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【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。
【Aim】 Recent studies have shown that CXCL12 / CXCR4 pathway is highly expressed in esophageal cancer tissue. This study was designed to investigate the effect of CXCL12 / CXCR4 on the expression of CXCR4 in esophageal cancer cell line Eca109 by lentivirus-mediated RNA interference Effect of Pathway on the Metastasis of Esophageal Carcinoma Cell Line Eca109. 【Method】 The human CXCR4 mRNA coding sequence was used as the interference target to construct the vshRNA expression plasmid targeting CXCR4, and the negative control vshRNA expression plasmid not targeting any known mRNA was constructed and transfected into the corresponding group of cells. The study was divided into control group, negative control group and silent group. Real-time quantitative PCR and Western blot were used to detect the expression of CXCR4 in esophageal cancer cells. The invasion ability of Eca109 esophageal cancer cells in each group was evaluated by the number of transmembrane cells in Transwell invasion cells. The adhesion ability of Eca109 cells to Matrigel was observed by MTT assay. The migration ability was detected by scratch assay. 【Results】 The CXCR4 shRNA lentiviral vector CXCR4-RNAi-LV was successfully constructed. The results of QPCR and Western blot showed that the expression of CXCR4 mRNA and protein in Eca109 cells was significantly lower than that in the negative control group and the blank group (P <0.05). After effective silencing of CXCR4, the adhesion, invasion and migration of esophageal cancer Eca109 cells were significantly decreased (P <0.05). 【Conclusion】 Lentivirus-mediated shRNA can effectively silence the CXCR4 gene expression in esophageal cancer cells, block the biological effects of CXCL12 / CXCR4 pathway and effectively inhibit the metastatic potential of esophageal cancer Eca109 cells, suggesting that CXCL12 / CXCR4 pathway may play an important role in esophageal squamous cell carcinoma The development process has an important role.