 杨燕[1] Liu Yuhua;Mao Aihong;Wang Jia;Yang Yan(Department of Digestive Surgery,Gansu Cancer Hospital,Lanzhou 730050,China;Medical Molecular Biology Research Center,Gansu Medical Academy,Lanzhou 730050,China)

机构地区：[1]甘肃省肿瘤医院消化外科,兰州730050 [2]甘肃省医科院医学分子生物研究中心,兰州730050

出　　处：《中华实验外科杂志》2019年第5期858-860,共3页Chinese Journal of Experimental Surgery

摘　　要：目的探讨微小RNA(miR)-520b靶向调节肝脏磷酸果糖激酶(PFKL)在胰腺癌细胞侵袭及凋亡中的机制。方法将细胞分为空白组、阴性对照组、miR-520b阳性组、miR-520b抑制组和siRNA-PFKL组,每组重复3次。实时荧光定量聚合酶链反应(FQ-PCR)检测miR-520b和PFKL基因相对表达量;蛋白质印迹法(Western blot)检测PFKL及上皮-间充质转化(EMT)相关分子蛋白分子水平;细胞体外侵袭实验(Transwell)和流式细胞技术分别检测细胞侵袭和凋亡能力。结果荧光素酶报告基因检测发现,在野生型PFKL中,miR-520b阳性组(0.25±0.04)的荧光素酶活性较阴性对照组(0.92±0.09)显著降低(P<0.05),而在突变型PFKL中差异无统计学意义(P>0.05)。与空白组[PFKL:1.72±0.13,N-钙黏蛋白(N-cadherin):1.36±0.10,E-钙黏蛋白(E-cadherin):1.27±0.09、β-连环蛋白(β-catenin):0.36±0.04、Snail:1.35±0.12、侵袭:(23.27±3.18)个/HF、凋亡:(9.89±0.72)%]和阴性对照组[PFKL:1.61±0.12,N-cadherin:1.40±0.11,E-cadherin:1.26±0.08、β-catenin:0.38±0.05、Snail:1.30±0.10、侵袭:(21.92±3.65)个/HF、凋亡:(10.67±0.69)%]比较,miR-520b阳性组[PFKL:0.12±0.01,N-cadherin:0.30±0.03,E-cadherin:2.42±0.21、β-catenin:0.19±0.02、Snail:0.40±0.07、侵袭:(12.13±1.39)个/HF、凋亡:(25.83±2.15)%]和siRNA-PFKL组[PFKL:0.11±0.01,N-cadherin:0.25±0.02,E-cadherin:2.53±0.24、β-catenin:0.26±0.05、Snail:0.22±0.04、侵袭:(13.91±1.10)个/HF、凋亡:(23.91±1.99)%]E-cadherin蛋白和CFPAC-1细胞凋亡水平显著增高,PFKL、N-cadherin、β-catenin、Snail蛋白和细胞侵袭水平显著减低(P<0.05);miR-520b抑制组[PFKL:3.47±0.25,N-cadherin:3.39±0.20,E-cadherin:0.49±0.03、β-catenin:2.11±0.15、Snail:2.05±0.22、侵袭:(43.22±7.85)个/HF、凋亡:(2.41±0.18)%]E-cadherin蛋白和CFPAC-1细胞凋亡水平显著降低,PFKL、N-cadherin、β-catenin、Snail蛋白和细胞侵袭水平显著增高(P<0.05)。结论miR-520b可抑制胰腺癌细胞的侵袭并促进其凋亡,其机制与调控靶Objective To investigate the mechanism of microRNA(miRNA,miR)-520b targeting the regulation of hepatic phosphofructokinase liver type(PFKL)in pancreatic cancer cell invasion and apoptosis.Methods Cells were divided into blank group,negative control group,miR-520b positive group,miR-520b inhibition group and siRNA-PFKL group,and repeat 3 times per group.Real-time fluorescent quantitative polymerase chain reaction(FQ-PCR)was used to detect the relative expression of miR-520b and PFKL genes.Western blotting was used to detect the levels of PFKL and epithelial-mesenchymal transition-related molecular proteins.Cell in vitro invasion assay(Transwell)and flow cytometry were used to detect cell invasion and apoptosis,respectively.Results The luciferase reporter gene assay showed that the luciferase activity of the miR-520b positive group(0.25±0.04)was significantly lower than that of the negative control group(0.92±0.09)in wild-type PFKL(P<0.05),but there was no significant difference in the mutant PFKL(P>0.05).Compared with the blank group[PFKL:1.72±0.13,N-cadherin:1.36±0.10,E-cadherin:1.27±0.09,β-catenin:0.36±0.04,Snail:1.35±0.12,invasion:(23.27±3.18)/HF,apoptosis:(9.89±0.72)%]and the negative control group[PFKL:1.61±0.12,N-cadherin:1.40±0.11,E-cadherin:1.26±0.08,β-catenin:0.38±0.05,Snail:1.30±0.10,invasion:(21.92±3.65)/HF,apoptosis:(10.67±0.69)%],the levels of E-cadherin protein and apoptosis were significantly increased in the miR-520b positive group[PFKL:0.12±0.01,N-cadherin:0.30±0.03,E-cadherin:2.42±0.21,β-catenin:0.19±0.02,Snail:0.40±0.07,invasion:(12.13±1.39)/HF,apoptosis:(25.83±2.15)%]and the siRNA-PFKL group[PFKL:0.11±0.01,N-cadherin:0.25±0.02,E-cadherin:2.53±0.24,β-catenin:0.26±0.05,Snail:0.22±0.04,invasion:(13.91±1.10)/HF,apoptosis:(23.91±1.99)%],and the levels of PFKL,N-cadherin,β-catenin,Snail protein and cell invasion were significantly reduced(P<0.05).However,the level of E-cadherin protein and apoptosis in the miR-520b inhibition group[PFKL:3.47±0.25,N-cadherin:3.39±0

关 键 词：胰腺癌 微小RNA 侵袭 凋亡 肝脏磷酸果糖激酶 

分 类 号：R735.9[医药卫生—肿瘤]