干扰CXCR4基因抑制胰腺癌经典Wnt通路活化的体外实验研究

目的观察CXCR4 shRNA转染胰腺癌细胞后经典Wnt通路的活性变化.方法构建CXCR4 shRNA表达载体转染胰腺癌CXCR4高表达细胞Miapaca-2并应用G418筛选出稳定表达株,通过Realtime-PCR、WesternBlot实验观察RNAi对CXCR4基因的抑制效率;通过PGL3-OT/OF荧光素酶检测方法观察CXCR4基因沉默后对胰腺癌Wnt/β-catenin经典通路活性的影响,并通过Realtime-PCR、Western Blot实验观察CXCR4 shRNA对Wnt/β-catenin通路下游靶基因的影响.结果特异性CXCR4基因沉默能够在基因和蛋白水平稳定、高效、特异地抑制CXCR4的表达,抑制效率达70%以上,同时,特异性CXCR4干扰后能够显著抑制PGL3-OT转染后的荧光素活性（P〈0.05）以及Wnt/β-catenin下游靶基因的mRNA及蛋白的表达.结论CXCR4基因沉默能抑制Wnt/β-catenin经典通路在胰腺癌中的活性,阻遏Wnt/β-catenin通路下游靶基因表达,为CXCR4作为胰腺癌治疗的分子靶点提供依据.

Inhibition of Canonical Wnt Signaling Pathway in Pancreatic Cancer by Interfering CXCR4 in Vitro

Objective To observe the influence of CXCR4 knockdown by tranfecting CXCR4 shRNA into pancreatic cancer cells on Wnt/β-catenin signaling （canonical Wnt pathway） in pancreatic cancer. Methods CXCR4 shRNA expression vector was constructed and transfected into pancreatic cancer cell Miapaca-2 with high expression of CXCR4, G418 was used to screen the stably transfected cells and Reahime-PCR and Western Blot were used to observe the inhibitory effect of RNAi on CXCR4 expression. PGL3-OT/OF luciferase activity was used to observe the influence of CXCR4 shRNA on Wnt activity. Reahime-PCR and Western Blot were applied to observe the influence of specific CXCR4 shRNA on Wnt/β-catenin signaling target gene expression. Results Specific CXCR4 silencing stably, effectively, and specifically inhibited the CXCR4 mRNA and protein expression, and the inhibitory efficiency was over 70%. Meanwhile, CXCR4 gene interference effectively inhibited Wnt pathway activity （P 〈 0.05）, and Wnt/β-catenin target gene expression in pancreatic cancer cell lines were downregulated. Conclusion CXCR4 knockdown can inhibit Wnt/β-catenin pathway and its target gene expression in pancreatic cancer, which can result in the decrease of pancreatic cancer cell growth, cell tumorigenesis and invasive ability, and this study may provide evidence for CXCR4 as a molecular target in pancreatic cancer treatment.