转录因子MafB在非小细胞肺癌中的表达及对CD14^+单核细胞分泌Ⅰ型干扰素的影响

Expression of Transcriptional Factor MafB in Non-small Cell Lung Cancer Patients and Its Effect on Type Ⅰ Interferon by CD14^+ Monocytes

[目的]研究非小细胞肺癌(NSCLC)患者中转录因子MafB的表达变化,并评估MafB对NSCLC患者CD14^+单核细胞分泌Ⅰ型干扰素和诱导CD4^+T细胞分化的影响。[方法]入组41例NSCLC患者(28例鳞癌和13例腺癌)和21例健康志愿者。收集外周血和支气管肺泡灌洗液(BALF),分离血浆和外周血单个核细胞(PBMC),纯化CD14^+单核细胞和CD4^+T细胞。酶联免疫吸附实验检测血浆和BALF干扰素(IFN)-α和IFN-β的水平,反转录实时定量PCR检测外周血和BALF中MafBm RNA相对表达量,Western blot检测MafB蛋白水平。利用MafBsi RNA转染CD14^+单核细胞,观察抑制MafB对CD14^+单核细胞分泌IFN-α和IFN-β的影响,检测干扰素调节因子3(IRF3)磷酸化水平。建立CD14^+单核细胞和CD4^+T细胞的直接接触和间接接触培养系统,通过ELISA法检测培养上清中IFN-γ、白细胞介素(IL)-4、IL-17和IL-21表达水平评估抑制MafB对CD14^+单核细胞调控CD4^+T细胞分化的影响。[结果]血浆IFN-α和IFN-β水平在健康志愿者和NSCLC患者之间差异无统计学意义,但肿瘤部位BALF中IFN-α(242.5±59.98pg/ml vs 282.5±45.24pg/ml,P=0.013)和IFN-β(12.40±2.81pg/ml vs 34.42±7.83pg/ml,P<0.0001)水平均显著性低于非肿瘤部位。PBMC中MafBm RNA相对表达量和蛋白水平在健康志愿者和NSCLC患者之间差异亦无统计学意义,但肿瘤部位BALF中MafB m RNA和蛋白水平则显著性高于非肿瘤部位(P<0.0001)。MafBsi RNA转染可显著性抑制CD14^+单核细胞中MafBm RNA和蛋白的表达。MafBsi RNA转染可促进CD14^+单核细胞IFN-β的分泌(16.09±5.79pg/ml vs 6.73±1.78pg/ml,P<0.0001),增加IRF3磷酸化(P<0.0001),但对IFN-α表达无明显影响(P>0.05)。而抑制NSCLC患者CD14^+单核细胞中MafB对CD14^+单核细胞和CD4^+T细胞共培养系统中IFN-γ、IL-4、IL-17和IL-21的分泌水平并无显著性影响(P>0.05)。[结论]NSCLC患者肿瘤部位过度表达的MafB可能诱导了Ⅰ型干扰素抑制,但MafB对肿瘤部位CD14^+单核细胞的免疫调控功能可能无影响。

[Objective]To investigate the expression of transcriptional factor MafB in non-small cell lung cancer(NSCLC)patients,and to evaluate its effect on typeⅠinterferon production by CD14^+ monocytes.[Methods]Forty-one NSCLC patients(28 cases with squamous carcinoma and 13 cases with adenocarcinoma)and 21 healthy individuals were enrolled in this study.Peripheral bloods and bronchoalveolar lavage fluid(BALF)were collected.Plasma and peripheral blood mononuclear cells(PBMC)were isolated,while CD14^+ monocytes and CD4^+ T cells were purified.Interferon(IFN)-αand IFN-βexpression in plasma and BALF were measured by ELISA.MafB mRNA relative level and protein expression were assessed in peripheral bloods and BALF by real-time RT-PCR and Western blot,respectively.CD14^+ monocytes were transfected by MafB siRNA.IFN-α/IFN-βproduction by CD14^+ monocytes and interferon regulatory factor 3(IRF3)phosphorylation was investigated in response to MafB inhibition.The direct contact and indirect contact co-culture system of CD14^+ monocytes and CD4^+ T cells was set up.The effect of MafB inhibition on CD14^+ monocyte regulation for CD4^+ T cells differentiation was investigated by measurements of IFN-γ,IL-4,IL-17,and IL-21 expression in culture supernatant.[Results]There were no significant differences in plasma IFN-αand IFN-βexpression between healthy individuals and NSCLC patients.IFN-α(242.5±59.98 pg/ml vs 282.5±45.24 pg/ml,P=0.013)and IFN-β(12.40±2.81 pg/ml vs 34.42±7.83 pg/ml,P<0.0001)expression in BALF were significantly down-regulated in tumor site when compared with non-tumor site.There were also no significant differences of circulating MafB mRNA relative level and protein expression between healthy individuals and NSCLC patients.However,MafB mRNA relative level and protein expression in BALF were significantly elevated in tumor site when compared with non-tumor site(P<0.0001).Maf B si RNA transfection significantly inhibited Maf B m RNA and protein expression in CD14^+ monocytes.Maf B si RNA transfection promoted IFN-βproduction by CD14^+ monocyets(16.09±5.79 pg/ml vs 6.73±1.78 pg/ml,P<0.0001)and increased IRF3 phosphorylation(P<0.0001),however,did not affect IFN-αproduction(P>0.05).Moreover,Maf B inhibition in CD14^+ monocytes from NSCLC patients did not significantly change IFN-γ,IL-4,IL-17,or IL-21 expression in CD14^+ monocytes and CD4^+ T cells co-cultured supernatant(P>0.05).[Conclusion]Overexpression of Maf B in NSCLC patients may suppress type Ⅰ interferon production,however,it may not affect the immunoregulatory activity of CD14^+ monocytes.