| NK/T-cell lymphoma?NKTCL?belongs to non Hodgkin's lymphoma,accounting for 2%-10%of all lymphoma.This type of lymphoma is a rare lymphomawhichrelated to EBV infection.Research shows the incidence of NK/T cell lymphoma is associated with various factors,including the abnormalities of gene,chromosome,signaling pathway and protein expression.The pathogenesis of NK/T-cell lymphoma is complex,this makes it more difficult for clinicians to diagnose,classify and treat the disease.The course NK/T-cell lymphoma progresses rapidly,the survival time of patients is short,the disease is prone to recurrence and drug resistance,and the prognosis of the disease is poor.At present,the molecular mechanism of multidrug resistance of NK/T cell lymphoma is not clear.Therefore,it is necessary to find the reason and the mechanism of drug resistance.The ABC transporter family 4?ATP-binding cassette transporter family class C4,ABCC4?belongs to the ABCC family,which regulates the molecular distribution in cells.It has been found that ABCC4 may be a key regulator of chemosensitivity in various tumors,including human NK/T cell lymphoma,by transporting drugs outside the cell and reducing the amount of drugs in the cell.Current studies have found that ABCC4 is associated with the occurrence of malignant tumors and multidrug resistance.Overexpression of ABCC4 has been found in glioma,retinoblastoma,colon cancer,neuroblastoma,rectal cancer and NK/T cell lymphoma.In addition,it was found that ABCC4 was positively correlated with drug resistance of leukemia,ovarian cancer and other cells.IL-13 is a cytokine derived from T cells,which can inhibits the production of inflammatory cytokines.IL-13 is an immunomodulatory center that inhibits the monitoring of tumor immunity.IL-13 plays crucial roles in many physiological processes,such as inflammation regulation,mucus production,tissue recovery,immune response,and autoimmune disease.IL-13 can also induce many chemokines,metalloproteinases,and cathepsin,thereby promoting the occurrence of inflammation.Currently,there is no study on the expression and role of IL-13 in NK/T cell lymphoma.In order to investigate the role and mechanism of IL-13 in regulating ABCC4 in NK/T cell lymphoma cell function and multidrug resistance,the expression of IL-13in peripheral serum of NK/T cell lymphoma was detected by ELISA,the expression of ABCC4 in NK/T cell lymphoma was detected by immunohistochemistry,and the relationship between the expression of IL-13 and clinicopathological parameters was analyzed.In addition,the lentiviral vector of ABCC4 interference was constructed by RNAi,and the effects of exogenous IL-13 on proliferation,apoptosis and drug resistance of YTS cells were examined.Finally,we will construct a nude mouse model of NK/T cell lymphoma in vivo to further study the mechanism of drug resistance mediated by IL-13 and ABC-4 in vivo.Objective:1.To detect IL-13 expression in peripheral serum of patients with NK/T cell lymphoma and ABCC4 expression in NK/T cell lymphoma,and to analyze the relationship between IL-13 expression level and clinicopathological parameters.2.To detect the effects of IL-13 on proliferation,apoptosis and drug resistance of YTS cells by regulating ABCC4.3.To further study the effects of IL-13 on the growth and drug resistance of ABCC4 transplanted tumors in mice.Main content:The first part:The expression of ABCC4 and IL-13 in NK/T cell lymphomaMethods1.The expression level of IL-13 in the peripheral serum of patients with NK/T cell lymphoma and rhinitis was measured by ELISA method.2.The expression level of ABCC4 in NK/T cell lymphoma and rhinitis tissues was determined by immunohistochemistry.Results1.ELISA results exhibited that the expression level of IL-13 in the peripheral serum of patients with NK/T cell lymphoma was significantly higher than that in patients with rhinitis.There was no obvious relationship between the positive expression rate of IL-13 and patients'gender,age,or location in NK/T cell lymphoma,but there was obvious difference in IL-13 expression of patients at clinical stages?-?and?-?.2.Immunohistochemical results demonstrated that the positive expression rate of ABCC4 in NK/T cell lymphoma tissues was 77%?23/30?,which was significantly higher than that in rhinitis tissues 42%?5/12?.The second part:The impact of IL-13 on YTS cells by regulating ABCC4 in vivoMethods1.The construction of Lenti-sh-ABCC4 lentiviral vector:the pLVX-shRNA1lentiviral vector after Age?and EcoR?double enzyme digestion was connected to ABCC4 shRNA fragment,and then sent to Shanghai Sangon Biotech Company for sequencing analysis after identification of colony PCR.The recombinant lentivirus vector which was sequenced and confirmed correct was packaged.Then the recombinant lentiviruses were titrated and were used to infecte YTS cells.The stable YTS-sh-ABCC4 cell lines were gained by screening with puromycin.2.The expression of ABCC4,AKT,p-AKT,mTOR,p-mTOR,P70S6K,and p-P70S6K proteins in YTS,NK and YTS-sh-ABCC4 cells was detected by Western blot.3.CCK-8 assay was implemented to determin the effect of IL-13 regulating ABCC4 on the proliferation and drug-resistance of YTS cells.4.Flow cytometry assay was performed to examine the effect of IL-13regulating ABCC4 on the apoptosis of YTS cells.Results1.The identification of colony PCR and sequencing results showed that the recombinant lentivirus vectors were successfully constructed.The titration value of LV-sh-ABCC4-1 and LV-sh-ABCC4-2 