The Study On The Genetic Changes And Mechanism Of NLRP3 Inflammasome In Multiple Myeloma

Background:Multiple myeloma(MM)is a life-threatening plasma cell malignancy whose pathogenesis is not fully understood and standard treatment is not yet well established.Up to now,many studies have proved that genetic and immunological changes are involved in the pathogenesis of MM.It is important to further explore the pathogenesis of MM and find the therapeutic target for MM.Inflammasome plays a crucial role in the immune system,and it can trigger innate immune defenses and promote adaptive immune responses[1].When activated,inflammatory cytokines such as IL-1?,IL-18,IL-22 and TNF-a are released,and the downstream NF-?B signaling pathway is activated,which can cause the tissue injury and tumor development.However,with further studies,it has been proved that inflammasome is a double-edged sword for the occurrence of tumors,and have both promoting and inhibiting effects.At present,the most studied inflammasome is Nod like receptor protein 3(NLRP3).NLRP3 inflammasome is a kind of protein complexes composed of NOD-like receptors(NLRs),apoptosis-associated speck-like protein containing a CARD(ASC)and cysteine aspartate protease(Caspase-1)composition.It is responsible for the maturation and secretion of pro-inflammatory cytokines,such as IL-? and IL-18[2].Caspase recruitment domain containing protein 8(CARD8)controls caspase-1 activation and caspase-1-mediated IL-1? and IL-18 activation[3].Some studies have shown that inflammasome are involved in the pathophysiology of MM[4].The up-regulation of NLRP3 expression depends on the activation of NF-?B signaling pathway.NF-?B is also a major inflammatory molecule involved in the growth and survival of myeloma cells in bone marrow microenvironment.IL-6 is also important for the survival of myeloma cells.The correlation between il-6 and inflammatory corpuscles is rarely reported.It has been confirmed that single nucleotide polymorphisms(SNPs)of the related genes of NLRP3 inflammasome are associated with the development of many diseases,such as rheumatoid arthritis,ankylosing spondylitis,melanoma,liver cancer,lung cancer and ovarian cancer.However,in MM,the role of single nucleotide polymorphisms(SNPs)of NLRP3 inflammasome related genes has not been clearly elucidated.We studied the NLRP3 inflammasome-associated genes SNPs:CARD8-C10X(rs2043211),NF-?B-94 ins/del(rs28362491),IL-1?(rs16944);IL-18(rs1946518),and gene expressions(CARD8,NF-?B,IL-1?,IL-18 mRNA)in patients with multiple myeloma,and explored their correlation with the clinical features of multiple myeloma.Objective:1.To search for the susceptibility factors of multiple myeloma in the SNPs of NLRP3 inflammasome related genes;2.To study the correlation between the SNPs of NLRP3 inflammasome related genes and the clinical features of MM;3.To study the correlation between the NLRP3 inflammasome related genes SNPs and genes expressions.Methods:1.PCR was used to detect the SNPs of CARD8-C1 OX(rs2043211),NF-?B-94 ins/del(rs28362491),IL-1p(rs16944)and IL-18(rs194651 8)in 355 MM patients and 350 normal controls.2.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expressions of NF-?B,CARD8,IL-1? and IL-18 mRNA in 42 MM patients.3.ELISA was used to determine the concentration of IL-1?,IL-18 and IL-6 in bone marrow supernatant of 42 MM patients.Results:1.CARD8-C10X(rs2043211)AT genotype may be a susceptibility factor to MM;Patients with TT genotype of CARD8-C10X(rs2043211)had earlier stages than those with AA and AT;The white blood cell counts of CARD8-C10X(rs2043211)genotypes showed an increasing trend(AA<AT<TT).2.Patients with del/del genotype of NF-?B-94 ins/del(rs28362491)have higher percentage of myeloma cells than ins/ins and ins/del.The hemoglobin concentrations of three genotypes of IL-18(rs 16944)showed an increasing trend(GG<GT<TT).3.The relative expression of CARD8 mRNA in patients with CARD8-C10X(rs2043211)TT genotype was significantly higher than that in AA and AT genotypes.Patients with NF-?B-94 ins/del(rs28362491)ins/del genotype have significantly higher relative expression of NF-?B mRNA than patients with del/del genotype.4.The expression of IL-1? in bone marrow supernatant of patients with three genotypes of CARD8(rs2043211)showed an increasing trend(TT<AT<AA),while the relative expression of IL-1? mRNA showed an decreasing trend(TT>AT>AA).The relative expression of IL-1? mRNA in patients with NF-?B-94 ins/del(rs28362491)ins/ins and