Preliminary Study Of Non-canonical Nf-κb Signaling Pathway In B-NHL Resistance

Background and ObjectiveB-cell non-Hodgkin’s lymphoma (B-NHL) is a group of heterologous malignant fatal disease of malignant B-cell monoclonal amplification. In recent years, with increasing levels of research and treatment, especially Rituximab in clinical applications, the survival period is gradually extended, and some patients can be cured. However, tumor cell resistance often leads to ineffective treatment or relapse, and the proportion of patients with poor prognosis. The present study showed that the activating of nuclear factor kappa B (NF-κB) signaling pathway may be one of the main reasons of B-NHL resistance. In peripheral blood of patients with B-NHL, B cell activating factor (BAFF) significantly increased with disease progression, severity, and treatment sensitivity [2,3,4]. BAFF receptor (BAFF-R) is a specific receptor for BAFF-specific regulation of B cell survival, and then, non-canonical NF-κB signaling pathway activated by BAFF/BAFF-R mainly mediated pathway. Early in the yeast three-hybrid experiments, we found interactions exsit among the receptor-interacting serine/threonine kinase2(RIPK2), BAFF-R and tumor necrosis factor receptor-associated factor (TRAF3), of whichTRAF3, as negative regulator molecule of the non-canonical NF-κB signaling pathway, has an important pivotal role. Those prompt RIPK2as an intracellular protein, may have the potential linkage with BAFF/BAFF-R mediated the non-canonical NF-κB signaling pathway activation leading to drug-resistance of lymphoma.This topic through detecting the non-canonical NF-κB signaling pathways related purpose gene mRNAs expression level of B-NHL patients peripheral blood mononuclear cells (PBMC) and lymph node of the tumor, study its correlations with B-NHL resistance and disease progression. MTT assay and stimulating Raji cells with different concentrations of recombinant human BAFF to show BAFF promoting proliferation of Raji cells in the human Burkitt lymphoma (BL), to detect expression of non-canonical NF-κB signaling pathways associated signaling molecules, and therefore, to provide clues for further study of the B-NHL resistance mechanisms. While preliminary explore RIPK2with BAFF/BAFF-R mediated signaling pathway.Materials and MethodsThe subject of peripheral blood specimens of36cases of B-NHL patients, from the hospitalization Affiliated Hospital of Academy of Military Medical Sciences in November2010to November2011, and over the same period seven healthy blood donors. Lymph node tumor tissue specimens from20cases of B-NHL patients hospitalized from January2009to November2011, the Academy of Military Medical Sciences.Extraction of peripheral blood and tissue samples of total RNA, and UV spectrophotometer and a1%agarose gel electrophoresis detection of RNA concentration, purity and integrity. Used PrimerScript Reverse Transcriptase into RNA by reverse transcription of cDNA and stored at-20℃.Amplification of the internal reference GAPDH by Semi-quantitative reverse transcription polymerase chain reaction (SqRT-PCR), then amplification products through1.5%agarose gel electrophoresis. Detecting PBMC and tumor tissue of non-canonical NF-κB signaling pathway relate target gene and RIPK2mRNAs expression levels by real-time fluorescence quantitative polymerase chain reaction (FQ-PCR), of which results calculate with2-△△Ct method. According to the clinical features group, with SPSS13.0software for statistical analysis, and application of the Pearson linear correlation analysis, to study target gene mRNAs expression level and mutual corelationship; application of Spearman rank correlation analysis, to study correlation between target gene mRNAs expression levels with disease progression degree.Application MTT assay of BAFF Raji cells proliferation by stimulating Raji cells with different concentrations (62.5ng/mL,125ng/mL,250ng/mL,500ng/mL) of recombinant human BAFF at24h,48h, and72h. Stimulating Raji cells by different concentrations (20ng/mL,100ng/mL,500ng/mL) of recombinant human BAFF respectively, using the FQ-PCR and Westen Blot technology to detect non-canonical NF-κB signaling pathway signaling molecule gene and protein expression. With Pearson linear correlation analysis to study the correlation between BAFF-R, NIK, p52, Bcl-xL, and RIPK2with BAFF mRNAs expression level, and with Spearman rank correlation analysis to study target gene mRNAs expression with the level of recombinant human BAFF concentration. At the same time, preliminary discuss relevance of RIPK2in BAFF/BAFF-R mediated the classic the NF-kappa B pathway signal.Results1B-NHL PBMC purpose gene mRNAs expression:B-NHL patients grouped according to clinical characteristics, the relative expression levels of BAFF-R and Bcl-xL and RIPK2mRNAs