pcDNA3.1-Annexin A1真核表达载体对结直肠癌细胞迁移、侵袭的影响

Effects of eukaryotic expression vector pcDNA3.1-Annexin A1 on migration and invasion of colon cancer cells

目的探讨Annexin A1对结直肠癌细胞迁移、侵袭能力的影响。方法构建重组质粒pcDNA3.1-AnnexinA1,转染至结直肠癌细胞株SW480中,G418筛选稳定表达株,实时荧光定量PCR、蛋白质印迹检测转染前后Annexin A1mR-NA及蛋白表达;以空载体转染组及未转染SW480细胞作为对照,通过划痕修复实验和Transwell细胞侵袭实验对转染前后细胞的迁移和侵袭能力进行比较观察和分析。结果成功构建重组质粒pcDNA3.1-Annexin A1载体;实时荧光定量PCR和蛋白质印迹检测结果均显示,重组体pcDNA3.1-Annexin A1稳定转染细胞株中Annexin A1mRNA及蛋白表达均明显高于空载体转染及未转染组细胞(P<0.01),而后两者间比较差异无统计学意义(P>0.05);重组体pcDNA3.1-Annexin A1转染组SW480细胞的迁移率(0.415±0.002)明显高于空载体转染组(0.267±0.003)及未转染组细胞(0.271±0.002),差异具有统计学意义(P<0.05),而后两者间差异无统计学意义(P>0.05);与空载体转染组(162.80±12.07)及未转染组(164.25±9.50)相比,重组体pcDNA3.1-Annexin A1转染组穿膜细胞数(221.75±12.07)增多,差异具有统计学意义(P<0.05).结论人Annexin A1基因表达上调能提高结直肠癌细胞迁移和侵袭能力。

Objective To evaluate the effects of Annexin A1 on migratory and invasive ability of colon cancer cells.Methods Eukaryotic expression vector pcDNA3.1-Annexin A1 was constructed and analyzed by restriction analysis and sequencing.The recombinant plasmid pcDNA3.1-Annexin A1 was transfected into SW480 cells by liposome-mediated gene transferred method.The stable transfectants were obtained after screening with G418.Real-time PCR and Western blotting analysis were used to examine the expression of Annexin A1 mRNA and protein in SW480 cells before and after transfection.Wound-healing experiment and Transwell invasion assays were used to study the effects of Annexin A1 on the migratory and invasive ability of SW480 cells.Results The recombinant plasmid pcDNA3.1-Annexin A1 was successfully constructed.The results of real-time PCR and Western blotting analysis showed that the Annexin A1 expression was significantly higher in cells transfected with pcDNA3.1-Annexin A1 than in un-transfected cells or those transfected with empty vectors（P0.01）,while there was no significant difference between the last two groups（P0.05）.The migratory rate of SW480 cells in pcDNA3.1-Annexin A1 group was significantly higher than those in un-transfected cells or transfected with empty vectors（／[0.415±0.002） vs ／[0.267±0.003／] and ／[0.271±0.002／],P0.05）,and there was no significant difference between the latter two groups.The migrating SW480 cells in pcDNA3.1-Annexin A1 group was significantly more than those in the other two groups（／[221.75±12.07／] vs ／[162.80±12.07／] and ／[164.25±9.50／],P0.05）.Conclusion Overexpression of Annexin A1 can increase the migratory and invasive ability of SW480 cell line.