ins/del are higher than that in patients with del/del genotype.5.There was no significant correlation between the expression of IL-6 cytokines and the SNPs of the four genes.Conclusion:1.Card8-c10x(rs2043211)AT genotype is a susceptible factor of MM,which contributes to the diagnosis of MM and serves as a new therapeutic target.2.The SNPs of NLRP3 inflammasome related genes are related to the clinical features of MM.Correlation between polymorphisms of NLRP3 inflammasome related genes and Th subsets in multiple myelomaIn recent years,with the development of new drugs and stem cell transplantation,the therapeutic effect of multiple myeloma has been improved rapidly,and the survival period of MM has been extended effectively,but MM is still a stubborn incurable disease.The role of abnormal immune function in the pathogenesis of MM is receiving increasing attention.CD4+T cells are at the center of the immune response.A large number of studies have shown that Th subgroups expression imbalance in MM patients may be involved in the pathogenesis of MM,but the specific mechanism is not completely clear.Studies have shown that the NLRP3 inflammasome are closely related to Th cells,and they interact with each other and participate in the pathogenesis and progress of inflammatory,immunological and neoplastic diseases.So far,there is no illumination of the relationship between NLRP3 inflammasome and Th subgroups in MM.The first part of our study,suggested that NLRP3 inflammasome related genes polymorphisms were associated with susceptibility of multiple myeloma and some clinical manifestations,indicated that the polymorphisms of NLRP3 inflammasome related genes were associated with the occurrence and development of MM.Therefore,we further analyzed the gene polymorphisms associated with NLRP3 inflammasome:CARD8-C10X(rs2043211),NF-kappa B-94 ins/del(rs28362491),IL-1?(rs 16944),IL-18(rs 1946518)and the expression of peripheral blood Th cell subsets(Th1/Th2/Th17/Treg/Th22),to explore the effect of NLRP3 inflammasome related genes SNPs on the role of Th cells in MM.Functional study on activation of NLRP3 inflammasome in MMOne of the main causes of relapse,refractory treatment and progression of MM patients is the drug resistance of myeloma cells.Therefore,it is very important for us to study the mechanism of chemotherapeutic drug resistance in MM patients,find the target and reverse the drug resistance effectively.Combination chemotherapy with glucocorticoids and bortezomib is a first-line treatment for MM.Dexamethasone is one of the most basic and important combined chemotherapy drugs.One of the mechanisms of dexamethasone in the treatment of multiple myeloma is to induce apoptosis of MM cells.Bortezomib(BTZ),trade name is Velcade(Vel),is a proteasome inhibitor,which can induce apoptosis of myeloma cells through a variety of mechanisms.The advent of the drug significantly improved the symptoms of MM patients and delayed the progression of disease.Nevertheless,almost all patients will inevitably relapse or drug resistance.Studies have suggested that NLRP3 inflammasome can promote apoptosis.In our study,LPS+ATP was applied in BMMCs of MM patients and KM3 cell line to verify whether the NLRP3 inflammasome were activated.If activation is successful,further study on whether activated NLRP3 inflammasome is involved in the apoptosis of myeloma cells induced by dexamethasone or bortezomib,in the hope of finding new ways for the drug resistance mechanism of multiple myeloma and providing theoretical basis for the study of drug resistance targets.Objective:To study the relationship between the SNPs of NLRP3 inflammasome related genes and Th subsets in MM.To investigate whether the activation of inflammasome of NLRP3 is involved in the apoptosis of myeloma cells induced by dexamethasone and bortezomib.The aim is to provide a theoretical basis for finding new therapeutic targets of MM.Methods:1.(1)The ratio of peripheral blood Th cell subsets(Thl,Th2,Th17,Treg,Th22)to CD4+T in 24 newly diagnosed MM patients(case group)and 20 healthy volunteers(control group)was measured by Flow cytometry.(2)Polymerase chain reaction(PCR)was used to detect the SNPs of NLRP3 inflammasome in bone marrow cells of 24 newly diagnosed MM patients,including CARD8-C10X(rs2043211),NF-?B-94 ins/del(rs28362491),IL-1?(rs16944)and IL-18(rs 1946518).(3)The relationship between the expression of SNPs and Th subgroup was analyzed.The corresponding transcription factors were detected by RT-PCR and the relationship between the transcription factors and SNPs was analyzed.2.