The Study Of The Molecular Mechanism On MicroRNA363-associated Drug Resistance In Diffuse Large B-cell Lymphoma

BackgroundDiffuse large B-cell lymphoma(DLBCL)is the most common subtype of non-Hodgkin’s lymphoma.The first-line immunochemotherapy containing anti-CD20 monoclonal antibody and anthracycline-based multidrug chemotherapy(R-CHOP)can induce complete remission or even cure in more than half of patients,but those with recurrent or refractory disease are lack of effective treatment approach.The mechanism of DLBCL resistance to first-line immunochemotherapy is very complicated and challenging.Anti-CD20 monoclonal antibody targets CD20 molecules on the surface of B lymphoma cells and exerts its anti-tumor effect by inducing antibody-dependent cytotoxicity,complement-dependent cytotoxicity and apoptosis.Thus,the reduction or loss of CD20 expression in tumor cells is the main reason for resistance to anti-CD20 monoclonal antibody.MicroRNA(miR)-363 is an intrinsic microRNA that located in miR-106a-363 cluster,which belongs to miR-17-92 family and is highly expressed in patients with R-CHOP resistance.Our previous study showed that miR-363 was significantly overexpressed in patients with resistance to R-CHOP regimen,and significantly correlated with poor prognosis.However,the mechanism of miR-363 regulation and resistance remains unclear.UCSC database was used to analyze miR-106a-363 cluster and its upstream sequence,we found that there were H3K4me3,H3K27Ac and CpG island structures at 4kb upstream of miR-363 which suggested that there may be potential promoter elements near CpG island.Meanwhile,reporter gene assay also confirmed that the upstream sequence of miR-106a-363 cluster has the function of promoter.Further study revealed that transcription factor c-Myc can bind to the promoter region of miR-106a-363 cluster,and the binding capacity is regulated by the level of promoter methylation.In a conclusion,the study demonstrated that miR-106a-363 cluster has a unique promoter region,and its CpG island methylation level negatively regulates c-Myc-mediated miR-363 expression.Previous studies indicated that CD20 molecules have the function on calcium channel or directly regulating the activity of calcium channel.Calcium influx is very important for apoptosis mediated by anti-CD20 monoclonal antibody.However,some studies found that CD20-deficient B cells still sustain calcium influx,suggesting that there are other calcium regulatory mechanisms in B cells.It has been reported that B cells express a series of calcium channel proteins,which are involved in the regulation of intracellular calcium concentration.Our study found that CACNA1C,a member of the L-type calcium channel family belonging to the L-type calcium channel a subunit,is lowly expressed in R-CHOP-resistant DLBCL patients.Our study demonstrated that CACNA1C stabilizes the structure of CD20 protein by interacting with CD20 molecules.The interaction between CACNA1C and CD20 inhibits degradation of CD20 through ubiquitin-proteasome pathway,which maintains the expression and stability of CD20 molecules on the surface of DLBCL cells,and affects the sensitivity of tumor cells to CD20 monoclonal antibody.Further studies showed that miR-363 negatively regulates the expression of CACNA1C,and then decreases the expression of CD20 on tumor B cells and induces resistance to anti-CD20 monoclonal antibody.A variety of small molecular substances can regulate the activity of L-type calcium channel,affecting calcium influx and intracellular calcium concentration,and then mediating the process of apoptosis.Our studies by in-vitro and mouse model experiments confirmed that CACNA1C-related L-type calcium channel agonists can promote calcium influx and increase the apoptosis of tumor cells induced by anti-CD20 monoclonal antibody.The expression of CACNA1C protein was not affected.These findings suggested that L-type calcium channel agonists have the potential to increase the efficacy of anti-CD20 monoclonal antibody and overcome its drug resistance,while L-type calcium channel antagonists can reduce the efficacy of anti-CD20 monoclonal antibody and induce drug resistance.In summary,our study explored that the mechanism of miR-363 regulation and its-associated resistance to first-line immunochemotherapy in diffuse large B-cell lymphoma.The hypermethylation of miR-106a-363 cluster promoter was confirmed to reduce the positive regulation of transcription factor c-Myc on its expression.L-type calcium channel protein CACNA1C can promote the stability of CD20 on the surface of DLBCL cells by interacting with CD20 molecules.MiR-363 negatively regulates the expression of CACNA1C which leads to resistance to anti-CD20 monoclonal antibody.L-type calcium channel modulator can regulate calcium influx,which affects the efficacy of anti-CD20 monoclonal antibody and becomes a potential method to improve the efficacy.Part 1:The study of the regulatory mechanism of miR-363 in diffuse large B cell