was 109.32±0.159 and 109.49±0.312 TU/mL,respectively.2.Western blot analyses showed that compared with the control cells,the expression level of ABCC4 protein in the YTS-sh-ABCC4-1 and YTS-sh-ABCC4-2cells was significantly decreased and ABCC4 interference efficiency in YTS-sh-ABCC4-2 group was significantly higher than that in YTS-sh-ABCC4-1cells.These results suggested that the stable YTS-sh-ABCC4 cells were successfully constructed.3.Western blot detection demonstrated that the expression of ABCC4 protein in NK/T cell lymphoma cell line YTS was significantly higher than that of NK cells.4.Compared with YTS cells,the expression of ABCC4 protein was significantly decreased after ABCC4 interference.After IL-13 treatment,the expression level of ABCC4 was significantly increased in YTS cells.Compared with YTS-sh-ABCC4group,ABCC4 expression level was significantly increased after IL-13 treatment.These results showed that IL-13 could promote the expression of ABCC4 in YTS cells.5.Compared with YTS cells,cell proliferation was increased after IL-13treatment,and cell proliferation was significantly decreased after ABCC4 interference.Compared with YTS-sh-ABCC4 group,cell proliferation was significantly increased after IL-13 treatment.It is suggested that IL-13 could promote the proliferation of YTS cells by regulating ABCC4.6.Compared with YTS cells,the apoptosis rate of YTS cells was significantly decreased after the addition of IL-13,and apoptosis rate was significantly increasedin YTS-sh-ABCC4.IL-13 treatment promoted the apoptosis rate of YTS-sh-ABCC4cells.The results demonstrated that IL-13 could observably inhibit the apoptosis of YTS cells by regulation of ABCC4.7.By Graphpad software,the results showed that the IC50 value of adriamycin in YTS cells was 2.014,the IC50 value of doxorubicin in YTS+IL-13 cells was 4.103,the IC50 value of doxorubicin in YTS-sh-ABCC4 cells was 0.589,and the IC50 value of doxorubicin YTS-sh-ABCC4+IL-13 cells was 1.649.Compared with parental YTS cells,the resistance of YTS-IL-13 cells to doxorubicin increased to 2.04 times,the resistance of YTS-sh-ABCC4 cells to doxorubicin decreased to 0.29 times,and the resistance of YTS-sh-ABCC4+IL-13 group to doxorubicin decreased to 0.82 times.8.The analyses by graphpad software showed that gemcitabine could inhibit cell growth of YTS cells,and the inhibitory effect was enhanced with the increase of dose.The IC50 value of gemcitabine in the YTS group was 9.263,,the IC50 value of gemcitabine in the YTS-IL-13 group was 17.520,the IC50 value of gemcitabine in the YTS-sh-ABCC4 group was 3.360,and the IC50 value of gemcitabine in the YTS-sh-ABCC4+IL-13 group was 6.648.Compared with parental YTS cells,the resistance of cells in the YTS-IL-13 cells to gemcitabine increased to 1.89 times,that in the YTS-sh-ABCC4 cells decreased to 0.36 times,and that in the YTS-sh-ABCC4+IL-13 cells decreased to 0.717 times.9.Western blot analyses showed that there was no significant difference in the expression of AKT,mTOR and P70S6K in each group.However,compared with YTS group,the expression of p-AKT,p-mTOR and p-P70S6K was increased in cells treated with IL-13.The expressions of p-AKT p-mTOR and p-P70S6K were significantly decreased in YTS+sh-ABCC4 group.Compared with YTS-sh-ABCC4group,p-AKT,p-mTOR and p-P70S6K expression were increased in YTS-sh-ABCC4+IL-13 group.The third part:The impact of IL-13 on NK/T cell lymphoma by regulating ABCC4 in vitroMethods1.The mouse model of NK/T cell lymphoma was constructed with YTS cells,and the volume of the transplanted tumor was detected regularly with vernier caliper.After 30 days,the weight of YTS transplanted tumors was weighed.2.Immunohistochemistry assays were conducted to detect the expression of ABCC4 in YTS transplanted tumor.3.Western blot analyses of the expression of the AKT/mTOR/P70S6K signaling pathway-related proteins AKT,p-AKT,mTOR,p-mTOR,P70S6K and p-P70S6K in YTS transplanted tumors of each group.Results1.The weight and volume of the transplanted tumors in mice showed that Il-13regulating ABCC4 may improve the drug resistance of YTS transplanted tumor and promote the growth of transplanted tumors.2.Immunohistochemical detection exhibited that IL-13 could promote the expression of ABCC4.3.Western blot analyses showed there was no significant difference in the expression of AKT,mTOR and P70S6K in the transplanted tumors of each group.However,compared with the YTS group,the expression of p-AKT,p-mTOR and p-P70S6K was increased in the IL-13 treatment group.The expressions of p-AKT p-mTOR and p-P70S6K were significantly decreased in YTS+sh-ABCC4 group.Compared with YTS+sh-ABCC4 group,p-AKT expression,p-mTOR and p-P70S6K expression were increased in the YTS+sh-ABCC4+IL-13 group.Conclusion1.IL-13 was highly expressed in peripheral blood serum of patients with NK/T cell lymphoma,which was closely related to clinical stage;ABCC4 expression in NK/T cell lymphoma tissue was increased.2.The expression level of ABCC4 in YTS cells was obviously higher than that in NK cells.IL-13 could activate AKT/mTOR/P70S6K pathway by positively regulating ABCC4,thereby promoting cell proliferation,inhibiting apoptosis,and improving resistance of YTS cells to adriamycin and gemcitabine.3.IL-13 could activate P70S6K/AKT/mTOR pathway by regulating ABCC4,thereby increasing the drug resistance of NK/T cell lymphoma,and promoting the growth of nude mouse transplantation tumor. |
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