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目的验证miR-338-5p对核因子κB1(NF-κB1)水平的影响,研究其对B细胞生物学功能的调控作用。方法利用双荧光素酶报告基因检测系统进行miR-338-5p靶基因的验证;采用抗Ig M抗体和/或重组人B细胞活化因子(rh BAFF)蛋白刺激的B细胞培养体系,将miR-338-5p激动剂、miR-338-5p拮抗剂、NF-κB1小干扰RNA(si NF-κB1)及其对应的阴性对照制剂转染CD20+B淋巴细胞,利用实时定量PCR法检测各转染组NF-κB1 mRNA水平,Western blot法检测NF-κB1蛋白(p105、p50)的水平,ELISA检测培养上清中Ig G含量。结果靶基因验证实验显示,miR-338-5p模拟物与报告载体共转染组的hRluc/h Luc萤光素酶活力比值显著升高;体外培养实验结果显示,在抗Ig M抗体与rh BAFF蛋白共培养体系,miR-338-5p激动剂转染组NF-κB1 mRNA、p105蛋白、p50蛋白、Ig G水平均显著升高,si NF-κB1的作用与miR-338-5p激动剂相反;miR-338-5p拮抗剂转染组NF-κB1 mRNA、Ig G水平均显著降低;相关性分析结果表明,NF-κB1 mRNA水平与Ig G水平呈显著正相关。结论 miR-338-5p靶向正调控NF-κB1、间接作用于BAFF信号参与调控B细胞生物学功能。
Objective To verify the effect of miR-338-5p on the expression of nuclear factor κB1 (NF-κB1), and to study the regulation of miR-338-5p on the biological function of B cells. Methods The dual-luciferase reporter assay was used to validate miR-338-5p target gene. The B cell culture system stimulated by anti-Ig M antibody and / or recombinant human B cell activating factor (rhBAFF) 338-5p agonist, miR-338-5p antagonist, NF-κB1 small interfering RNA (si NF-κB1) and its corresponding negative control were transfected into CD20 + B lymphocytes by real-time quantitative PCR The level of NF-κB1 mRNA was detected by Western blot. The level of NF-κB1 protein (p105, p50) was detected by Western blot. The content of Ig G in culture supernatant was detected by ELISA. Results The results of target gene validation showed that the luciferase activity of hRluc / h transfected with miR-338-5p mimics and the reporter vector was significantly increased. The results of in vitro culture showed that the anti-Ig M antibody and rhBAFF Protein co-culture system, miR-338-5p agonist transfection group NF-κB1 mRNA, p105 protein, p50 protein, Ig G levels were significantly increased, si NF-κB1 role and miR-338-5p agonist opposite; The levels of NF-κB1 mRNA and Ig G in miR-338-5p transfection group were significantly decreased; the correlation analysis showed that there was a significant positive correlation between NF-κB1 mRNA level and Ig G level. Conclusion Targeting miR-338-5p positively regulates NF-κB1 and indirectly affects BAFF signaling in the regulation of B cell biology.