WWOX基因对卵巢癌PE01细胞黏附能力的影响

Effects of WWOX on ovarian cancer cell attachment in vitro

目的观察WWOX基因转染细胞对卵巢癌PE01细胞黏附能力的影响，并探讨其作用机制。方法利用已构建的WWOX转染细胞、质粒转染细胞及PE01母细胞进行黏附实验，观察不同细胞对纤粘连蛋白（Fn）介导的细胞黏附性。利用整合素仅和B介导的细胞黏附实验，筛选出与卵巢癌细胞黏附有关的整合素。利用筛选出的整合素，进行封闭卵巢癌细胞表面整合素的功能实验，流式细胞检测整合素的表达情况。结果在Fn包被的培养孔中培养2h后，WWOX转染细胞对Fn的细胞黏附性明显低于PE01母细胞和质粒转染细胞（均P〈0．01）。在质粒转染细胞中，整合素以和α3的表达明显高于WWOX转染细胞（均P〈0．05），而整合素β1、β2的表达无明显变化（P〉0．05）；封闭整合素α3后，所有转染细胞对Fn的黏附性明显降低，与非特异性抗体IgG相比，差异有统计学意义（P〈0．05），而封闭整合素以后，与非特异性抗体IgG相比，差异无统计学意义（P〉0．05）。流式细胞检测结果显示，在WWOX转染细胞中，整合素013的表达明显低于质粒转染细胞（P〈0，05）。结论WWOX基因通过调节卵巢癌细胞与细胞外基质的相互作用，降低整合素α3活性，进而降低卵巢癌细胞对Fn介导的黏附性。

Objective To observe the effects of WWOX on cell attachment in ovarian cancer, and to explore its mechanisms of action. Methods Attachment assay was used to assess the adhesion of wwox- transfected PEO1 cells and vector-transfected PEO1 cells that were constructed, as well as PEO1 parent cells. Alpha/beta integrin-mediated cell adhesion assays were designed to identify cells surface integrins in PEO1 clone cells, lntegrin function blocking experiments were designed to further determine integrins in PEO1 clone cells according to the integrin that was selected in integrin expression profiling. FACS analysis was used to further detect the level of integrin alpha3 on the cell membrane. Results Attachment assay showed that adhesion of WWOX-transfected PEO1 cells to fibronectin was significantly slower than that in vector-transfected controls or PEO1 parent cells, cultured on the pre-coated fibronectin for 2 hours （P 〈 0.01 ）. The level of membranous integrins α2 and α3 in the WWOX-transfected PEO1 cells was significantly decreased, as compared with that in veetor-transfected controls （ P 〈 0.05 ）, but there was no association with the level of functioning integrins betal or beta2 in clone cells （P 〉 0.05）. The attachment assays were repeated after pre-incubating the cells with integrin α2 or α3 function-blocking antibodies. These results showed that blocking integrin a3 significantly reduced the binding to fibronectin of all the PEO1 clonal lines, as compared with cells pre-incubated with a non-specific IgG antibody （ P 〈 0.05 ）. In contrast, proincubation with α2 blocking antibody had very little effect on fibronectin binding in these cells （ P 〉 0.05 ）. FACS analysis showed that membranous integrin α3 expression revealed a marked reduction in WWOX- transfected cells than that in vector-transfected cells. Conclusion WWOX acts as an ovarian tumor suppressor by modulating the interaction between tumor cells and the extracellular matrix, decreasing integrin activity and adhesion of tumor cells to fibronectin. This suggests an important role for loss of WWOX tumor suppressor in promoting attachment and adhesion of ovarian cancer cells on locoregional peritoneum, and further resulting in enhancing locoregional peritoneal tumor spread.