| Background:Lymphoma is a malignant tumor derived from lymphoid hematopoietic system.With the continuous development of medical treatment,the survival and quality of life of patients have been greatly improved.However,there are still many lymphoma patients that will relapse or be resistant to chemotherapy,and even lead to death.Lymphoma is an immune system tumor,and the occurrence of tumors is a multi-factor,multi-link,multi-gene interaction results which are related to genetic,immune microenvironment abnormalities,apoptosis,tumor suppressor gene inactivation and abnormal cell proliferation.Recent studies have shown that tumor cells in the development or chemotherapy treatment could produce inflammasome which was regarded as an important part of immunity.NLRP3 inflammasome is the most studied inflammasome which occupies an important position in the occurrence,development and chemoresistance of tumor.Inflammasome can be recruited,assembled and activated by exogenous pathogen invasion or in vivo cell damage and death.Inflammasomes release a large number of IL-1 and IL-18 and other inflammatory cytokines through the NLRP3/caspase axis to promote tumorigenesis.IL-18 is the main effector molecule of NLRP3 inflammasome which could inhibit the growth of NK cells to promote tumor growth and metastasis by inducing the expression of PD-1.Single nucleotide polymorphisms(SNPs)affect the inflammatory microenvironment nesting malignant tumors,and SNPs may also provide a direct effect on the malignant cells.Genetic SNPs associated with NLRP3 inflammasome were found to be related to the development of various cancers.NLRP3 inflammasome and its products could directly promote the growth of tumor cells and drug resistance.The inhibition of inflammatory response or the neutralization of its products has a far-reaching inhibitory effect on canceration and tumor progression,therefore targeting NLRP3 inflammasome is expected to become an important means of tumor therapy.Objective:The aim of this study was to investigate the polymorphism and expression of NLRP3 inflammasome in patients with lymphoma and evaluate its clinical significance,and further to explore its mechanism in lymphoma by using functional experiment of lymphoma cell line.Methods:1.SNPs of NLRP3-related gene in lymphoma patients:390 patients with lymphoma and 385 healthy controls were selected.Bone marrow smear was taken from patients with lymphoma.EDTA anticoagulated whole blood was extracted from healthy controls,and then extracted by DNA sample extraction kit DNA.PCR(FISH)were used to detect the genotypes.2.The expression of NLRP3-related genes in lymphoma patients and controls:Sixty eight patients with lymphoma(46 newly diagnosed patients and 22 patients with remission)and 40 healthy controls were used to extract peripheral blood mRNA and reverse transcribed into cDNA.The expressions of IL-18,IL-1?,caspase-1,NLRP3,ASC and NF?B mRNA in peripheral blood mononuclear cells of patients with lymphoma and controls were detected by RT-PCR.The levels of IL-18 and IL-1? in peripheral blood of patients with lymphoma and controls were detected by ELISA.The protein expressions of IL-18,NLRP3 and ASC in lymphoma tissues and normal lymph nodes were detected by immunohistochemistry.3.The expression levels of ASC and NF?B protein were detected by western blot after adding LPS and ATP in Pfeiffer culture system,while GAPDH was used as internal reference.4.Pfeiffer cells were added with different concentrations of dexamethasone or after adding LPS and ATP,and then were detected for drug sensitivity.The experimental groups were divided into control,LPS+ATP,DEX and DEX+LPS+ATP.The cells and supernatants were collected for determination.The mRNA expressions of IL-18,IL-1?,caspase-1,NLRP3,c-myc,TP53,bcl-2 and bax in cells were detected by RT-PCR.The levels of IL-18 and IL-? in supernatant were measured by ELISA.5.IL-18 and anti-IL-18 were added to Pfeiffer culture system for 48h.The experimental group was divided into control,DEX,IL-18+DEX and anti-IL-18+DEX.The expressions of c-myc,TP53,bcl-2 and bax mRNA were detected by RT-PCR.6.CCK8 method was used to detect the cell proliferation and the drug sensitivity analysis.According to the cell cycle kit,cell cycle analysis was performed by flow cytometry.Apoptosis of each group was detected using flow cytometry by Annexin V/PI apoptosis detection kit.7.Statistical analysis:The continuous and categorical variables were compared by ANOVA or nonparametric Kruskal-Wallis test.The standard chi-square test was used to test the genotype frequencies of Hardy-Weinberg equilibrium.The association between genotype and disease risk was assessed by calculating the odds ratio(OR)and the corresponding 95%confidence interval(CI).The Mann-Whitney U test was used to perform mRNA and ELISA analysis to compare the expression of each group.The Kaplan-Meier curve was used to describe survival.The cell experiments were tested three times,and the quantitative data were expressed as the mean ąSD deviation and analyzed using the unpaired t test.P<0.05 was considered statistically significant.Results:1.Relationship between SNPs of NLRP3 inflammasome and susceptibility and prognosis of lymphoma(1)The genotype distribution of IL-18(rs1946518)and NF?B 94 ins/del(rs28362491)in lymphoma patients was significantly different from that in normal controls.