PCDH10通过下调Wnt/β-catenin信号通路抑制多发性骨髓瘤细胞的增殖  

PCDH10 inhibits cell proliferation of multiple myeloma through down-regulating Wnt/β-catenin signaling

作　　者：许永会[1] 陈建斌[1] 袁海汀 杨泽松[2] 李珍[2] 李颖[2] 汤为学[3] 黄宗干[1] 

机构地区：[1]重庆医科大学附属第一医院血液科,重庆400016 [2]重庆医科大学附属第一医院血液科实验室,重庆400016 [3]重庆医科大学附属第一医院实验研究中心,重庆400016

出　　处：《第三军医大学学报》2015年第14期1471-1477,共7页Journal of Third Military Medical University

基　　金：重庆市卫生局医学科研计划重点项目(2011-1-032);重庆市医学科研计划(2010-2-108)~~

摘　　要：目的探讨原钙粘蛋白-10(procadherin-10,PCDH10)基因是否通过调控Wnt/β-catenin信号通路抑制多发性骨髓瘤细胞的增殖及其相关机制。方法采用脂质体转染法将空载体pc DNA3.1(+)TP53质粒和pc DNA3.1(+)PCDH10质粒分别转染至人多发性骨髓瘤细胞KM3细胞中。实验分为对照组、空载体组、转染组。采用RT-PCR和Western blot检测3组细胞PCDH10基因的表达。RTPCR检测不同浓度的Li Cl(0、5、10、15、20μmol/m L)处理KM3细胞48 h后β-catenin的表达,选取Li Cl的最佳浓度分别作用于3组细胞。应用CCK-8法检测细胞增殖能力。采用流式细胞仪检测细胞周期。质粒双荧光素酶报告基因检测系统分析PCDH10转染前后对KM3细胞LEF/TCF基因的影响。实时荧光定量PCR(qRT-PCR)和Western blot检测β-catenin、c-Myc、Cyclind1基因mRNA、蛋白水平及β-catenin蛋白在细胞核内的表达。免疫荧光观察PCDH10对β-catenin核转位的影响。结果 PCDH10基因转染后在细胞中获得稳定表达。CCK-8检测结果发现转染组细胞的增殖能力明显受抑(P<0.05),流式细胞仪检测结果显示转染组细胞周期阻滞于G1期、增殖指数降低(P<0.05);荧光素酶活性分析显示PCDH10显著抑制LEF/TCF基因活性(P<0.05);qRT-PCR和Western blot结果显示,转染组细胞中β-catenin、c-Myc、Cyclind1表达明显降低(P<0.05),细胞核内β-catenin的蛋白表达量明显下调;免疫荧光观察到转染组细胞核内β-catenin荧光强度减弱。RT-PCR检测发现20μmol/m L的Li Cl为KM3细胞的最佳作用浓度,经Li Cl处理3组细胞后,与对照组、空载体组比较,转染组细胞增殖能力及β-catenin、c-Myc、Cyclind1的表达也受到抑制(P<0.05)。结论 PCDH10基因能抑制KM3细胞的增殖,其机制可能主要与下调LEF/TCF基因活性及抑制β-catenin核转位有关。Objective To explore whether the effect of procadherin-10（ PCDH10） on inhibiting cell proliferation is related with Wnt signaling in multiple myeloma（ MM） and the underlying mechanism.Methods Human MM cell line KM3 was stably transfected with pc DNA3. 1（ ＋） or pc DNA3. 1（ ＋）PCDH10 by lipofectamine2000. The expression of PCDH10 gene was confirmed by RT-PCR and Western blottig. Different concentrations（ 0,5,10,15 μmol/m L and 20 μmol/m L） of Li Cl was used to treat KM3 cells for 48 h,and then RT-PCR was adopted to determine the proper concentration of Li Cl. CCK-8 assay was applied to examine the cell proliferation. Dual luciferase reporter assay was performed to assess the activity of LEF/TCF gene. Cell cycle was analyzed by flow cytometry. The expression of relative genes was detected by q PCR and Western blotting. Results The expression of PCDH10 gene was obviously increased after transfection. It was observed that the proper concentration of Li Cl was 20 μmol/m L. PCDH10 over-expression highly inhibited the cell proliferation,even when the cells were treated with Li Cl（ P〈0. 05）. Dual luciferase reporter assay showed that the activity of LEF/TCF gene was significantly impaired by PCDH10 forced expression（ P〈0. 05）. Flow cytometry revealed that PCDH10 arrested cell cycle in G1phase（ P〈0. 05）,and the cells treated with Li Cl showed the same trend（ P〈0. 05）. In addition,the expression of c-Myc and Cyclind1 were down-regulated at both mRNA and protein levels. The mRNA expression of β-catenin was not blocked evidently,buy the protein expression and nuclear accumulation of β-catenin were inhibited obviously even when the KM3 cells were treated with Li Cl（ P〈0. 05）. Conclusion PCDH10 can inhibit cell proliferation through inhibiting Wnt/β-catenin signaling in MM.

关 键 词：原钙粘蛋白-10 WNT/Β-CATENIN信号通路 多发性骨髓瘤 细胞增殖 

分 类 号：R374.3[医药卫生—病原生物学] R730.23[医药卫生—基础医学]