Analysis And Verification Of Differential Genes Expression Of Gemcitabine-resistant Cell Line Of Human NK/T Lymphoma

NK cell lymphomas(NKCL)is a very seldom type of non-Hodgkin's lymphoma(NHL).NKCL is approximately accounting for 5%-18% of NHL.These diseases mostly occur in Asia and Latin America,such as Japanese,Chinese and korean [1].According to the classification of World Health Organization(WHO),NKCL includs two subtypes:the aggressive NK cell leukemia(ANKI)and NK/T cell lymphoma[ nasal type(ENKL)and extranodal.Malignancies putatively derived from cytotoxic cells of either natural killer(NK)cell or T-cell lineages are aggressive neoplasms [1].They occur most commonly in the nasal,paranasal and oropharyngeal areas.In the current World Health Organization classification,most of them are regarded as a distinct clinicopathologic entity,extranodal NK/T-cell lymphoma,nasal type [2].Some of these lymphomas are located outside the nasal area and upper aerodigestive tract,occurring in the gastrointestinal tract,salivary glands,skin and testis.These cases have been referred to as “non-nasal” NK/T-cell lympho-mas,with an apparent implication that they might be clinically different[3].NK/T cell lymphoma is active malignancy,and application of CHOP or CHOP-like scheme has a poor prognosis,which is due to high multi-resistant genes expression in NK/T cell lymphoma,coding for P glycoprotein,therefore curative effect of the traditional chemotherapy regimens including anthracycline-based chemotherapy drugs to this lymphoma,is poorer.Gemcitabine is a P glycoprotein non-dependent drug.The regimen of containing Gemcitabine significantly improved its prognosis,which is now considered the standard treatment of NK/T cell lymphoma.The latest we firstly establish DDGP regimen(containing Gemcitabine,Cisplatin,Dexamethasone and Pegaspargase)of NKTCL in our He nan province center of lymphoma,in which PR was 24%,CR was 71%.The regimen is more effective than traditional chemotherapy regimens[6].But it is a kind of highly aggressive malignant disease with poor prognosis and some patients are resistant to Gemcitabine.Drug resistance mechanism is complex,involved resistant concrete mechanism and process are unclear.So as far as possible,it is important that make it clear the drug-resistant mechanism for the choice of drugs and improvement of the prognosis of the disease.With the progress of the biological technology,high-throughput sequencing technology has been very widely used in life science and emergency medical study.The application of high-throughput sequencing technologies,improve the accuracy of the sequencing,the speed and efficiency,and promote the ways of tumor diagnosis and treatment.Digital gene expression profiles is a methods from RNA transcription to RNA expression of quantitative detection by a high-throughput sequencing technology,based on the different samples for testing,which can identify differentially expressed genes.It can analyze the differences between genes at the same time and then find out significant differentially expressed genes.It is of great significance with studying the function of differentially expressed genes for the prognosis of disease development,and the choice of therapeutic targets.This study intends to find the differential genes between NK/T cell tumor and analyzes the possible mechanisms of drug resistance by high throughput sequencing.Objective To screen differentially expressed genes between the two cell lines of YTS(NK/T lymphoma cell lines)and YTSR(Gemcitabine-resistat NK/T lymphoma cell lines),and verify the differentially expressed genes by PCR and western-blots.Methods RNA of non-Gemcitabine-resistat NK/T lymphoma cell line and Gemcitabine-resistat NK/T lymphoma cell line was extracted by Trizol.Differences of digital gene expression profile was detected by high throughput sequencing.The expression of one drug-resistance related gene(AQP3)were detected by Real-time PCR and western-blots in the cell lines.The results 1.Obvious difference between gene expression was Selected between non-Gemcitabine-resistat NK/T lymphoma cell line and Gemcitabine-resistat NK/T lymphoma cell line.Tthe possible mechanism of Gemcitabine-resistat NK/T lymphoma cell lines was predicted.2.There were 142 up-regulation and 96 down-regulation genes.3.MAPK and Adhere junction in pathway had a significance difference by diferential gene cluster and functional analysis.4.The expression of one drug-resistance related gene(AQP3)were detected by Real-time PCR and western-blots in the cell lines.Outcome of Real-time PCR and western-blots was consistent with the DGE results.Conclusion 1 A large number of significant differences in expression between non-Gemcitabine-resistat NK/T lymphoma cell line and Gemcitabine-resistat NK/T lymphoma cell line.2 The AQP3 gene expression of the Gemcitabine-resistat NK/T lymphoma was up-regulation,which maybe one of the drug resistance mechanism of the Gemcitabine-resistat NK/T lymphoma.