对头颈鳞状细胞癌中高表达基质金属蛋白酶基因具有激活作用的人类膜蛋白基因筛选

摘要

Objective To screen out the human membrane protein genes activating matrix metalloproteinases (MMPs) (MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, and MMP13) highly expressed in squamous-cell carcinoma of head and neck.Methods Seven alternative MMP promoter reporter gene plasmids were constructed.PMA, a kind of protein kinase C activator, was used to stimulate them;Dual luciferase assay system was used to detect the basic and stimulated activities of MMP promoter reporters.The relative luciferase activity (RLA) values were measured as the ratio of the basic divided by the stimulated ones;the highest RLA was selected MMP promoter reporter gene plasmid.A total of 722 candidate genes encoding potential human transmembrane proteins were screened in a dual luciferase based assay system.The candidate genes with RLA greater than 10 were considered as the activator to MMP promoter reporters in the first round, and the second and third rounds followed the same way.Finally, the efficacious human membrane protein genes were ascertained.Results The RLAs of MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, and MMP13 promoter reporters stimulated by PMA were 4.53±0.72, 1.47±0.28, 23.28±1.96, 2.37±0.46, 8.81±1.35, 2.15±0.51, and 3.47±0.99.The MMP3 reporter was selected for a high through-put screening for the sensitivity (P<0.05).G protein-coupled receptor 65 (GPR65), AXL receptor tyrosine kinase (AXL), and tumor necrosis factor receptor superfamily 10b (TNFRSF10B) were confirmed as significant activators to MMPs by screening all 722 plasmids in 3 rounds.Conclusion GPR65, AXL, and TNFRSF10B could activate MMPs highly expressed in head and neck squamous-cell carcinoma.%目的 对头颈部鳞状细胞癌中高表达的基质金属蛋白酶(MMP)基因(包括MMP1、MMP2、MMP3、MMP9、MMP10、MMP12 和 MMP13)具有激活作用的人类膜蛋白基因进行筛选.方法 构建上述7个备选MMP启动子报告基因质粒.用豆蔻酸佛波酰乙酯(PMA,一种蛋白激酶C激活剂)刺激7个备选MMP启动子报告基因质粒.用双荧光素酶检测系统检测各备选MMP启动子报告基因质粒PMA刺激前的活性及PMA刺激后的活性,以后者除以前者得出各备选MMP启动子报告基因质粒的相对荧光素酶活性值(RLA),RLA最大者选定为MMP启动子报告基因质粒.用人类膜蛋白基因质粒库中的722种质粒依次刺激MMP启动子报告基因质粒,刺激前后MMP启动子报告基因质粒RLA均数超过10确定为对MMP启动子报告基因质粒活性有明显激活作用的人类膜蛋白基因质粒.将筛选出的人类膜蛋白基因质粒以相同方法进行第二轮筛选,第二轮筛选出的人类膜蛋白基因质粒以相同方法进行第三轮筛选,最终确定对头颈部鳞状细胞癌中高表达的MMP有激活作用的人类膜蛋白基因.结果 PMA刺激后MMP1、MMP2、MMP3、MMP9、MMP10、MMP12、MMP13启动子报告基因质粒的RLA分别为4.53±0.72、1.47±0.28、23.28±1.96、2.37±0.46、8.81±1.35、2.15±0.51、3.47±0.99.MMP3启动子报告基因质粒的RLA明显高于其他MMP启动子报告基因质粒(P均<0.05),被选为MMP启动子报告基因质粒.全部722个质粒经3轮筛选,确定AXL受体酪氨酸激酶(AXL) 、肿瘤坏死因子受体超家族成员 10b(TNFRSF10B)和G蛋白耦联 受体65(GPR65)质粒对MMP启动子报告基因质粒均有明显的激活作用.结论 GPR65、 AXL、TNFRSF10B基因对头颈部鳞状细胞癌中高表达的MMP基因有激活作用.