The Regulatory Effect And Underlying Mechanism Of JMJD3in Promoting Progress Of Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent non-Hodgkin lymphoma (NHL) in adulthood, accounting for30-40%of diagnoses. DLBCL represents a biologically heterogeneous group of tumors with various clinical symptoms, it is highly heterogeneous in morphology, genetics, immune phenotype and prognosis. DLBCL were mainly grouped into two categories that represented different stages of B cell differentiation:germinal center B-cell-like (GCB) DLBCL and activated B-cell like (ABC) DLBCL. These two subtypes of DLBCL derive from cells with discrete biological programs during normal B-cell differentiation and have unique biological characteristics respectively:The BCL2gene translocations was characteristic of GCB DLBCL, whereas ABC DLBCL exist many gene mutation leading to the constitutive activation of NF-кB signal pathway which is important for tumor cell survival.The study of DLBCL genome discovered that aberrant expression of epigenetic regulation factor affecting histone methylation were ubiquitous and impact the tumorigenesis and progression of DLBCL. Among these epigenetic regulation factors, JMJD3is a member of JmjC domain containing enzyme family. JMJD3mainly possesses demethylase activity to different histone substrates and regulates the expression of target genes. Based on the current knowledge, JMJD3may have a important role in antigen-driven B-cell differentiation and aberrantly expressed in Hodgkin’s Lymphoma (HL). However insight into of the biological effect and mechanism of JMJD3in DLBCL progression remains poorly understood. The aim of our study is to confirm the regulatory effect and underlying molecular mechanism of JMJD3in promoting progress of DLBCL.To adjust the biological effect of JMJD3in DLBCL progression, firstly we investigate the expression level of JMJD3in DLBCL. We performed immunohistochemistry analysis of JMJD3levels on a DLBCL tissue microarray composed of100surgical DLBCL samples. Relative to normal lymph node controls, which expressed low levels of JMJD3, expression was strong in92%of the DLBCL cells. Then we examined the expression level of JMJD3in HMy2.CIR and seven DLBCL cell lines. Similarly, JMJD3expression was elevated in most DLBCL cell lines, but not a normal B cell line.To investigate the role of JMJD3in the biology of DLBCL, we assessed the effect of JMJD3on the viability and apoptosis of cell line models of DLBCL. Our results showed that the expression of JMJD3increased the viability of normal B cells and DLBCL cells, while suppression of JMJD3expression decreased the viability of DLBCL cells and induced apoptosis in DLBCL cells. Next, we explored the insight mechanism of the anti-apoptotic effect of JMJD3in DLBCL. Firstly we characterized the effects of JMJD3depletion on the regulation of the IRF family genes in both ABC and GCB subtype DLBCL cells. The results revealed that JMJD3regulated the expression of IRF4and chromatin immunoprecipitation (ChIP) assays confirmed that JMJD3expression influenced H3K27trimethylation in the IRF4promoter region. But then we found JMJD3promotes the survival of different subtypes of DLBCL via distinct mechanismsIn ABC DLBCL, the elevated expression of IRF4in JMJD3-depleted DLBCL cells rescues survival of DLBCL cells, suggesting that IRF4mediates the anti-apoptosis effect of JMJD3in ABC DLBCL. Further study found that NF-κB signaling is critical for survival mediated by the JMJD3-IRF4axis in ABC cells, the transcriptional factor IRF4also potently up-regulated the expression of JMJD3in an NF-κB dependent manner, suggesting JMJD3-IRF4-NF-κB form a positive feedback mechanism perpetuates this pro-survival pathway in ABC cells.In GCB DLBCL, the anti-apoptotic protein BCL2mediates the anti-apoptotic effect of JMJD3in GCB DLBCL cells. Firstly we confirmed that JMJD3expression influenced H3K27trimethylation in the BCL2promoter region via chromatin immunoprecipitation (ChIP) assays, indicating BCL2is another downstream target gene of JMJD3. The elevated expression of BCL2in JMJD3-depleted DLBCL cells rescues survival of DLBCL cells, suggesting that BCL2mediates the anti-apoptotic effect of JMJD3in GCB DLBCL. In ABC DLBCL, the elevated expression of BCL2in JMJD3-depleted DLBCL cells partially rescues survival of DLBCL cells, suggesting that BCL2participate in the anti-apoptotic effect of JMJD3in ABC DLBCL.In established ABC and GCB subtype DLBCL tumor mouse model, Therapeutic delivery of JMJD3siRNA via intratumor multi-point injection inhibits DLBCL tumor growth and induces tumor cell death.Based on above experiments, we could get the following conciusions:JMJD3mainly sustains the survival of DLBCL cells via anti-apoptotic effect. In ABC DLBCL, JMJD3regulates the expression of IRF4and then influence the activity of NF-kB signal pathway to sustain the survival of DLBCL cells. JMJD3-IRF4-NF-kB forms an oncogenic positive feedback loop. In GCB DLBCL, the anti-apoptotic protein BCL2mainly mediates the anti-apoptotic effect of JMJD3. Therapeutic delivery of JMJD3siRNA via intratumor multi-point injection inhibits DLBCL tumor growth and induces tumor cell death.In conclusion, as JMJD3plays an important role in sustaining the survival of DLBCL cells, suggesting maybe it is a potential therapeutic target of DLBCL. Designing specific small molecule inhibitors targeting JMJD3may provide new horizons for clinical targeted therapy of DLBCL.