Construction Of Adenovirus Targets Human SOST Gene And Its Affection On MG-63 Cells In Indirectly Cocultrue System With MDA-MB-231 Cells

ObjectiveTo construct a recombinant adenovirus expressing si RNA target sites for human SOST,and evaluate the role of SOST in osseous metastasis of breast cancer. Methods1.Designed two pairs of primer containing 3 siRNA target sites for human SOST using Dharmacon’s siDESIGN program and got the overlapping inserts by PCR amplifications. The correctly identified recombinant plasmid was linearized with Pme Ⅰ and transformed into BJ5183-Ad-easy competent cells containing adenovirus backbone vector to produce recombinant adenovirus DNA by homologous recombination. Then the recombinant adenovirus DNA was linearized with Pac Ⅰ and transfected into 293 cells to make adenovirus. The silencing efficiency of recombinant adenovirus was assess by RT-PCR and ELISA in MDA-MB-231 cell line.2. In order to mimic the osseous metastasis of breast cancer,we set up an indirect co-culture syetem of MDA-MB-231 and MG-63.And tested the osteoblast function by PCR assay after block the expression of sclerostin by adenovirus. Results1.Three pairs of oligonucleotides targeting human SOST were subcloned into shuttle plasmid pAdTrace-OK to make recombinant adenovirus by “on-step”.After infected with the recombinant adenovirus SOST-siRNA, the expression of SOST sharply decrease in the human breast cancer cell line MDA-MB-231.2.The osteogenic ability of osteoblast cell line MG-63 decreased after co-culture with MDA-MB-231 cell;and increased while block the expression of sclerosin in MDA-MB-231. Conclusions1.The recombinant adenovirus expressing siRNA target sites for human SOST has been successfully constructed and may be used to silence the expression of SOST in MDA-MB-231 cell line effectively.2.Sclerostin was shown to involved in osseous metastasis of breast cancer,and block its expression would improve the osteoblastic level.