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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。
Esophageal cancer is a common gastrointestinal tumor, the mechanism of cell invasion and migration is unclear. The purpose of this study was to investigate the effect and possible mechanism of long non-coding RNA (AFNA) AFAP1-AS1 on invasion and migration of esophageal squamous carcinoma cell line EC9706. Methods The AFAP1-AS1 eukaryotic expression vector was constructed and the pcDNA3.1-AFAP1-AS1 expression vector and the pcDNA3.1 empty vector were transfected into EC9706 by lipofectamine2000. The EC9706-AFAP1-AS1 cell line stably expressing AFAP1-AS1 RNA was established by G418 selection. The changes of invasion and migration of the two cell lines were detected by Millicell chamber. The mRNA expression of VEGF-C and Artemin in two cell lines was detected by Real-time PCR. Western blotting was used to detect the expression of VEGF- Changes of VEGF-C and Artemin Protein Expression in Two Strains. Results The EC9706 cell line stably expressing AFAP1-AS1 was successfully constructed. The migration of EC9706 cells was observed after AFAP1-AS1 was overexpressed (data distribution was in accordance with the normal distribution, SW1 = 0.912, SW2 = 0.878, all P> 0.05) (T = 0.002, P <0.001) and invasion (data distribution in line with the normal distribution, SW1 = 0.945, SW2 = 0.972, all P> 0.05; (P <0.001). The distribution of VEGF-C (normal distribution, SW1 = 0.931, SW2 = 0.922, all P> 0.05). There was no significant difference between the two groups (t = 0.14, P> 0.05) There was no significant difference in mRNA expression between Artemin and normal control (SW1 = 0.964, SW2 = 0.981, P> 0.05; no significant difference between two groups, t = 0.29, P> 0.05) However, the distribution of VEGF-C (normal distribution data, SW1 = 0.762, SW2 = 0.82, P> 0.05; mean difference between the two groups was statistically significant, t = 0.005, P <0.001) SW1 = 0.892, SW2 = 0.853, all P> 0.05; the difference between the two groups was statistically significant, t = 0.004, P <0.001). Conclusion AFAP1-AS1 may enhance its invasion and migration by increasing the expression of VEGF-C and Artemin in EC9706 cells, which may play an important role in the invasion and progression of esophageal cancer.