(1)BMMCs were isolated by Ficoll density gradient centrifugation(Ficoll density gradient centrifugation)in 6 MM patients on appeal.Each patient's cells were divided into two groups:the control group(the unstimulated group)and the experimental group(the stimulated group).The expression of NLRP3,ASC,Caspase-1 and IL-1 beta were detected by Western blot.RT-PCR was used to detect mRNA expression of NLRP3,ASC,Caspase-1,IL-1(3 and IL-18 in two groups.The levels of IL-1? and IL-18 in the bone marrow supernatant of both groups were detected by ELISA.The purpose is to confirm the activation of inflammasome.(2)After culture and passage of MM cell line KM3,LPS + ATP stimulation was added,and Western blot,RT-PCR and ELISA were used to detect the same indexes.(3)Flow cytometry was used to detect the effect of activation of NLRP3 Inflammasome on the apoptosis of BMMCs and KM3 cells induced by dexamethasone and bortezomib.Results:1.The proportion of Thl cells in the case group was significantly lower than that in the control group(median:12.6%in the control group,9.15%in the case group;P<0.001),and the proportion of Th17 cells was significantly higher(median:2.30%in the control group,3.07%in the case group;P = 0.016),and the proportion of Treg cells was significantly lower(median:3.22%in the control group,0.95%in the case group;P<0.001).The Thl/Th2 ratio in case group was lower than that in normal control group(median:control group 54.25,case group 43.49;P= 0.048),and the Th17/Treg ratio was higher than that in normal control group(median:control group 0.74,case group 2.90;P= 0.034).2.The expression of Thl cells and their transcription factor T-bet mRNA in peripheral blood of patients with CARD8-C10X(rs2043211)TT,AT and AA genotypes showed an increasing trend(TT<AT<AA),and the difference was statistically significant.The expression rate of Thl cells in patients with NF-?B-94 ins/del(rs28362491)del/del was significantly lower than that of del/ins and ins/ins(P<0.05).However,there was no significant difference in the expression of T-bet mRNA among the above three genotypes.In addition,SNPs of the four inflammasome related genes were not significantly correlated with the expression of other Th cells(Th2,Thl7,Th22,Treg).3.After LPS+ATP stimulation,the levels of NLRP3 and IL-1?-pl7 protein in BMMCs lysate were significantly higher than those in control group(P<0.05),the expressions of Caspase-1 mRNA and IL-1? mRNA were significantly higher than those in control group(P<0.05),and the levels of IL-1? and IL-18 in bone marrow supernatant after stimulation were all significantly higher than those in control group(P<0.05).After LPS+ATP stimulation in KM3 cells,the protein expression levels of NLRP3 and IL-1?-pl7 were increased compared with the control group(P<0.05).The expression of caspase-1 mRNA(P=0.016)and IL-1?mRNA(P=0.037)were increased compared with the control group.The expression level of IL-1? in the cultured supernatant was higher than that of the control group(P=0.024),and the expression level of IL-1 8 was higher than that of the control group(P=0.055).4.Addition of Dex(P<0.001),Vel(P<0.001)or Dex+Vel(P<0.001)to BMMCs significantly increased the early apoptosis rate compared with the control group.The addition of Dex(P=0.017),Vel(P=0.026)or Dex+Vel(P=0.045)after LPS+ATP stimulation significantly reduced the early apoptosis rate of each group compared with that of the non-stimulated group.In KM3 cells,addition of Dex(P<0.001),Vel(P<0.001)or Dex+Vel(P<0.001)significantly increased the early apoptosis rate compared with the control group;The addition of Dex(P=0.03),Vel(P=0.025)or Dex+Vel(P=0.055)after LPS+ATP stimulation reduced the early apoptosis rate of each group compared with that of the unstimulated group.Conclusion:1.In the peripheral blood of MM patients,the proportion of Th1 and Treg cells decreased,the proportion of Th17 cells increased,the ratio of Th1/Th2 decreased,and the ratio of Th17/Treg increased.Therefore,Th subgroup imbalance existed in MM patients.2.The SNPs of CARD8-C10X(rs2043211)may affect the expression of Thl cells in multiple myeloma.3.LPS+ATP can activate NLRP3 inflammasome in multiple myeloma cells.4.Activation of inflammasome of NLRP3 inhibited early apoptosis of myeloma cells induced by dexamethasone and bortezomib,suggesting that activation of inflammasome of NLRP3 may be involved in resistance mechanisms of dexamethasone and bortezomib.