关键词: BMP4, siRNA, 腺病毒, 乳腺癌, 骨转移

Abstract: Objective To construct a recombinant adenovirus expressing siRNA target sites for human BMP4. Methods six pairs of oligonucleotides containing siRNA target sites for human BMP4 using Dharmacon’s siDESIGN program were subcloned into the Sfi I site of pSOS,which has been inserted the coding regions of human BMP4,resulting in pSOS-sirBMP4.Then the pSOS-sirBMP4 were transfected into HEK-293 cells, and GFP signal levels were used to assess the silencing efficiency of different siRNA target sites. Four pairs of oligonucleotides were chosed and subcloned into shuttle plasmid pSES-HUS and authenticity of PCR amplified sequences and the oligonucleotide cassettes were verified by DNA sequencing. The correctly identified recombinant plasmid was linearized with Pme1 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 Pac1 and transfected into 293 cells to make adenovirus. the silencing efficiency of recombinant adenovirus was assess by RT-PCR and Western blot in MDA-MB-231 cell line. MTT assay was used to detect the proliferation of MDA-MB-231 cells transfected with pAd-sirBMP4. Results Four pairs of oligonucleotides with high silencing efficiency were chosed and subcloned into shuttle plasmid pSES-HUS to make recombinant adenovirus . After infected with the recombinant adenovirus BMP4-siRNA, the expression of BMP4 sharply decrease in the human breast cancer cell line MDA-MB-231 . Conclusion The recombinant adenovirus expressing siRNA target sites for human BMP4 has been successfully constructed and could be used to silence the expression of BMP4 in MDA-MB-231 cell line effectively.