慢病毒介导shRNA静默CXCR4对Eca109细胞转移特性的影响

Effect of CXCR4 Gene Silencing by Lentivirus-shRNA on Metastatic Characteristics of Human Esophageal Carcinoma Cell Line Eca109

【目的】新近研究显示CXCL12/CXCR4通路在食管癌组织中有较高的表达,本研究探讨应用慢病毒介导的RNA干扰技术,有效静默食管癌细胞株Eca109细胞CXCR4的表达,研究CXCL12/CXCR4通路对食管癌Eca109细胞转移能力的影响。【方法】以人CXCR4mRNA编码序列作为干扰靶点,构建靶向CXCR4的vshRNA表达质粒,另构建不针对任何已知mRNA的阴性对照vshRNA表达质粒,转染相应组别细胞。研究分为控制组、阴性对照组及静默组。应用实时定量PCR及Westernblot检测转染后食管癌细胞CXCR4表达的变化。Transwell侵袭小室实验以穿膜细胞的数量评估各组Eca109食管癌细胞的侵袭能力,MTT法观察Eca109各组细胞与Matrigel胶黏附能力的变化,划痕法检测迁移能力改变。【结果】成功构建CXCR4shRNA慢病毒载体CXCR4-RNAi-LV。QPCR及Westernblot结果显示静默组Eca109细胞CXCR4mRNA及蛋白的表达水平较阴性对照组及空白组显著降低(P<0.05)。CXCR4有效静默后,食管癌Eca109细胞黏附、侵袭及迁移能力显著下降(P<0.05)。【结论】慢病毒介导的shRNA能有效地静默食管癌细胞CXCR4基因的表达,阻断CXCL12/CXCR4通路生物学效应,有效抑制食管癌Eca109细胞的转移潜能,提示CXCL12/CXCR4通路在食管鳞癌发展过程中具有重要作用。

【Objective】 CXCL12 / CXCR4 is highly expressed in esophageal squamous cell carcinoma （Escc）. To study the effect of CXCR4 down-regulation by lentivirus-mediated RNA interference （RNAi） silencing technique on metastatic characteristics of human esophageal carcinoma cell line Eca109. 【Methods】 One lentiviral vetor for RNAi of CXCR4 containing a sequence of human CXCR4 mRNA coding region was constructed,the other lentiviral vetor for RNAi of CXCR4 containing a sequence of no significant and homology to human gene sequences was constructed,and transfected into Eca109 cells,respectively. There were control group,NC-GFP-RNAi-LV group,and CXCR4-RNAi-LV group in our study. The expression of CXCR4 in the cells was detected by QRT-PCR and Western blot after transfection. The adhesive ability of Eca109 cells to extracellular matrix matrigel was evaluated by MTT assay. The invasive ability was measured by Transwell experiment. The locomotion ability was measured by scrape assay. 【Results】 The lentivirus RNAi vector of CXCR4-RNAi-LV was constructed successfully. Compared with NC-GFP-RNAi-LV group and Control group cells,CXCR4 mRNA and protein expression in CXCR4-RNAi-LV group cells were down-regulated （P 0.05）. The number of Eca109 cells infiltrated Transwell membrane in NC-GFP-RNAi-LV group was decreased （P 0.05）. The adhesion rate of CXCR4-RNAi-LV group cells was obviously reduced （P 0.05）. The locomotion ability of CXCR4-RNAi-LV group cells was obviously reduced （P 0.05）. 【Conclusion】 CXCR4-vshRNA can inhibit CXCR4 expression specificallyand effectively in esophageal squamous cell carcinoma cell line Eca109,CXCR4-vshRNA can inhibit metastatic potential of Eca109 cells through intervening the expression of CXCR4. Suggesting that CXCL12 / CXCR4 might have an oncogene role in the progression of Escc.