Regulatory Mechanism Of MiRNA-223-3p/CHUK/NF-κB Pathway Axis On Survival And Ibrutinib Susceptibility Of Chronic Lymphocytic Leukemia

Part one:Regulatory mechanism of miRNA-223-3p/CHUK/NF-κB pathway axis on survival of chronic lymphocytic leukemiaBackground and objective:Chronic lymphocytic leukemia is a kind of incurable malignant tumor and is the most common leukemia type in western countries.At the same time,it is also a common hematological tumor in middle-aged and elderly people in China.Our previous studies demonstrated that miRNA-223-3p was significantly low-expressed in patients with CLL and was an independent poor prognostic factors.However,the detailed role of miRNA-223-3p in CLL has not been clarified.Besides,studies showed that the abnormal NF-κB pathway activation played an important role in CLL development and progression.miRNA-223-3p affected macrophage differentiation through regulating CHUK.Based on this,we will explore miRNA-223-3p’s role in CLL pathogenesis.Methods:1)After lentivirus infection to B cell lymphoma cell lines(Granta-519,Jeko-1)to establish stable cell lines,we explored the effects of overexpression and low expression of miRNA-223-3p on cell proliferation,apoptosis and cycle by CCK8 assay,flow cytometry assay and flow cytometry assay.Western blot was performed to detect Bcl-2 and Bax protein expression level.2)After lentivirus infection to CLL primary cells,we explored the effects of overexpression of miRNA-223-3p on cell proliferation,apoptosis by CCK8 and flow cytometry assay.3)The regulation effect of miRNA-223-3p on CHUK in HEK293 T cells was determined by luciferase reporter assay and verified in B cell lymphoma cell lines by q PCR and western blot.The regulation effect of miRNA-223-3p on NF-κB non-classical pathway activation in B cell lymphoma cell lines was determined by western blot and immunofluorescence assay.4)After lentivirus infection to B cell lymphoma cell lines(Granta-519,Jeko-1)to establish stable cell lines,we explored the effects of overexpression and low expression of CHUK on cell proliferation,apoptosis and cycle by CCK8,flow cytometry and flow cytometry assay.Western blot was performed to detect Bcl-2 and Bax protein expression level.5)After lentivirus infection to CLL primary cells,we explored the effects of low expression of CHUK on cell proliferation,apoptosis by CCK8 and flow cytometry assay.6)After lentivirus infection to miRNA-223-3p overexpression/low expression B cell lymphoma stable cell lines,we explored the reversible effect of CHUK on miRNA-223-3p overexpression/low expression B cell lymphoma stable cell lines proliferation.7)NC group and miRNA-223-3p overexpression group of Jeko-1 xenograft tumor model were established in BALB/c nude mice and effect of miRNA-223-3p overexpression on NF-κB non-classical pathway was examined by western blot.Tumor growth was monitored and apoptosis was detected by TUNEL staining.8)The expression levels of CHUK m RNA in CLL primary cells and normal CD19+ cells were detected by q PCR.The expression levels of miRNA-223-3p and CHUK m RNA in CLL primary cells were detected by q PCR and the correlation was analysed.Results:1)Overexpression of miRNA-223-3p in B cell lymphoma cell lines significantly inhibited cell proliferation,induced cell apoptosis,but no effect on cell cycle G1 arrest.Cells overexpressing miRNA-223-3p stimulated lower Bcl-2 and higher Bax protein expression compared with NC cells.Low expression of miRNA-223-3p in B cell lymphoma cell lines significantly promoted cell proliferation,inhibited cell apoptosis,but no effect on cell cycle G1 arrest.Cells silencing miRNA-223-3p stimulated higher Bcl-2 and lower Bax protein expression compared with NC cells.2)Overexpression of miRNA-223-3p in CLL primary cells significantly inhibited cell proliferation and induced cell apoptosis.3)The results of luciferase reporter assay,q PCR and western blot showed that CHUK was targeted and negatively regulated by miRNA-223-3p.Overexpression of miRNA-223-3p in B cell lymphoma cell lines was associated with down-regulated protein expression level of CHUK,phospho-CHUK,the level of NF-κB2 p100 processing into p52 and nuclear translocation of Rel B.Low expression of miRNA-223-3p in B cell lymphoma cell lines was associated with up-regulated protein expression level of CHUK,phospho-CHUK,the level of NF-κB2 p100 processing into p52 and nuclear translocation of Rel B.4)Overexpression of CHUK in B cell lymphoma cell lines significantly promoted cell proliferation,inhibited cell apoptosis,but no effect on cell cycle G1 arrest.Cells overexpressing CHUK stimulated higher Bcl-2 and lower Bax protein expression compared with NC cells.Low expression of CHUK in B cell lymphoma cell lines significantly inhibited cell proliferation,promoted cell apoptosis,but