The Role Of Nanog Gene In Side Population Cells Of NK/T-cell Lymphoma

Background and ObjectiveLymphoma is a malignant tumor that originated from the lymphatic hematopoietic system,including Hodgkins Lymphoma?HL?and non-hodgkin's Lymphoma?NHL?.Natural killer/T-cell lymphoma?NKTCL?is a subtype of non-Hodgkin's lymphoma,which mainly occur in nasal,sinus and oropharyngeal regions and is closely related to Epstein-Barr virus.NKTCL has the characteristics of high invasiveness,insensitivity to chemotherapy,and poor prognosis in clinical practice.However,its cellular origin and biological characteristics are not yet clear,and the exploration of these mechanisms may provide a potential target for the clinical treatment of NKTCL.Cancer stem cells?CSC?is defined as a small subpopulation?usually<1%?of cells within a tumor that is capable of self-renewal,generating progenitor cells through asymmetric cell division,and retain the potential to differentiate.It has been suggested that conventional chemotherapy is able to kill most tumor cells but rarely CSC,which is regarded as the fundamental cell population that establishes and maintains a tumor.This results in the selection of CSCs with chemoresistance,which in turn gives rise to more aggressive tumors.A lot of research illustrate that molecules important in CSC biology could be promising targets for novel cancer therapies.Side population?SP?cells is a small amount of cells which is found by Goodell on the separation of bone marrow stem cells and is occupied by a side of a large amount of cells.SP cells have the ability of multi-directional differentiation,infinite proliferation and self-renewal and other stem cell-like characteristics.Currently,SP cells have been found in a variety of tumor tissues.Some studies confirm the presence of stem cell-like SP cells in some lymphomas,however,there are no reports on the related aspects of NKTCL.Our previous study demonstrated the presence of doxorubicin-resistant SP cells in NKTCL cell line SNK-6.At the same time,the stem cell gene Nanog was expressed significantly,but the mechanism of Nanog gene on this part of SP cells was not clear.Therefore,in this experiment,to investigate the effect of Nanog on malignant biological behaviors such as proliferation,apoptosis,migration and clone formation ability of SNK-6/ADM-SP in the SP cells of NKTCL,Nanog gene was selected as the target gene to construct a lentiviral expression vector carrying the full-length cDNA sequence and the shRNA sequence of Nanog mRNA coding region.So that we can elucidate the biological effects of Nanog gene on SP cells of NKTCL,and to provide a possible new target for the treatment of NKTCL.Part?:The Construction and Transfection of Nanog Lentiviral VectorObjectiveTo construct a stable high/silent Nanog of NK/T cell lymphoma side population cell line by recombinant lentiviral expression vector.Materials and methodsWe used the lentiviral vector plasmid pCDH-CMV-MCS-EF1-copGFP?DCE?as a vector to link the Nanog template PCR product was cloned,forming a high expression lentiviral vector DCE-Nanog.The plasmids pMAG-Nanog-shRNA1 and pMAG-Nanog-shRNA2 were reconstructed by interference of lentivirus pMAGic7.1.The recombinant plasmids that overexpressed and silently expressed Nanog gene were packaged,concentrated and titrated.Lipo6000TM method was used to transfect lymphoma cells and then the infection efficiency was determined.According to Real-Time PCR and Western Blot assays to screen out effective RNA interference sequences.ResultsDigestion and sequencing showed that the recombinant overexpression of Nanog lentivirus DCE-Nanog and interference Nanog expression lentivirus pMAG-Nanog-shRNA1 and pMAG-Nanog-shRNA2 were built successfully.The viral titer was up to 10⁹IFU/ml.Each plasmid was transfected into SNK-6/ADM-SP cell line and named SNK-6/ADM-SP-con?empty vector sub-strain?,SNK-6/ADM-SP-N?overexpression sub-strain?,SNK-6/ADM-SP-shN1?silent expression sub-strain?andSNK-6/ADM-SP-shN2?silentexpressionsub-strain?respectively.SNK-6/ADM-SP-shN2 had the best interference effect which was chosen for the second part of the follow-up experiments.The relative expression level of Nanog protein was detected by Western Blot,which showed that Nanog expression was the lowest in SNK-6/ADM-SP-shN group,highest in SNK-6/ADM-SP-N group,no significant difference between SNK-6/ADM-SP and SNK-6/ADM-SP-con group.ConclusionWe successfully reconstructed the stably overexpressing and interference Nanog of NK/T cell lymphoma side population cell subline.Part?:The biological characteristics of Nanog gene in SNK-6/ADM-SP cellsObjectiveTo investigate the biological characteristics of Nanog gene on the SP cells of NK/T lymphomaMaterials and methods1.The impact of Nanog on SNK-6/ADM-SP cell proliferative activity were observed by CCK-8 method.2.The effect of Nanog on the cell cycle distribution and apoptosis rate of SNK-6/ADM-SP assayed by flow cytometry?FCM?.3.The impact of Nanog on SNK-6/ADM-SP migration analyzed by tanswell assay.4.The clone capacity of Nanog gene to SNK-6/ADM-SP was determined by methylcellulose colony-forming assay.Results1.The results of CCK-8 assay showed that:there is no significant difference in proliferation between SNK-6/ADM-SP group and SNK-6/ADM-SP-con group?P>0.05?,the proliferation of SNK-6/ADM-SP-N group was significantly enhanced?P<0.05?,and the proliferation of SNK-6/ADM-SP-shN group was significantly decreased?P<0.01?.2.FCM results showed that:compared with SNK-6/ADM-SP-con empty plasmid group,there were no significant difference in apotosis rate and cycle distribution of cells in SNK-6/ADM-SP group?P>0.05?.As to SNK-6/ADM-SP-N group,the proportion of cells in G₀/G₁ phase decreased?P<0.05?,in S phase increased?P<0.01?,at the same time,the proportion of apoptosis decreased significantly?P<0.01?.As to SNK-6/ADM-SP-shN group,the proportion of cells in G₀/G₁ phase was significantly increased?P<0.01?and that in S phase decreased?P<0.01?,at the same time the apotosis rate increased significantly?P<0.01?.3.The transwell assay showed that:compared with SNK-6/ADM-SP-con empty plasmid group,the ability of migration had no significant difference in SNK-6/ADM-SP group?P>0.05?;while markedly increased in SNK-6/ADM-SP-N group?P<0.01?and decreased in SNK-6/ADM-SP-shN group?P<0.01?.4.Methylcellulose colony-forming assay showed that:there was no significant difference in the ability of cloning between SNK-6/ADM-SP group and SNK-6/ADM-SP-con group?P>0.05?,the ability of cloning of SNK-6/ADM-SP-N group was significantly enhanced?P<0.05?,and the ability of cloning in SNK-6/ADM-SP-shN group was significantly decreased?P<0.05?.Conclusion1.SP cells are the cells with special biological characteristics in NKTCL.The high expression of Naong gene can promote the malignant biological characteristics of NKTCL.The specific mechanism still needs further study.2.High expression of Nanog can promote the proliferation,migration,and clonal capacity of SNK-6/ADM-SP cell line,as well as the cell cycle progression.