Aquaporin 3 Facilitates Chemoresistance In Extranodal Natural Killer/T Cell Lymphoma To Gemcitabine

Lymphoma?Malignant lymphoma,ML?is a hematological malignant tumor derived from lymph nodes or other lymphohematopoietic tissues such as thymus,bone marrow and spleen.Lymphoma can be divided into Hodgkin's lymphoma?Hodgkin'S lymphoma,HL?and non-Hodgkin's lymphoma?non-Hodgkin'S lymphoma,NHL?.Non-Hodgkin's lymphoma can be divided into B-cell lymphoma,T-cell lymphoma and NK/T-cell lymphoma.NK/T cell lymphoma?NK/T-Cell Lymphoma,NKTCL?tumor cells have both T-cell phenotype and NK cell phenotype.The disease is rare in western countries.The incidence is slightly higher in Asia but it is also a rare disease.NK/T cell lymphoma often invades tissues and organs other than lymph nodes,such as nasal cavity and nasopharynx,because its tumor cells secrete perforin and granzyme,which often cause tissue festering and perforation in the lesion site.In some patients,perforation of palate is a symptom,in addition,it can also invade esophagus,skin,testis,center and bone marrow.The etiology of NK/T cell lymphoma is not clear.some studies have suggested that it may be closely related to the chronic infection of EB virus,but its specific mechanism is not clear.NK/T cell lymphoma can be divided into early stage and extranodal stage.For early NK/T cell lymphoma,it is found that radiotherapy combined with chemotherapy is superior to radiotherapy alone or chemotherapy alone,and most of the patients have a good prognosis.Extranodal NK/T-cell lymphoma has strong invasiveness,poor response to chemotherapy and poor prognosis.At present,there is no unified treatment standard in the world.Advanced extranodal invasion of NK/T cell lymphoma,Gemcitabine-based DDGP?Gemcitabine,Pectinase,Cisplatin,Dexamethasone?regimen has achieved good results in patients with relapsed and refractory extranodal NK/T cell lymphoma,but there are still patients with primary drug resistance or acquired drug resistance which leads to treatment failure,thus affecting the overall prognosis of the disease.Aquaporin?Aquaporins,AQPs?is a water and glycerol transporter,which is widely expressed in a variety of tumors and plays an important role in cell migration,angiogenesis,tumor formation and tumor development.AQP3 has been studied in many tumor cells,and AQP3 is overexpressed in lung cancer,colon cancer,esophageal and oral squamous cell carcinoma.In addition,AQP3 and AQP5 were co-expressed in hepatocellular carcinoma and pancreatic ductal adenocarcinoma.The overexpression of AQP3 is related to lymph node metastasis,the histological classification,and lymphatic invasion of gastric adenocarcinoma,resulting in lower survival time and shorter survival time than that of patients with lower expression of AQP3.Similarly,an increase in AQP3 expression was observed in patients with invasive ductal carcinoma of the breast.This is associated with higher histological grade and increased lymph node spread,emphasizing the importance of AQP3 in the progression of breast cancer.In a study of patients with bladder cancer,higher tumor stages were associated with lower AQP3 expression.We screened the differentially expressed genes between human NK/T cell lymphoma cell line?YTS?and gemcitabine resistant cell line?YTSR?by high throughput sequencing.The results showed that the expression of AQP3 was up-regulated in gemcitabine resistant cell line.However,the role of AQP3 in the drug resistance of NK/T cell lymphoma to gemcitabine is not clear.In this study,the overexpression of AQP3 plasmid was constructed and transfected into NKTCL cell line YTS,and the empty plasmid group was used as blank control,Western blot and PCR to verify the overexpression effect.Different concentrations of gemcitabine were used to act on the cells of blank control group and overexpression group.The sensitivity of the two groups to gemcitabine was detected by CCK8?Cell Counting Kit-8 assay?.FCM?Flow Cytometer?was used to detect the sensitivity of the two groups of cells to gemcitabine.AnnexinV-PI was used to detect the effect on apoptosis.The expression of apoptosis and autophagy related proteins were detected by Western blot.It is hoped that this study will find a new therapeutic target