no effect on cell cycle G1 arrest.Cells silencing CHUK stimulated lower Bcl-2 and higher Bax protein expression compared with NC cells.5)Low expression of CHUK in CLL primary cells significantly inhibited cell proliferation and induced cell apoptosis.6)CHUK overexpression reversed up-regulated miRNA-223-3p-inhibited cell proliferation in B cell lymphoma cell lines.CHUK knockdown reversed downregulated miRNA-223-3p-promoted cell proliferation B cell lymphoma cell lines.7)In nude mice xenograft tumor model of Jeko-1,miRNA-223-3p overexpression group significantly inhibited tumor growth in volume and weight,while promoted apoptosis.Overexpression of miRNA-223-3p in nude mice xenograft tumor model is associated with down-regulated protein expression level of CHUK,phospho-CHUK and the level of NF-κB2 p100 processing into p52.8)CHUK m RNA level in CLL primary cells was significantly higher than that in normal CD19+ cells.There was a significant negative correlation between miRNA-223-3p and CHUK m RNA level in CLL primary cells.Conclusion:1.miRNA-223-3p affects proliferation and apoptosis of B cell lymphoma cell lines through regulating CHUK/NF-κB non-classical pathway in vitro and in vivo.2.miRNA-223-3p affects proliferation and apoptosis of CLL primary cells through regulating CHUK/NF-κB non-classical pathway.Part Two:Regulatory mechanism of miRNA-223-3p/CHUK/NF-κB pathway axis on ibrutinib susceptibility of chronic lymphocytic leukemiaBackground and objective:With the widespread application of BTK inhibitors including ibrutinib in patients with chronic lymphocytic leukemia(CLL),it becomes more and more important to clarify ibrutinib resistance mechanism.miRNA is involved not only in tumor development and progression,but also in tumor multi-drug resistance.Our previous study showed that the expression levels of miRNA-223-3p and CHUK m RNA in patients with refractory/ drug-resistant CLL are significantly lower and higher than those in previously untreated patients.Besides,the targeted regulating role of miRNA-223-3p in CHUK has been demonstrated(seen in this study’s first part).Based on this,we explore miRNA-223-3p’s detailed role in ibrutinib resistance in CLL,which could provide a new way to prevent drug resistance and provide effective salvage treatment.Methods:1)After lentivirus infection to B cell lymphoma cell lines(Granta-519,Jeko-1)to establish stable cell lines,we explored the effects of overexpression and low expression of miRNA-223-3p on ibrutinib susceptibility by CCK8 assay.2)After lentivirus infection to B cell lymphoma cell lines(Granta-519,Jeko-1)to establish stable cell lines,we explored the effects of overexpression and low expression of CHUK on ibrutinib susceptibility by CCK8 assay.3)After lentivirus infection to CLL primary cells,we explored the effects of overexpression of miRNA-223-3p and low expression of CHUK on ibrutinib susceptibility by CCK8 assay.4)After lentivirus infection to miRNA-223-3p overexpression/low expression B cell lymphoma stable cell lines,we explored the reversible effect of CHUK on miRNA-223-3p overexpression/low expression B cell lymphoma stable cell lines ibrutinib susceptibility by CCK8 assay.5)NC group and miRNA-223-3p low expression group of Jeko-1 xenograft tumor model were established in BALB/c nude mice and treated with ibrutinib.The effect of miRNA-223-3p low expression on Jeko-1 xenograft tumor model ibrutinib susceptibility was detected by monitoring tumor growth in volume and weight.Results:1)Overexpression of miRNA-223-3p in B cell lymphoma cell lines significantly increased ibrutinib susceptibility.Low expression of miRNA-223-3p in B cell lymphoma cell lines significantly decreased ibrutinib susceptibility.2)Overexpression of CHUK in B cell lymphoma cell lines significantly decreased ibrutinib susceptibility.Low expression of CHUK in B cell lymphoma cell lines significantly increased ibrutinib susceptibility.3)Overexpression of miRNA-223-3p significantly increased CLL primary cells ibrutinib susceptibility.Low expression of CHUK significantly increased CLL primary cells ibrutinib susceptibility.4)CHUK overexpression reversed up-regulated miRNA-223-3p-promoted ibrutinib susceptibility in B cell lymphoma cell lines.CHUK knockdown reversed down-regulated miRNA-223-3p-inhibited ibrutinib susceptibility in B cell lymphoma cell lines.5)In nude mice xenograft tumor model of Jeko-1 which were treated with ibrutinib,miRNA-223-3p low expression group significantly inhibited ibrutinib susceptibility compared with NC group.Conclusion:1.miRNA-223-3p affects B cell lymphoma cell lines ibrutinib susceptibility through regulating CHUK/NF-κB non-classical pathway in vitro and vivo.2.miRNA-223-3p affects CLL primary cells ibrutinib susceptibility through regulating CHUK/NF-κB non-classical pathway.