下调MDA-MB-231乳腺癌细胞骨硬化蛋白(SOST)可增加间接共培养的MG-63成骨细胞的成骨作用  

Knockdown of SOST in MDA-MB-231 breast cancer cells increase MG-63 osteoblast-like cell function in co-culture system

作　　者：程俊雄 郭丹[2] 陈茜[1] 王婷[1] 黄佳祎[1] 

机构地区：[1]重庆医科大学基础医学院病理生理学教研室,分子医学与肿瘤研究中心,重庆400016 [2]重庆医科大学第二附属医院乳腺外科,重庆400016

出　　处：《细胞与分子免疫学杂志》2016年第11期1481-1485,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：重庆市科委课题(cstc2013jcyj A10097)

摘　　要：目的构建靶向抑制骨硬化蛋白(SOST)基因的小干扰RNA(siRNA)腺病毒,感染MDA-MB-231乳腺癌细胞并与MG-63成骨细胞共培养,检测对成骨相关分子表达的影响。方法针对SOST基因RNA序列设计带3个干扰片段的2对引物,以p B2B为模板,扩增"背靠背"排列的H1和U6启动子。两个PCR产物以Gibson DNA Assembly的方式同时连接腺病毒穿梭质粒p Ad Trace-OK,经同源重组、HEK293细胞包装获得SOST-siRNA腺病毒。感染MDA-MB-231乳腺癌细胞,实时定量PCR检测SOST mRNA的表达水平,ELISA检测SOST蛋白水平。将转染该腺病毒的MDA-MB-231细胞与MG-63成骨细胞间接共培养,实时定量PCR检测护骨因子(OPG)、骨钙蛋白(OCN)、整合素结合涎蛋白(IBSP)、核因子κB受体激活蛋白配体(RANKL)的表达情况。结果成功获得带3个干扰片段的腺病毒穿梭质粒,经同源重组和包装后,获得AdR-si SOST腺病毒。用该病毒感染MDA-MB-231细胞后,明显抑制SOST的表达。在乳腺癌骨转移体外模型中感染Ad-si SOST腺病毒,能导致MG-63细胞的OPG、OCN、IBSP mRNA表达上升,RANKL mRNA降低。结论在乳腺癌骨转移体外模型中感染Ad-si SOST腺病毒,能促进MG-63细胞的成骨分化,提高OPG/RANKL的比例。Objective To construct a recombinant adenovirus expressing siRNA targeting human sclerostin（SOST）gene,and test the function of MG-63 cells while co-cultured with MDA-MB-231 cells infected by Ad-si SOST.Methods According to the RNA sequence of SOST gene,two pairs of primers which contained 3 siRNA sequences were designed,and a p B2 B plasmid was taken as template to amplify 2 DNA sequences.Both of the 2 DNA sequences were ligated to p Ad Trace-OK by Gibson DNA Assembly way.After homologous recombination between recombinant shuttle plasmid and adenovirus vector plasmid,the adenovirus was packaged in HEK-293 cells.PCR and ELISA were used to test the expression of SOST in MDA-MB-231 cells which were infected with Ad-si SOST.In the co-culture system of MG-63 cells and MDA-MB-231 cells infected Ad-si SOST,osteoprotegerin（OPG）,osteocalcin（OCN）,integrin binding sialoprotein（IBSP）and receptor activator of nuclear factor-κB ligand（RANKL） were tested by quantitative real-time PCR.Results Recombinant shuttle plasmid which contained 3 interfering fragments was constructed successfully,and Ad-si SOST was obtained after homologous recombination and packaging.SOST expression in MDA-MB-231 cells was downregulated significantly after infeceted with Ad-si SOST.The mRNA levels of OPG,OCN,IBSP in MG-63 cells increased significantly,while the level of RANKL mRNA decreased significantly, and all 4 gene expressions were reversed after the infection of Ad-si SOST.Conclusion Knockdown of SOST in MG-63 cells increases osteogenesis and ratio of OPG/RANKL in vitro.

关 键 词：SOST SIRNA 腺病毒 乳腺癌 骨转移 

分 类 号：Q279[生物学—细胞生物学] B737.9[哲学宗教—外国哲学]