for the treatment of NK/T cell lymphoma and make a breakthrough in the drug resistance of NK/T cell lymphoma to gemcitabine,so as to improve the therapeutic effect of the disease.ObjectiveTo investigate the role of aquaporin 3?AQP3?in the resistance of NK/T cell lymphoma cells to gemcitabine.The possible molecular mechanisms are investigated.Method1.Construction and verification of AQP3 overexpression plasmid:AQP3 open reading frame?ORF?was amplified by genomic DNA to ligate PCR product into vector digested by EocR1 and Not1,and AQP3 overexpression plasmid was constructed.The overexpression plasmid of AQP3was verified by real-time fluorescence quantitative PCR and sequencing.2.Lentivirus packaging and transfection of NK/T cell lymphoma cell?YTS?:were carried out by recombinant packaging with verified successful AQP3 overexpression plasmid.293T cells were transfected with Lipo6000 and then the virus was collected.The transfection efficiency of YTS cells was measured by flow cytometry after transfection with Polybrene gene transfection enhancer.YTS cells stably overexpressing AQP3 were obtained and labeled as YTS-AQP3^high,and empty vector group as YTS-Ctrl.The experimental groups were divided into two groups:YTS-Ctrl group and YTS-AQP3^highigh group.3.Cell culture:YTS-Ctrl cell and YTS-AQP3high cell were cultured in 1640 medium containing 10%fetal bovine serum,respectively.The new culture medium was replaced by centrifugation every 2 or 3 days.293T cells belong to adherent cells,cultured in 10cm petri dish.The cell culture medium contains 90%high glucose DMEM+10%fetal bovine serum,which is subcultured every 2 or 3 days according to cell count and status.The cells were placed in a incubator at 37?and 5%CO2.4.CCK8 assay was used to detect the inhibition rate of cell proliferation in the two groups:CCK 8 assay was used to detect the inhibitory levels of cell proliferation at different concentrations of GEM?0,2.5,5,10,20 nM/L?at YTS-Ctrl and YTS-AQP3^highigh after 48 hours and 72 hours,respectively.Three complex holes are provided under each condition.The absorbance of each hole was set as the absorbance of wavelength at 450nm,and the OD value of each hole was obtained.The data of each group were recorded and the average±standard deviation was calculated.5.Apoptosis was detected by AnnexinV-PI:after treated with different concentrations of GEM?0,5,10,20 nM/L?for 48 hours,the level of apoptosis induced by gemcitabine was detected by AnnexinV-PI.6.Western blot assay was used to detect the expression of protein:YTS-Ctrl and YTS-AQP3^highigh cells were treated with the same dose of GEM?20nM/L?for 48 hours to extract the proteins of each group.The expression of apoptosis-related and autophagy related proteins were detected.GAPDH was used as internal reference.7.The data were analyzed and plotted by SPSS21.0 and GraphPad Prism 5.01 software.The data of each group were expressed as mean±standard deviation.The comparison between the two groups of data,if the variance is using t test,if the variance is uneven using t'test.There was significant statistical difference between the two groups?p<0.05?.Result1.NK/T cell lymphoma cells(YTS-AQP3^high)stably overexpressing AQP3 were successfully constructed.2.The expression level of AQP3 in NK/T lymphoma cells was related to its sensitivity to gemcitabine:after high expression of AQP3,YTS-AQP3^highigh developed a certain resistance to gemcitabine.The inhibitory rate of GEM at the same concentration on cell proliferation in YTS-Ctrl group was significantly higher than that in YTS-AQP3high group?p<0.05?,and the apoptosis rate in YTS-Ctrl group was significantly higher than that in YTS-AQP3^highigh group after48 h treatment with the same concentration of GEM?p<0.05?.Overexpression of AQP3 can promote the proliferation and inhibit the apoptosis of NKTCL cells.3.After overexpression of AQP3,the expression of YTS-AQP3^highigh apoptosis pathway related protein Cleaved caspase-3 decreased?p<0 05?,but there was no significant difference in the expression of autophagy associated protein LC3-II/LC3-I?p>0 05?.ConclusionOverexpression of aquaporin 3 can induce drug resistance of NKTCL cells to gemcitabine,which may be induced by down-regulation of Cleaved caspase-3.