Inhibition of MMP-1 expression in oteosarcoma cell line by RNA interference

Objective To construct the specific short hairpin RNA(shRNA)eukaryotic expres- sion vector targeting human matrix metalloproteinase-1(MMP-1)gene,further to observe its effect on inhibiting MMP-1 gene expression in human osteosarcoma cell line MG63.Methods According to MMP-1 cDNA coding sequence,the specific RNA interference(RNAi)fragments targeting MMP-1 gene were designed and synthesized,which were cloned into pSilencer 3.0-H1 neo plasmid vector,and the shRNA eukaryotic expression vector siRNA MMP-1targeting MMP-1 gene was constructed..Af- ter the vector was constructed,MG63 cells were transfected with negative control vector siRNA neg or siRNA MMP-1 and selected by G418.Expression of mRNA and protein of MMP-1 in the stable trans- fected cells was investigated separately by RT-PCR and Western blot.Cell invasion assay was utilized to evaluate invasiveness of the stable transfetants.Results The specific shRNA eukaryotic expression vector siRNA MMP-1 targeting MMP-1 gene was constructed successfully.The stable transfectants containing negative control vector siRNA neg or siRNA MMP-1 were obtained.Expression of mRNA and protein of MMP-1was inhibited significantly in MG63/siRNA MMP-1 cells,whereas MMP-1 gene expression levels were hardly changed in control group.A significant reduction in cell invasion was measured in MG63/siRNA MMP-1 cells as compared with negative control cells.Conclusions MMP- 1 gene expression can be suppressed markedly by specific shRNA in MG63 cells,the current results establishing the experimental foundation for further studying the biological functions and its mecha- nisms of MMP-1 in MG63 cells.