ENO1在胃癌干细胞中表达及其与胃癌侵袭转移的相关性研究

Expression of ENO1 in Gastric Cancer Stem Cells and Its Relation to Invasion and Metastasis of Gastric Cancer

[目的]探讨ENO1在胃癌干细胞中的表达及其对胃癌侵袭转移的影响。[方法]以胃癌SNU-5模型,实时荧光定量PCR和双色细胞免疫荧光检测ENO1在其分选的CD44^+细胞中的表达情况。运用慢病毒载体稳定干扰SUN-5中ENO1的表达,并用实时荧光定量PCR和Western blot检验干扰效率。流式细胞术分选稳定干扰ENO1的SNU-5中CD44^+细胞后(shRNA-ENO1),实时荧光定量PCR检测其Oct-4、Sox 2以及干性相关基因Nanog的表达,同时分别进行细胞成球实验、体外侵袭与体内致瘤的胃癌干细胞生物学特性研究,Western blot检测ENO1对胃癌干细胞侵袭转移影响的相关分子机制。[结果] SNU-5的CD44^+细胞中ENO1基因和蛋白表达明显高于CD44-细胞,并建立稳定干扰ENO1的SUN-5。shRNA-ENO1细胞中干性基因Oct-4、Sox 2和Nanog中m RNA的表达显著低于PLV-Ctr细胞(P<0.05)。与PLV-Ctr细胞相比较,shRNA-ENO1细胞的自我更新能力、侵袭能力和肿瘤重量显著降低。Western blot检测shRNA-ENO1细胞中Vimentin、Snail和N-cadherin下调表达,同时E-cadherin上调表达,并伴随AKT和PI3K的磷酸化水平降低,提示ENO1的作用可能通过PI3K/AKT信号通路激活。[结论] ENO1在胃癌干细胞中高表达,其在调控胃癌侵袭转移能力中发挥重要作用。

[Purpose] To investigate the expression of enolase-1(ENO1) in gastric cancer stem cells(GCSCs) and its relation to the invasion and metastasis of gastric cancer. [Methods] CD44^+ cells were sorted from human gastric cancer SNU-5 cells,the expression of ENO1 mRNA and protein was detected by RT-PCR and two-color immunofluorescence method. SUN-5 cells and sorted CD44^+ cells were transfected with short hairpin RNA(shRNA) to knock down the expression of ENO1. The expression of Oct-4,Sox 2 and stemness-related gene Nanog was detected by RT-PCR,and the biological characteristics of GCSCs were examined by sphere colony formation assay,invasion assay and in vivo tumorigenicity. [Results] The expression of ENO1 mRNA and protein in CD44^+ cells from SNU-5 was significantly higher than that in CD44^- cells. The mRNA expression of Oct-4,Sox 2 and Nanog from shRNA-ENO1 cells was significantly lower than that of control PLV-Ctr cells(P<0.05). Compared with the PLV-Ctr cells,the shRNA-ENO1 cells showed significantly decreased self-renewal capacity and invasion ability,and the tumor growth in vivo was inhibited. Western blot showed that Vimentin,snail and N-cadherin was up-regulated in shRNAENO1 cells,while E-cadherin was down-regulated and the phosphorylation of AKT and PI3K was decreased,suggesting that ENO1 may be activated by AKT/PI3K signaling pathway. [Conclusion]ENO1 is highly expressed in GCSCs,which may be involved in the invasion and metastasis of gastric cancer.