Inhibition Of Antisense Heparanase CDNA On Proliferation And Invasion Of Lung Cancer Cells

Tumor cell invasion and subsequent metastasis are the leading causes of cancer-related mortality. It is generally accepted that the invasion of the basement membrane(BM) and extracellular matrix (ECM) is one of the critical steps for cancer cellmetastasis. Heparan sulfate proteoglycans(HSPGs), composed of a protein corecovalently linked to heparan sulfate (HS) side-chains, represent a principal componentof theECM and, in particular, BM. Cell surface HS can also serve as coreceptors, alongwith the other cell surface molecules, to form functional receptor complexes thatfacilitate signal transduction. Furthermore, HS side chains bind a large number ofbioactive molecules and regulate their availability and function. These include growthfactors, chemokines, cytokines, and enzymes that are essential for angiogenesis, celladhesion, and locomotion. Accordingly, enzymatic degradation of HS may play acritical role in cancer cell invasion and metastasis. Heparanase (Hpa) is anendoglycosidase that cleaves HS side chains of HSPGs at a limited number of sites,thus facilitating disassembly of the ECM and enhancing cell invasion. At the sametime, HS degradation also results in the release of bound bioactive molecules fromECM depots, and promote tumor invasion, metastasis and angiogenesis. In the presentstudy, we investigated the espression of Hpa in human lung cancer cell lines withdifferent metastatic potentials; analyzed the relationship between Hpa and metastaticpotential of lung cancer cell lines; contructed a recombinant eukaryotic expressionplasmid pEGFP-C1 containing human antisense Hpa cDNA gene, and then anylazedthe effects of antisense RNA technology targerting Hpa protein expression on thebiological traits of human lung cancer cell.Materials and Methods1. Three human lung cancer cell lines with different metastatic potential (highly metastatic cell subline of human lung giant cell cancer (PG) BE1, weakly metastaticcell subline of human lung giant cell cancer (PG) LH7, weakly metastatic cell line ofhuman lung adenocarcinoma A2) were grown in RPMI 1640 supplemented with 10%fetal calf serum(FCS). The expression levels of Hpa mRNA and protein were detectedwith immunohistochemistry, Western blot and reverse-transcription polymerase chainreaction(RT-PCR).2. The plasmid pcDNA-Hpa contains human Hpa cDNA of 1758bp whichinserted as a EcoR I/Xho I fragment. The fragment was cut and antisense insertedEcoR I/Xho I restriction enzyme sites of the empty vector pEGFP-C1 in order toconstructed Hpa antisense recombinant eukaryotic expression plasmid pEGFP-C1-AShpa and then transfected into lung cancer cell BE1 which expression of Hpa washigher. The plasmid pEGFP-C1 transfected as the same time. Trasfected BE1 cells wereselected with 600μg/ml G418. The expression of Hpa in the different transfected cellclones was assessed by RT-PCR, Western blot and immunofluorescence in order toselected the; the cell clone which the expression levels of Hpa mRNA and protein wasthe weakest was named BE1-ASHpa.The cell that was transfected empty plasmid(pEGFP-C1) was named BE1-Em. BE1 and BE1-Em were used as control groups.The proliferation ability of BE1-ASHpa, BE1 and BE1-Em was tested bycytometry and MTT. The cell cycle and apoptosis of lung cancer cells before and aftertransfection were examined with flow cytometry. Changes of invasive capacity andlocomotory ability of BE1-ASHpa, BE1 and BE1-Em were measured by Transwellinvasion chamber coated with Matrigel or not. The protein expressions of VEGF,VEGF-C, Akt and pAkt in BE1-ASHpa, BE1 and BE1-Em were assessed by Westernblot.3. The SPSS 13.0 software was employed to analyze the data. P＜0.05 wasconsidered as statistical significance.Results1. The expression levels of Hpa protein and mRNA were all in the three humanlung cancer cell lines. But the expression quantities of Hpa protein and mRNA wereboth significantly higher in BE1 cells than in LH7 or A2 cells (P=0.002, P=0.007for Hpa protein; P=0.002, P=0.005 for Hpa mRNA). Immunohistochemical staining of Hpa in LH7 and A2 grown on coverslips was lower than in BE1.2. The plasmid pcDNA3 containing Hpa total length cDNA was cut by restrictionenzyme EcoRâ… and Xhoâ… , then recovered Hpa cDNA. The empty vector pEGFP-C1was linearized by EcoRâ… and Xhoâ… , linked with Hpa cDNA and transformed. At last,we gained Hpa antisense recombinant expression plasmid pEGFP-C1-ASHpa.3. pEGFP-C1-AShpa transfected BE1 cell which the expression levels of Hpaprotein and mRNA in it were higher than in the other two. And stable transfectant poolswere obtained after selection with G418. The expression of Hpa mRNA and proteinin BE1-ASHpa cells (0.211±0.009, 0.539±0.012) was reduced 57.8% and 57.5% thanEB1 cells(0.496±0.013, 1.277±0.017). Immunofluorescence staining of Hpa in it wasweaker than in BE1 and BE1-Em.4. Transfected group cells were significantly slower than other group cells in therate of cell proliferation(P＜0.05). The apoptosis rate of BE1-ASHpa cells wassignificantly higher(7.72±0.11%) than BE1 cells (0.02±0.01%)(P=0.000). Thepercentage of G₁ stage cells in transfected group cells was significantlyhigher(63.27±2.06%) than BE1 cells (41.28±0.98%)(P=0.006).5. The invasion capacity and locomotory ability of BE1 cells was decreased aftertransfected pEGFP-C1-ASHpa plasmid into the cell line. The cell numbers of invasioncapacity and locomotory ability (12.00±1.41, 20.67±1.63) were significantly lower inBE1-ASHpa than in BE1 (49.71±3.48, 62.83±3.60) (P=0.00, P=0.000).6. The expression of VEGF, VEGF-C, pAkt protein significantly decreased(P＜0.01) and the expression of Akt protein was not changed(P＞0.05) in transfectedgroup cells.Conclusions1. The expression of Hpa mRNA and its protein was both in the three human lungcancer cell lines presently examined and the levels of Hpa mRNA and its proteincorrelated positively with the metastatic potential of lung cancer cell lines.2. Construction of Hpa cDNA antisense recombinant eukaryotic expressionplasmid pEGFP-C1-ASHpa was successful. The expression levels of Hpa mRNA andprotein could be reduced in human lung cancer cells by pEGFP-C1-ASHpa.3. Lung cancer cells were decreased proliferation and induced apoptosis with transfected pEGFP-C1-ASHpa plasmid into them. These results maybe correlate withPI3K/Akt signal pathway.4. Inhibition of Hpa protein expression by antisense Hpa cDNA (pEGFP-C1-ASHpa) could decrease invasion capacity and loeomotory ability of lung cancercells.