LKB1调控Peutz-Jeghers综合征错构瘤及肠上皮细胞中的上皮间质转化

LKB1 regulates epithelial-mesenchymal transition in Peutz-Jeghers hamartoma and intestinal epithelial cells

目的探讨LKB1在Peutz-Jeghers综合征(PJS)错构瘤及肠上皮细胞中对上皮间质转化(EMT)调控作用。方法采用免疫组化法检测20例PJS错构瘤标本及10例正常肠道组织中LKB1、ecadherin、vimentin蛋白的表达。Masson三色染色法分析胶原纤维沉积。选取NCM460人肠上皮细胞株,慢病毒转染后建立LKB1敲低及空载体稳定表达株。以CCK8实验和transwell迁移实验评价LKB1敲低对细胞增殖、迁移的影响。运用WB、q PCR、免疫荧光检测LKB1敲低后对细胞中EMT相关蛋白ecadherin、vimentin、ncadherin、snail、slug表达的影响。结果与正常肠组织相比,PJS错构瘤中LKB1和ecadherin蛋白表达下降,而vimentin蛋白表达增加。Masson染色提示错构瘤中胶原纤维沉积增多,沉积面积扩大。与空载体组相比,LKB1敲低的NCM460细胞增殖、迁移能力明显增强(P<0.01),细胞中ecadherin表达降低,ncadherin、vimentin、snail、slug表达增高。结论LKB1可能通过调控EMT从而影响PJS错构瘤的形态改变及纤维化进程,提示EMT可能成为PJS治疗的新靶点。

Objective To investigate the molecular mechanism by which LKB1 regulates epithelial-mesenchymal transition （EMT） in Peutz-Jeghers hamartoma and intestinal epithelial cells. Methods Immunohistochemistry was used to detect gene expression of LKB1, E-cadherin, and vimentin in 20 hamartoma tissues and 10 normal intestinal tissues, and collagen fiber deposition was analyzed using Masson trichrome staining. Normal intestinal epithelial NCM460 cells were transfected with LKB1 shRNA plasmid or negative control via lentiviral vectors, and the role of LKB1 in cell polarization and migration were determined using CCK8 and Transwell assays. Western blotting, quantitative real-time PCR （qPCR） and immunofluorescence were used to assess the alterations of EMT markers in the cells with LKB1 knockdown. Results Compared with normal intestinal tissues, hamartoma polyps showed significantly decreased LKB1 and E-cadherin expressions and increased vimentin expression with increased collagen fiber deposition. The cells with LKB1 knockdown exhibited enhanced cell proliferation and migration activities （P〈0.01）. Western blot analysis, qPCR and immunofluorescence all detected decreased E-cadherin and increased N-cadherin, vimentin, Snail, and Slug expressions in the cells with LKB1 knockdown. Conclusions LKB1 deficiency triggers EMT in intestinal epithelial cells and Peutz-Jeghers hamartoma, suggesting that EMT can serve as the therapeutic target for treatment of Peutz-Jeghers syndrome.