Studies On Digital Gene Expression Of Human NK/T Cell Lymphoma Cell Line By RNA Sequencing

BackgroundNK cell lymphomas (NKCL) is a special subtype of non-Hodgkin’s lymphoma (NHL), approximately accounting for2%～10%of NHL. According to the latest World Health Organization (WHO) classification, NKCL is divided into two subtypes: extranodal NK/T-cell lymphoma, nasal type (ENKL) and the aggressive NK cell leukemia (ANKL).These diseases mostly occur in Asia and Latin America populations. The neoplastic cells mostly derived from mature NK cells (CD2+CD56+, cytoplasmic CD3ε+and EBV+) and a few derived from NK like cytotoxic T cell (CD56+, EBV+).The existence of EB virus (EBV) could be detected on most of NKCL tumor cells and this indicted that EBV may plays an important role in the pathogenesis of this disease.Histologically, NKCL is characterized by vascular damage,tumor necrosis and apoptosis,so it is recognized to be an aggressive disease. According to different pathological regions, ENKL is mainly divided into two subtypes:the nasal cavity and extronasal areas, the former is called nasal NK cell lymphoma and the latter is NK cell lymphoma, nasal type. The clinical course of the advanced cases develope fast, especially in the NK cell lymphoma cases of nasal type which has poor prognosis similar to ANKL. It is well known that multiple drug resistance (MDR1) gene encoded product P-glycoprotein (P-gp) highly expressed in ENKL and ANKL tumor cells, so the treatment response of NKCL is poor with regimes including anthracyclines. So NKCL is a malignance which characterized by strong aggressiveness, traditional chemotherapy regimens resistance and poor prognosis. As the low incidence and the significant difference in geography, the etiology and pathogenesis of NKCL has not been that clear.Recently, RNA sequencing technique plays an important role in the research of gene expression profiles in malignancies. Especially the emergence of high-throughput sequencing technique has greatly promoted the development of tumor diagnosis and treatment.By exon sequencing of a certain tumor tissue and normal tissue as controls, the current studies have found the gene mutations relative to the pathogenesis of ovarian, kidney, pancreatic, bladder transitional cell cancers and leukemia.There are cases that sequencing technology was used to detect the tumor-specific rearrangement genes in the plasma of cancer patients and these genes were used as biomarkers to improve the individual diagnosis of the tumor. Furthermore the residual disease after treatment could be monitored by these biological markers. Digital gene expression(DGE) is a high-throughput sequencing technique which quantify the mRNA expression from transcriptome level. Differential expression genes could be found and analyzed through detecting different samples by DGE. Thus significant differential expression genes were found. DGE is widely used in basic research in various industries,.especially in oncology, but there is no related research in NK cell lymphomas.ObjectiveTo detect the digital gene expression profile of the normal NK cell and NK/T cell lymphoma cell lines (YTS and SNK-6) and explore the different expressed genes among the three cell lines by DGE technology,we established three digital gene expression libraries,screened different-expressed genes in these three cell lines and defined the expression of common drug-resistance related genes in NK/T-cell lymphoma cell lines. To examine the accuracy of sequencing technique, quantitative Real-time PCR(qPCR) was used to amplify the two drug-resistance related genes in these three cell lines and this would provide experimental basis to analyze the etiology and drug-resistance mechanism in NK/T-cell lymphoma.MethodsDGE technology was used to detecte the digital gene expression and different expressed genes of normal NK cell, YTS and SNK-6; To verify the results of sequencing, Real-time PCR was used to investigate the expression of common drug-resistance related genes (ABCC5and ERBB4).Results1Digital gene expression (DGE) libraries of one normal NK cells and two NK/T cell lymphoma cell lines were established and significantly different expressed genes of three cell lines were screened.2The expression of two drug-resistance related genes (ABCC5and ERBB4) in the three cell lines were detected by Real-time PCR,which was consistent with the result of DGE.Conclusion1There are quantities of significantly different-expressed genes between NK/T cell lymphoma cell lines and normal NK cell, which may be related to the oncogenesis,prognostic,remove and drug-resistance of NK/T cell lymphoma.2The drug resistance of NK/T cell lymphoma may be related to certain different expressed drug-resistance related genes.3DGE technology is accurate and reliable,so it could provide experimental basis to the tumor relative research.