(2)The risk of lymphoma carrying IL-18(rsl 946518)allele "G" or NF?B 94 ins/del(rs28362491)allele "ins" was significantly increased.(3)IL-18(rsl946591)genotype was associated with LDH levels.CARD8(rs2043211)genotype was associated with gender,B symptoms and extranodal infiltration.And NF?B 94 ins/del(rs28362491)genotype was associated with LDH levels,typing and bone marrow infiltration.8(4)IL-18(rsl946518)gene and NF?B 94 ins/del(rs28362491)gene were significantly associated with susceptibility to B-NHL.IL-18(rs 1946518)gene was also significantly associated with susceptibility to T-NHL.The risk of T-NHL or B-NHL was significantly increased in the carriers with IL-18 allele "G",and the NF?B allele "ins" was associated with a significant increase in the risk of B-NHL.(5)Lymphoma patients with CARD8(rs2043211)AA genotype or higher LDH level have a poor survival.2.Expressions of NLRP3 inflammasome-related genes in patients with lymphoma and their related functions(1)The mRNA expressions of IL-18,IL-1?,caspase-1,NLRP3 and NF?B of patients with lymphoma were significantly higher than those of controls.However,only the expression of IL-18 mRNA in lymphoma patients after chemotherapy remission was lower than that in newly diagnosed lymphoma patients.(2)IL-18 serum levels in newly diagnosed lymphoma patients were significantly higher than those in controls,and decreased after chemotherapy.The serum levels of IL-1? in patients with newly diagnosed lymphoma were significantly higher than those in controls,but showed no significant change with those after chemotherapy.The serum levels of IL-18 in IL-18(rsl946518)GT genotype were significantly higher than those in GG genotype.(3)The protein expressions of ASC and NLRP3 in lymphoma tissues were significantly higher than those in controls,and the protein expressions of IL-18 in lymphoma patients were slightly higher than those in controls.(4)After adding LPS for 6 hours and ATP for 1 hour,the protein expression levels of ASC and NF?B were increased in Pfeiffer.Moreover,the expressions of IL-18,caspase-1,IL-1? and NLRP3 mRNA were increased,and the levels of IL-18 and IL-1? in culture supernatant were significantly increased,which suggested that NLRP3 inflammasome was activated.(5)After being treated with LPS and ATP,the inhibitory rate of dexamethasone on Pfeiffer cells decreased,and the IC50 value of dexamethasone increased significantly.So the activation of NLRP3 inflammasome enhanced the resistance of Pfeiffer cells to dexamethasone.(6)Dexamethasone inhibited the proliferation of Pfeiffer cells.The activation of NLRP3 inflammasome promoted the cell cycle from G1 to S phase,reduced the effect of dexamethasone on cell cycle,andpromoted the cell proliferation by increasing the expression of c-myc mRNA.(7)Dexamethasone induced the Pfeiffer cell apoptosis.The activation of NLRP3 inflammasome inhibited cell apoptosis and weakened the anti-tumor effect of dexamethasone by up-regulation of bcl-2 and down-regulation of bax.(8)The expressions of c-myc and bcl-2 mRNA were significantly increased,while the expressions of TP53 and bax mRNA were significantly decreased after rhIL-18 was added,which promoted the cell proliferation,inhibited the cell apoptosis,and inhibited the anti-tumor effect of dexamethasone.Moreover,anti-IL-18 antibody has the according opposite effects.Conclusion:1.The gene polymorphisms of IL-18(rs1946518)and NF?B 94 ins/del(rs28362491)were associated with the increasing susceptibility of lymphoma.The CARD8(rs2043211)polymorphism had a great effect on the survival of lymphoma.2.The expressions of NLRP3-related genes in patients with lymphoma were higher than those in normal subjects.NLRP3 inflammasome could promote the proliferation of lymphoma cells,inhibit the apoptosis of lymphoma cells,and reduce the anti-tumor effect of dexamethasone by its downstream genes such as IL-1? and IL-18.3.IL-18 could activate the NLRP3 inflammasome,promote the proliferation of lymphoma cells,inhibit the apoptosis of lymphoma cells,and reduce the anti-tumor effect of dexamethasone.Anti-IL-18 could play the opposite effect.4.NLRP3 inflammasome contributes to the susceptibility of lymphoma and plays a carcinogenic role through its effector cytokine IL-18. |
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