title: [Knockdown of SOST in MDA-MB-231 breast cancer cells increase MG-63 osteoblast-like cell function in co-culture system].
abstract: 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-siSOST. MethodsAccording to the RNA sequence of SOST gene, two pairs of primers which contained 3 siRNA sequences were designed, and a pB2B plasmid was taken as template to amplify 2 DNA sequences. Both of the 2 DNA sequences were ligated to pAdTrace-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-siSOST. In the co-culture system of MG-63 cells and MDA-MB-231 cells infected Ad-siSOST, 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-siSOST was obtained after homologous recombination and packaging. SOST expression in MDA-MB-231 cells was downregulated significantly after infeceted with Ad-siSOST. 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-siSOST. Conclusion Knockdown of SOST in MG-63 cells increases osteogenesis and ratio of OPG/RANKL in vitro.
authors: Research Center of Molecular Medicine and Cancer, Department of Pathophysiology, College of Basic Medicine, Chongqing 400016, China.
authors: Junxiong
authors: J
authors: Cheng
authors: Department of Breast Surgery, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors: Dan
authors: D
authors: Guo
authors: Research Center of Molecular Medicine and Cancer, Department of Pathophysiology, College of Basic Medicine, Chongqing 400016, China.
authors: Xi
authors: X
authors: Chen
authors: Research Center of Molecular Medicine and Cancer, Department of Pathophysiology, College of Basic Medicine, Chongqing 400016, China.
authors: Ting
authors: T
authors: Wang
authors: Research Center of Molecular Medicine and Cancer, Department of Pathophysiology, College of Basic Medicine, Chongqing 400016, China. *Corresponding author, E-mail: e738741@126.com.
authors: Jiayi
authors: J
authors: Huang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins / Q000172:deficiency / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D005819:Genetic Markers / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D058574:Integrin-Binding Sialoprotein / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis / Q000235:genetics / Q000502:physiology; D053244:Osteoprotegerin / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; D058574:Integrin-Binding Sialoprotein; D053244:Osteoprotegerin; D053245:RANK Ligand; C422903:SOST protein, human; C505688:TNFSF11 protein, human; D015675:Osteocalcin