Effects Of Inhibition Expression Of Heparanase By RNA Interference On The Biological Behavior Of Lung Carcinoma Cells

Invasive and metastasis is considered to be a major determinant of the malignant behavior of human lung cancer. The mechanism of tumor metastasis is not well illuminated so far, but the degradation of extracellular matrix(ECM) and basement membrane(EM) has been found to be an essential prerequisite for metastases of malignant tumor. The consequence of degradation of heparm sulfate proteoglycans (HSPG), a chief component of ECM, is considered to play a dominant role in tumor metastasis. Heparanase (Hpa) is the only endoglycosidase in mammal cell so far that degrades the heparin sulfate (HS) chains of HSPG. Elevated levels of heparanase have been indicated to have a strong association with the invasive phenotype in many tumors. In addition, HSPG is also the reservoir for a variety of bioactive molecules including angiogenic growth factors and degradation of HSPG would result in releasing of these growth factors which involved in angiogenesis. However, the mechanism of that heparanase in lung cancer has not been fully elucidated. In the present study, we construct shRNA expression vectors targeting human heparanase gene (pshRNA-Hpa) and to investigate the effects of gene silencing of heparanase by RNA interference on the biological behavior of lung carcinoma cells.Materials and MethodsCells lines and animalsThe human lung cancer cell line BE1 was routinely grown in RPMI1640, supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin and 100 U/ml streptomycin at 37℃in a 5% CO₂ incubator. Balb/c nu/nu mice (four weeks of age), provided by the Institute of Laboratory Animal Science, PUMC & CAMS, were maintained in a specific pathogen-free environment.PlasmidsTargeting human heparanase gene siRNA sequences were designed using siRNA Target Finder software program. The shRNA was ligated into the plasmid PGCsilencerTM H1 /Neo/GFP, which had been digested with BamHI and HindIII. The plasmid was verified by nucleotide sequence analysis.TransfectionBE1 cells were transfected with pshRNA-Hpa plasmid and nontargeting control plasmid vectors as control, using Lipofectamine 2000 reagent according to the protocol of the manufacturer. Colonies were selected in 600μg/ml G418. Heparanase expression was determined for each clone by RT-PCR and Western blotting.Proliferation assays1×10³ cells were seeded in a 96-well plate, incubated for 24 to 120 h, the MTT and dimethyl sulfoxide (DMSO) were added according to the protocol of the manufacturer. The OD value of each well was measured using a microplate reader with a test wavelength of 490 nm.Flow cytometryFlow cytometry were perfermed using Annexin V-FITC Apoptosis Detection Kit I according to the manufacturer's protocol on BD FACSCalibur^TM Flow Cytometer.Matrigel invasion assays100μl Cells were added to the upper chamber of an 8μm pore size coated with Matrigel. 600μl RPMI1640/10%FBS was added to the lower chamber and cells were allowed to migrate for 24 hours. Cells were fixed in 4% Paraformaldehyde and stained with hematoxylin. The migrated cells were counted in 20 random high-power fields. Tumorigenic assays and HE stainCells were injected into the right back region of 4-week-old nu/nu mice (n =5 per group per experiment). Mice were sacrificed after 5 weeks. Tumors and the organs were excised, fixed in 4% paraformaldehyde and paraffin embedded. The tissue sections of organs were stained with hematoxylin and eosin (HE) stain.Statistical analysisThe SPSS 14.0 software was employed to analyze the data. P < 0.05 was considered as statistical significance.ResultsRecombinant eukaryotic expression plasmid pshRNA-Hpa was constructedsuccessfullyDNA sequencing confirmed that the DNA fragments encoding heparanase targeted shRNA were cloned into the PGCsilencerTM H1 /Neo /GFP.Targeted down-regulation of heparanase expression using RNAinterferencepshRNA-Hpa transfected clones showed inhibition of heparanase expression as judged by RT-PCR, whereas clones transfected with a nontargeting control vector showed similar levels of heparanase expression as the parental BE1 cells. These data were further confirmed by Western blotting and Immunofluorescence.Inhibition of heparanase expression decreases cell proliferation, invasionand induces cell apoptosis in vitroMTT assay showed that the proliferation ability of BE1 cells transfected by pshRNA-Hpa was significantly reduced in comparison with those in the control cells. The apoptotic rate of the BE1 cells transfected with pshRNA-Hpa was 11.28 %±1.05 %, significantly higher than those of the control cells(both P <0.01). The number of BE1 cells in control group that invade through Matrigel was 37.0±2.6. Inhibiting heparanase decreased significantly the number of the cells (8.7±3.5, P<0.01).Heparanase down-regulation inhibits tumor formation and metastasis invivoTreatment with pshRNA-Hpa transfected cells significantly suppressed tumor growth compared with the controls, the mice injected the pshRNA-Hpa cells exhibited about 40% reduction in tumor volume. There was no difference in the tumor growth between mock and scrambled vector controls.Metastasis occurred in 100% of the control animals, but only one pshRNA-Hpa transfected cells treated animal showed up liver metastasis.ConclusionsExpression silencing of heparanase by RNA interference could effectively.reduce lung carcinoma proliferation, invasive and metastasis, induce cell apoptosis.