LncRNA AFAP1-AS1对食管鳞癌细胞株EC9706转移潜能影响机制探讨

Effect and mechanism of LncRNA AFAP1-AS1 on migration and invasion of esophageal squamous cell carcinoma cell line EC9706

目的食管癌是常见的消化道肿瘤,其细胞侵袭与迁移机制尚不清楚。本研究旨在探讨长链非编码RNA(long non-coding RNA,LncRNA)AFAP1-AS1对食管鳞癌细胞株EC9706侵袭及迁移能力的影响及可能的作用机制。方法构建AFAP1-AS1真核表达载体,用脂质体2000将pcDNA3.1-AFAP1-AS1表达载体及pcDNA3.1空载体转染食管癌细胞EC9706。通过G418筛选,建立稳定表达AFAP1-AS1RNA的EC9706-AFAP1-AS1细胞系。Millicell小室检测2株细胞侵袭及迁移能力的变化,Real-time PCR法检测2株细胞中血管内皮生长因子C(vascular endothelial growth factor C,VEGF-C)及Artemin的mRNA表达情况,蛋白质印迹法检测2株细胞中VEGF-C及Artemin蛋白表达的变化。结果成功构建稳定表达AFAP1-AS1的EC9706细胞系,过表达AFAP1-AS1后,EC9706细胞的迁移(数据分布符合正态分布,SW1=0.912,SW2=0.878,均P>0.05;两组间均值差异有统计学意义,t=0.002,P<0.001)及侵袭(数据分布符合正态分布,SW1=0.945,SW2=0.972,均P>0.05;两组间均值差异有统计学意义,t=0.02,P<0.001)能力均增加,VEGF-C(数据分布符合正态分布,SW1=0.931,SW2=0.922,均P>0.05;两组间均值差异无统计学意义,t=0.14,P>0.05)和Artemin(数据分布符合正态分布,SW1=0.964,SW2=0.981,均P>0.05;两组间均值差异无统计学意义,t=0.29,P>0.05)的mRNA表达差异无统计学意义,但VEGF-C(数据分布符合正态分布,SW1=0.762,SW2=0.82,均P>0.05;两组间均值差异有统计学意义,t=0.005,P<0.001)及Artemin(数据分布符合正态分布,SW1=0.892,SW2=0.853,均P>0.05;两组间均值差异有统计学意义,t=0.004,P<0.001)的蛋白表达明显增加。结论 AFAP1-AS1可能通过增加EC9706细胞中VEGF-C及Artemin的蛋白表达来增强其侵袭及迁移能力,在食管癌侵袭及进展中发挥作用。

OBJECTIVE Esophageal cancer is a common gastrointestinal tumor, and the mechanism of cell invasion and migration is not clear. The purpose of this study was to explore the invasion and migration ability of long chain noncoding RNA AFAP1-AS1 in esophageal carcinoma cells EC9706, and further to explore the biological mechanisms. METHODS Eukaryotic expression vector pcDNA3.1-AFAP1-AS1 was constructed, pcDNA3.1-AFAP1-AS1 was transfected into EC9706 cells by lipofectamine 2000 for 24 h and selected with 500 μg/mL G418 for 3 weeks, transfected with pcDNA3.1 empty vector acted as a control. The positive clone was identified by reverse transcription polymerase chain reaction （RT-PCR） for AFAP1-AS1 RNA expression. Cell invasion and migration ability was assessed using millicell chamber and would healing assay. VEGF-C and Artemin mRNA level were detected by Real-time PCR. VEGF-C and Artemin protein level were detected by Western blotting analysis. RESULTS Stable cell line EC9706 expressing AFAP1-AS1 was successfully established. The migration （73.37±9.50 vs 28.05±5.01, t=0. 002, P〈0. 001） and invasion （34. 05±5.08 vs 21.33± 3.51, t= 0.02, P〈0. 001） ability were enhanced in EC9706 cell lines upregulated expression of AFAP1-AS1. VEGF-C （t=0.14, P〉0.05） and Artemin （t=0.29, P〉0.05） mRNA level had no remarkable difference between two groups. VEGF-C （t=0. 005, P〈0. 001） and Artemin （t=0. 004, P〈0. 001） protein expression were higher in EC9706 cell line expressing AFAP1-AS1 than that in the control group. CONCLUSION VEGF-C and Artemin protein expression increased in EC9706-AFAP1-AS1 cells compared with EC9706-BC cells, which suggested that AFAP1-AS1 RNA might play some role in EC9706 cells invasion and progression.