Silencing Of Breast Cancer Of Heparanase Gene-targeted By Small Interfering RNA

Recent studies have revealed that heparanase (HPSE) is an endoglycosidase which degrades cell surface heparan sulfate proteoglycans (HSPGs). Through degrading extracellular matrix (ECM) and basement membrane (BM), heparanase probably facilitates the metastasis of tumor. RNA interference (RNAi) is a new method which means the reduction of targeted gene by siRNA. In the transcription level RNAi can block the expression of heparanase, inhibit tumor invasion and metastasis. It may lay the theoretical foundation for the clinical treatment of tumor and provide some new ideas for the therapy of cancer diseases.According to the human heparanase gene sequences of gene bank, we constructed four sequences (HPSE-1: 5'-GCA ATG AAC CTA ACA GTT TCC-3'; HPSE-2: 5'-GCT TTA TGT GGC TGG ATA A-3'; HPSE-3: 5'-CTG TCA GCC CGA ATG GGA A-3'; HPSE-4: 5'-CTT GCC ACC TTT AAT GGA A-3'). The plasmid pGPU6/GFP/Neo veter was cuted to a long chain with some digestion treatment. DNA oligoes designed was dissolved respectively with Tris-EDTA buffer. The corresponding sense chain and anti-sense chain oligo solution was annealed in the PCR instrument. Two complementary oligonucleotide strands was synthesized and inserted into pGPU6/GFP/Neo vector. Reorganized plasmid was identified by Bam H I and Pst I enzyme. Positive recombinant vectors were sent Shanghai Invitrogen company to analysis whether they were right or not. MDA-MB-231 cell lines were maintained in growth medium (Leibovitz's L-15 with 10% fetal bovine serum) without antibiotics, and grown in a humidified 5% CO2, at 37°C atmosphere. Transient transfection was carried out with LipofectamineTM2000. Seven groups were designed, including HPSE-1 group, HPSE-2 group, HPSE-3 group, HPSE-4 group, NEGATIVE CONTRAL (NC) group, MOCK (added transfection reagent only) group, blank group. Fluorescence photographs were taken after 24 h and 48 h. Under the excitation wavelength MDA-MB-231 cells should display green color with green fluorescence microscope. The result showed that transfection was successful. Total RNA was isolated from cells using RNA Trizol Reagent with a single-step phenol-extraction method and used as a template. For heparanase, the amplification reaction involved denaturation at 95°C for 3 min, annealing at 95°C for 30 seconds, and extension at 62°C for 40 seconds (40 cycles). Proteins were analyzed by western blot method.Reorganized plasmid was successfully constructed. The result of RT-PCR and western blot showed that expression of heparanase was inhibited significantly. HPSE-1 group showed the best results. The result of MTT showed that the proliferation of interfering groups droped obviously compared to other groups. Therefore, this article may do some help for the development of mechanism of tumor metastasis. With RNAi method the epression of heparanase may be inhibited. Correspondly the metastasis of tumor may be stopped. The new method may provide theoretical basis and methods for the development of anti-tumor drugs in future.