[Construction and screening of specific short hairpin RNA vector targeting heparanase gene].
ABSTRACT To construct short hairpin RNA (shRNA) expression vectors of RNA for specific silencing of heparanase (HPA) gene.
The genomic sequence of HPA gene was retrieved from GenBank and the cDNA encoding shRNA for HPA gene silencing was designed. Five specific interference sequences and a random negative control sequence were inserted into the vector pGPU6/GFP/Neo. After verification by restriction enzyme digestion and sequence analysis, the recombinant vectors were transfected into MDA-MB-231 cells via lipofectamin. Fluorescent quantitative PCR and Western blotting were employed to detect the expression of HPA gene expressions in the transfected cells at the mRNA and protein levels, respectively.
Both restriction analysis and sequencing confirmed correct construction of the shRNA vectors. Transfected with the specific siRNA vectors HPSE-1 and HPSE-5 resulted in significantly decreased expression level of HPA protein in MDA-MB-231 cells, while negative control vector produced no significant changes in HPA expressions.
We have obtained two shRNA vectors which can significantly down-regulate HPA expressions in MDA-MB-231 cells, which facilitates further investigation of the role HPA may play in the invasiveness and metastasis of human breast cancer.