Rna Interference Of Heparanase Expression In Cne-2Affects Cell Proliferation And Motility

Background and ObjectiveNasopharyngeal carcinoma (NPC) is a disease with distinct ethnic and geographic distribution. This tumour is relatively rare in the Western world, but represents a significant disease burden in Southern China and Southeast Asia, with an annual incidence rate of about20per100,000people in endemic areas. Globally, NPC accounts for80,000new cases and50,000deaths annually. The aetiology of NPC is complex, and includes a host of viral, genetic and environmental factors. It is widely accepted that Epstein-Barr virus (EBV) infection plays a major role in the pathogenesis of NPC in both endemic and nonendemic areas. The association between NPC and EBV was initially discovered from serological studies and was later supported by demonstration of EBV DNA and nuclear antigen proteins (EBNA) in NPC tumour cells.Although antibodies against EBV viral capsid antigen (VCA) and plasma EBV DNA have been shown to have prognostic value, But in a few recent study they have been proven to be unstable and hard to manipulate at clinic trials. Several molecular aberrations in NPC cells are been intensely studied, especially the receptor-midiated mechanisms of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), mesenchymal-epithelial transition factor (c-MET), as well as intracellular mitogenic signals PI3K/AKT/mTOR inhibition, Hypoxia inducible factor1alpha (HIF-1a).Based on above analysis, this study uses RNAi technology to induce Hpa silencing in nasopharyngeal carcinoma cell line CNE-2, investigating the Hpa silenced CNE-2status on tumor growth, invasion and cell motility, providing theoretical basis for NPC target choosing of tumor gene therapy.Method1. Tumor tissue and adjacent normal tissue of a clinically verified NPC patient were studied for the expression of heparanase. Additionally, the expression of heparanase protein in a NPC cell line CNE-2was also exanmined by Immunocytochemistry2. Heparanase mRNA sequence is obtained from online database Genebank. Interference RNA sequences were obtained through on line design tool in web site, and screened by several parameters. We further designed dsDNA of interference sequences for synthesis. Then double restricted enzyme digested pGenesil-1was ligated with synthesized dsDNA. After being verified by sequencing we had it amplified and extracted.3. CNE-2nasopharyngeal carcinoma cells were stablely transfected with each recombinant interference plasmid by lipofectamine2000method. After4weeks of G418pressure screening, drug resistant clones were obtained from the plasmid-transfected cells and was named as:CNE-2/Hpa-.4. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were performed to verify the heparanase expression of the plasmid-transfected CNE-2/Hpa-subclone cells.5Biological behaviour, such as cell proliferation and growth, cell motility of CNE-2/Hpa-was investigated by a modified3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) technique and transwell invasion experiment.Result1. Significant heparanase protein expression was detected in NPC primary tumor tissue and in NPC cell line CNE-2cells, while there is no detectable heparanase expression in adjacent normal tissue of NPC patient.2. Interference sequences were designed by online tools:1214-1232:5’-GGCTATCTCTTCTGTTCAA-3’. Interference sequences dsDNA and control sequences were correctly cloned into pGenesil-1plasmid and confirmed by sequencing. The plasmid was named as pGenesil-1/Hpa-. 3. CNE-2cells were transfected with interference vector pGenesil-1/Hpa-and a GFP-expressing stable cell clone was ecreened out. RT-PCR and Western blot showed that heparanase mRNA or protein were significantly repressed in CNE-2/Hpa-subclone cells.4. Western blot was carried out and confirming that after transfected with interfering vector, CNE-2cells showed a significantly downregulated Hpa expression.5. MTT result and transwell invasion experiment indecated that after transfected with interfering vector, CNE-2cells showed a significantly downregulated cell proliferation ability and cell motility in ECM material.Conclusion1. By immunohistochemistry and immunocytochemistry, we manage to demonstrated that NPC tumor tissue express higher heparanase protein than its adjacent normal tissues2.We successfully designed a RNA interference sequences targetting Hpa RNA and constructed a RNAi plasmids. By lipofectamine transfection and G418screening, we obtained a CNE-2Hpa negative stable cell clone. And we managed to prove that this cell clone express a much lower level of Hpa RNA and protein.3. In vitro MTT and transwell studies showed that the CNE-2Hpa negative stable cell clone displayed a largly impaired proliferating ability and invasion ability, compared with the untransfected CNE-2cells.Our study showed that the heparanase was significantly up-regulated in nasopharyngeal carcinoma cells and it may play a key role in NPC malignant proliferation and metastasis. The RNAi sequences we designd can effectively inhibit the heparanase expression in NPC cell line. And the heparanase-inhibited CNE-2cells were compromised in proliferation and invasion ability. This result clearly indecating that Hpa is the main mechanism underlying the invasion and metastasis.