The Effect Of The Small Interference RNA Targeted Heparanase Gene Silencing On Malignant Biological Behavior Of Porstate Cancer Cells

Objective: Heparanase, a mammalian endo-B-D-glucuronidase, is capable of degrading heparin sulfate (HS), the main polysaccharide in the basement membrane (BM) and extracellular matrix (ECM), and is proposed to play a key role in invasion, metastasis and angiogenesis of cancer cells. The aim of our research is to evaluate whether heparanase gene-silencing strategies can inhibit the malignant characteristics of prostate cancer cells.Methods:We designed four small interfering RNAs(siRNAs) directed against the human heparanase mRNA, and two siRNAs targeted the human Argonaute(AGO1 and AGO2) mRNA. Human prostate cancer cell lines, PC3, which have been genetically engineered to overexpress heparanase and Argonaute, were transfected with both anti-heparanase and anti-Argonaute siRNAs. RT-PCR, real-time quantitative PCR and Western blot were used to confirm the efficient silencing of heparanase and Agronaute genes expression. Cell proliferation was detected by MTT colorimetry and colony formation assay, the in vitro invasion and metastasis of cancer cells were measured by cell adhesion assay and matrigel invasion assay. The angiogenesis capabilities of cancer cell were measured by tube formation of endothelial cells.Results: Transfection of siRNA against 1496-1514bp of coding regions resulted in profoundly reduced expression of heparanase, which started at 24 h and lasted for 96 h post-transfection. The siRNA-mediated gene silencing of heparanase supressed the cellular proliferation of PC3 cells.The invasion, metastasis, and angiogenesis of human prostate cancer cells in vitro were markedly inhibited after knock-down of heparanase. Additionally, human Argonaute-1(AGO-1) and Argonaute-2 (AGO-2) are involved in siRNA-mediated post-transcriptional gene scilencing in human prostate cancer cells. Our finding indicates that inhibition of AGO-1 and AGO-2 genes expression significantly reverses heparanase gene silencing by siRNA (siH4). Meanwhile, silencing of heparanase expression by siRNA(siH4) profoundly reduce in their mRNA and protein level of vascular endothelial growth factor(VEGF) and matrix metalloproteinases-9(MMP-9).Conclusions: Our results demonstrated that siRNA-mediated gene silencing of heparanase can efficiently inhibit the invasion, metastasis, and angiogenesis of human prostate cancer cells in vitro, suggesting that heparanase-specific siRNA may be of potential values as an efficient cancer therapeutic strategy for human prostate cancer. AGO-1 and AGO-2 have been involved in siRNA-mediated post-transcriptional gene silencing pathways, and shown necessary for efficient RNAi in prostate cancer cells. In addition, heparanase gene silencing by siH4 led to decrease the mRNA and protein levels of VEGF and MMP-9, indicating that endogenous heparanase is actively involved in VEGF and MMP-9 genes regulation.