lymphomaObjectiveTo study the mechanism of regulation of miR-363 expression in diffuse large B cell lymphoma.MethodsUCSC database was used to obtain the reference sequence of miR-106a-363 cluster and predict its potential promoter subsequence.Promoter function was verified by EGFP reporter gene assay.Western Blotting and ChIP were used to detect c-Myc expression and the binding of miR-106a-363 cluster promoter to c-myc.The relationship between miR-106a-363 cluster promoter methylation and miR-363 expression was detected by DNA methyltransferase inhibitors decitabine stimulation combined with BSP method.Results1.Independent promoter structure is located in approximately 4Kb upstream of the miR-363 coding region.Fluorescent reporter test confirmed that this sequence has active promoter function and regulates the transcription of miR-106a-363 cluster.2.There are four E-box structures in the CpG island of the miR-106a-363 cluster promoter region,and the transcription factor c-Myc regulates the expression of miR-363 by binding to the E-box 2 region.The methylation level in the E-box 2 region was negatively correlated with the expression of miR-363.3.DNA methyltransferase inhibitors decitabine reduces the methylation level in E-box 2 region of miR-106a-363 cluster promoter and upregulated miR-363 expression.Conclusions1.The miR-106a-363 gene cluster has an independent promoter sequence,and the transcription factor c-Myc binds to the E-box structure of the promoter region to regulate the transcription of the miR-106a-363 gene cluster.2.CpG island methylation in the promoter region of miR-106a-363 in DLBCL cells negatively regulates the expression of miR-363 by inhibiting the binding of c-Myc to E-box 2.Part 2:The study of the mechanism of miR-363-associated resistance in diffuse large B-cell lymphomaObjectivesTo explore the mechanism of miR-363-associated resistance to first-line immunochemotherapy in DLBCL patients.MethodsThe distinctive expression of calcium channel members was compared between patients with sensitive and resistant to RCHOP regimen.Reporter containing wild or mutated 3 ’UTR sequences of CACNA1C gene were constructed and transfected into 293T cells together with internal control vectors and miR-363 overexpression vectors,and then luciferase activity was detected 48 hours later.Primary DLBCL cell lines OCI-ly3,OCI-ly7,OCI-ly8 and their corresponding miR-363 overexpression or knockout cell lines were used in experiments.Western blot was used to detect the expression of CACNA1C protein,and the apoptosis was detected by flow cytometry.ShRNA vectors of CACNA1C was transfected into OCI-Ly7,OCI-Ly8 cell lines to generate CACNA1C knockdown cell lines.The finding was further validated through in vitro cell lines and in vivo mouse models.The relationship between the expression of CANCA1C and CD20 was investigated by quantitative fluorescence PCR,immunofluorescence,immunoprecipitati on and western blot.The proteasome inhibitor bortezomib was used to stimulate the CACNA1C knockdown cell lines,and western blot was used to detect the expression of CD20 protein.The L-type calcium channel agonist Bay K8644 and the inhibitor nimodipine were used to stimulate OCI-ly3,OCI-ly7 and OCI-ly8.Apoptosis was detected by flow cytometry.The PDX mouse model was constructed using tissues of DLBCL patients to observe the effect of L-type calcium channel modulator on the sensitivity of rituximab.MiR-363 overexpressed and knockout DLBCL cell lines were constructed to verify the regulation of miR-363 on CACNA1C and the effect on DLBCL cells in vitro.Results1.CACNA1C expression is negatively associated with R-CHOP resistance and survival of DLBCL patients following R-CHOP treatment.CACNA1C is an independent prognostic factor for R-CHOP regimen,whereas there is no significant correlation between CACNA1C expression and prognosis in DLBCL patients following CHOP regimen.CACNA1C expression is different among different stages of B cell development.2.Low expression of CACNA1C is associated with rituximab-mediated DLBCL cell apoptosis in vitro and tumor subduction in mouse xenograft models.3.Rituximab induced direct interaction between CACNA1C and CD20 on the surface of B cells,which inhibits CD20 degradation and enhances CD20 protein stability.4.In vitro and in vivo studies confirmed that L-type calcium channel protein modulators can regulate the function of CACNA1C,affect the calcium influx mediated by rituximab,and regulate the sensitivity of DLBCL cells to rituximab.5.MiR-363 negatively regulates the expression of CACNA1C and causes the decrease of CD20 protein,which reduces the apoptosis of DLBCL cells mediated by rituximab.Conclusions1.MiR-363 negatively regulates the expression of CACNA1C,increases the degradation of CD20 protein through the proteasome pathway,and mediates anti-CD20 monoclonal antibody resistance.2.CACNA1C-related L-type calcium channel modulators affect the efficacy of anti-CD20 monoclonal antibodies by regulating calcium influx.