compared with the normal control group was significantly increased (P<0.05); male group, IPI(Lymphoma International Prognostic Index) score of0～2Group, age≥the age of60groups, respectively, with the female group, the IPI score3to4group, age≤60years of age group, in PBMC relative expression level of the target gene mRNAs had no significant difference,(P>0.05); The relative expression levels of Ⅲ/Ⅳ stage and drug resistance groups PBMC target gene mRNAs, respectively, higher than the phase Ⅰ/Ⅱ and non-resistant group (P<0.05). Pearson linear correlation analysis showed that relative expression levels of every two target gene mRNAs each other was a positive correlation (P0.0001), indicate that the target gene expression trends are consistent. Spearman rank correlation analysised different clinical stage, and non-resistance/resistance group, results showed that the target gene mRNAs relative expression level and extent of disease progression was a positive correlation (P <0.0001), suggesting that as the disease progresses, the target gene increased expression along.2. B-the NHL tissues target gene mRNAs expression:The relative expression levels of mRNAs of Bcl-xL in the resistance group was significantly higher than non-resistant group (P<0.05);the expression level of of BAFF, BAFF-R, NIK, p52and RIPK2of resistant group tended to increase, compared with non-resistant group, however, no significant difference (P>0.05).3. MTT assay of three recombinant human BAFF stimulating Raji cells:Different concentrations of recombinant human BAFF to stimulate the cells at24h,48h, and72h, compared with the control group, the OD value of each treatment group increased in varying degrees, therefore, by different concentrations, at different time points, and the interaction were significant differences (F (4.24)=425.88, P<0.0001; F (2.24)=519.73, P<0.0001; F (8.24)=5.50, P<0.0001). BAFF can promote the proliferation of Raji cells, with recombinant human BAFF increasd the concentration and treatment time Raji cell proliferation rate gradually increased (P<0.05). The role of BAFF promoting proliferation was evident in dose-effect and time-effct dependence.4. Raji cell target gene FQ-PCR experiments:Different concentrations of recombinant human BAFF to stimulate Raji cells, BAFF, BAFF-R, NIK, p52, Bcl-xL, and RIPK2mRNA expression compared with negative control group were significantly increased, P<0.05. Among20ng/mL,100ng/mL,500ng/mL three groups, the mRNAs of the target gene relative expression levels were significantly different (P<0.05), and with the increased concentration of recombinant human BAFF expression level was an upward trend.Pearson linear correlation analysis showed that BAFF-R, NIK, p52, Bcl-xL, and RIPK2with BAFF mRNAs relative expression levels are positively correlated (P<0.0001). The Spearman rank correlation analysis results show that the target gene mRNAs relative expression levels is positively correlated with recombinant human BAFF concentration, that is, the recombinant human BAFF concentration increased, the elevated expression of target genes, P<0.0001.5. Westen Blot Experiment: With different concentrations recombinant human BAFF stimulate Raji cells, BAFF, BAFF-R, NIK, p52, Bcl-xL and RIPK2protein expression increased with recombinant human BAFF concentration increased. ConelusionsⅢ/Ⅳ stage, resistance B-NHL patients PBMC BAFF, BAFF-R, Bcl-xL and RIPK2mRNAs relative expression level higher than Ⅰ/Ⅱ, no-resistant group respectively, and normal control group, suggesting the target gene expression relate to disease progression and severity. Relative expression levels of BAFF, BAFF-R, NIK, p52, Bcl-xL, and RIPK2mRNAs in resistant B-NHL tumor tissues were higher than non-resistant group, suggesting that of BAFF/BAFF-R mediated non-canonical NF-κB signaling pathways associated with B-NHL cell resistance, and RIPK2may be associated with the signaling pathway.MTT assay results showed that the the BAFF could promote the proliferation of BL Raji cells, and BAFF in promoting proliferation of dose and time dependent manner. Recombinant human BAFF to stimulate Raji cell, can activate the non-canonical NF-κB signaling pathway, and the degree of activation is related to the concentration of the recombinant human BAFF, which could cause downstream target proteins Bcl-xL and other anti-apoptotic factor overexpression, the latter promoting tumor cell survival and proliferation. These suggest the proliferation of Raji cells could be regulated by BAFF through non-canonical NF-κB signaling pathway. BAFF can increase expression of RIPK2, which indicate that functional relationship may exist between the RIPK2and non-canonical NF-κB signaling pathway.In this study, to further explore the occurrence, development, and resistant biological mechanisms of the B-NHL, providing new clues.