应用RNA干扰技术抑制骨肉瘤细胞中基质金属蛋白酶-1基因表达的研究

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

目的构建针对人基质金属蛋白酶-1(MMP-1)基因的特异性短发卡RNA(shRNA)真核表达载体,并观察其在人骨肉瘤细胞MG63中对MMP-1基因表达的抑制作用。方法培养骨肉瘤细胞MG63,根据MMP-1基因cDNA编码序列,设计并合成针对MMP-1基因的特异性RNA干扰片断,将其克隆入pSilencer 3.0-H1 neo质粒,构建MMP-1基因shRNA真核表达载体siRNA MMP-1。与阴性对照质粒分别转染MG63细胞,G418筛选稳定转染的细胞系。采用RT-PCR、Western blot检测MMP-1 mRNA和蛋白的表达水平,同时进行体外侵袭试验。结果成功构建了MMP-1基因shRNA真核表达载体siRNA MMP-1。获得了稳定转染siRNA MMP-1载体和阴性对照载体siRNA neg的细胞系,并发现MG63/siRNA MMP-1细胞MMP-1mRNA和蛋白表达均受到明显抑制,细胞侵袭能力也显著下降。结论特异性shRNA能够明显抑制MMP-1基因在MG63细胞中的表达,这为进一步研究MMP-1在MG63细胞中的生物学功能和作用机制奠定了实验基础。

Objective To construct the specific short hairpin RNA （shRNA） eukaryotic expression 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-HI neo plasmid vector, and the shRNA eukaryotic expression vector siRNA MMP-ltargeting MMP-1 gene was constructed.. After the vector was constructed, MG63 cells were transfected with negative control vector siRNA neg or siRNA MMP-1 and selected by （3418. Expression of mRNA and protein of MMP-1 in the stable transfected 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-lwere obtained. Expression of mRNA and protein of MMP-lwas 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 mechanisms of MMP-1 in MG63 cells.