膜联蛋白A2质粒的构建及其在膀胱癌pumc-91细胞增殖和迁移中的作用

Construction of annexin A2 plasmid and its role in the proliferation and migration of bladder cancer pumc-91 cells

目的膜联蛋白A2(annexin A2,Anxa2)在多种肿瘤细胞中具有调节生物活性的作用。本研究旨在探讨Anxa2蛋白的表达与膀胱癌pumc-91细胞增殖和迁移的相关性。方法扩增ANXA2序列,插入pcDNA3.1(+)载体,制备pcDNA3.1(+)-ANXA2质粒。用脂质体lipo2000将pcDNA3.1(+)-ANXA2瞬时转染至pumc-91膀胱癌细胞中。Western blotting检测空白组、转染pcDNA3.1(+)空载的对照组和转染pcDNA3.1(+)-ANXA2质粒的实验组中Anxa2蛋白的表达。利用Cell Counting Kit-8(CCK8)检测pumc-91细胞的增殖能力,transwell实验检测pumc-91细胞的迁移水平。采用单因素方差分析或重复测量的方差分析比较各组间检测数据的差异。结果PcDNA3.1(+)-ANXA2质粒构建成功,测序完全正确。Western blotting检测显示,与空白组和对照组相比,实验组Anxa2蛋白表达水平增高。CCK8实验显示实验组增殖的pumc-91细胞数量相比于空白组(P<0.001)和对照组(P=0.001)明显增多;transwell实验中实验组穿膜的pumc-91细胞数量较空白组(P=0.011)和对照组(P=0.027)也明显增多。结论Anxa2蛋白的表达上调可能在膀胱癌pumc-91细胞增殖和迁移中起重要作用。

Objective Annexin A2(annexin A2,Anxa2)has been reported to regulate bioactivity in various tumors cells.The purpose of this study was to investigate the correlation between the expression of Anxa2 protein and the proliferation and migration abilities of bladder cancer pumc-91 cells.Methods The ANXA2 sequence was amplified and inserted into the pcDNA3.1(+)vector in order to prepare the pcDNA3.1(+)-ANXA2 plasmid.PcDNA3.1(+)-ANXA2 was transiently transfected into pumc-91 bladder cancer cells by lipofectamine 2000.Western blotting assay was performed to detect the expression of Anxa2 protein in the blank group,the control group transfected with pcDNA3.1(+),and the experimental group transfected with pcDNA3.1(+)-ANXA2 plasmid.The proliferation ability of pumc-91 cells was detected using Cell Counting Kit-8(CCK8),and the migration level of pumc-91 cells was detected by transwell assay.Differences in detection data among the groups were compared using one-way ANOVA or repeated measures ANOVA.Results The plasmid construction was successful and the sequencing was absolutely correct.Western blotting assay showed elevated Anxa2 protein expression level in the experimental group compared to the blank and control groups.CCK8 assay suggested that the number of proliferating pumc-91 cells was significantly higher in the experimental group than in the blank group(P<0.001)and the control group(P=0.001).Transwell assay also showed that the number of pumc-91 cells crossing the membrane was significantly higher in the experimental group than in the blank group(P=0.011)and the control group(P=0.027).Conclusion Our findings suggested that up-expression of Anxa2 may play a critical role in regulating proliferation and migration of bladder cancer pumc-91 cells.