NLS-RARα通过与p38α MAPK相互作用抑制ATRA对NB4细胞的效应

发布时间：2018-11-20 10:44

【摘要】：目的:带核定位信号的维甲酸受体(Nuclear localization signal retinoic acid receptor alpha:NLS-RARα)在急性早幼粒细胞白血病(Acute promyelocytic leukemia:APL)的发生发展中扮演着重要的角色,它是由中性粒细胞弹性蛋白酶(Neutrophil elastase:NE)切割早幼粒细胞白血病-维甲酸受体(Promyelocytic leukemia-retinoic acid receptor alpha:PML-RARα)融合蛋白形成的。然而,NLS-RARα对APL的作用机制尚未明确,本研究论文探讨NLS-RARα对APL细胞株NB4细胞的作用及其机制。方法:免疫印迹实验和CCK-8增殖实验分别检测全反式维甲酸(All-trans retinoic acid:ATRA)处理后的NB4细胞分化标志物C/EBPβ、CD11b,丝裂原活化蛋白激酶p38α(Mitogen-activated protein kinase p38α:p38αMAPK)表达水平和NB4细胞增殖水平;分子克隆技术构建p38αMAPK真核表达质粒,双荧光素酶实验检测p38αMAPK的转录激活活性;利用慢病毒介导的NLS-RARα基因过表达,进一步用免疫印迹实验验证过表达效率并检测NLS-RARα对NB4细胞分化标志物C/EBPβ、CD11b和p38αMAPK表达水平的影响,同时,CCK-8增殖实验检测NLS-RARα对NB4细胞增殖水平的影响;间接免疫荧光实验分析nls-rarα与p38αmapk的空间共定位,免疫共沉淀实验分析nls-rarα与p38αmapk的相互作用;应用p38αmapk活性抑制剂pd169316研究atra激活p38αmapk后再募集nls-rarα还是atra募集已与p38αmapk结合的nls-rarα后激活p38αmapk。结果:全反式维甲酸(all-transretinoicacid:atra)处理组nb4细胞分化指标c/ebpβ、cd11b,磷酸化的丝裂原活化蛋白激酶p38α(phosphorylated-mitogen-activatedproteinkinase:p-p38αmapk)的量增加(p0.05),细胞增殖水平下降(p0.05),p38αmapk表达水平无差异(p0.05);成功构建p38αmapk真核表达质粒,p38αmapk真核表达质粒转染组的荧光素酶活性增加(p0.05);成功在nb4细胞中过表达nls-rarα,当未用atra处理细胞时,过表达nls-rarα组nb4分化指标c/ebpβ和cd11b的表达水平下降(p0.05),p38αmapk和p-p38αmapk的量无改变(p0.05),当用atra处理细胞时,过表达nls-rarα组nb4分化指标c/ebpβ、cd11b和p-p38αmapk的量减少(p0.05),p38αmapk表达水平无差异(p0.05),细胞增殖水平上升(p0.05);免疫荧光实验和免疫共沉淀实验证实nls-rarα与p38αmapk直接相互作用;p38αmapk抑制剂pd169316的在lv-nls-rarα-nb4细胞中的应用发现,与atra处理组相比,atra+pd169316处理组lv-nls-rarα-nb4细胞中的分化指标c/ebpβ、cd11b和p-p38αmapk的量减少(p0.05),nls-rarα和p38αmapk表达水平无差异(p0.05),细胞增殖水平上升(p0.05),pd169316在293t细胞中的应用发现抑制剂处理组和未处理组中p38αMAPK和NLS-RARα都能发生相互作用。结论:ATRA募集已与p38αMAPK结合的NLS-RARα后,能通过激活p38αMAPK促进APL细胞株NB4细胞分化而抑制其增殖,然而在此过程中,NLS-RARα能够通过下调p-p38αMAPK而抑制ATRA对NB4细胞的效应。
[Abstract]:Objective: retinoic acid receptor (Nuclear localization signal retinoic acid receptor alpha:NLS-RAR 伪) with nuclear localization signal plays an important role in the pathogenesis and development of acute promyelocytic leukemia (Acute promyelocytic leukemia:APL). It is formed by neutrophil elastase (Neutrophil elastase:NE) cleavage of promyelocytic leukemia-retinoic acid receptor (Promyelocytic leukemia-retinoic acid receptor alpha:PML-RAR 伪) fusion protein. However, the mechanism of NLS-RAR 伪 on APL is not clear. In this study, the effect of NLS-RAR 伪 on APL cell line NB4 cells and its mechanism are discussed. Methods: Western blot assay and CCK-8 proliferation assay were used to detect the differentiation markers C/EBP 尾 and CD11b, of NB4 cells treated with all trans retinoic acid (All-trans retinoic acid:ATRA). The expression of mitogen-activated protein kinase p38 伪 (Mitogen-activated protein kinase p38 伪: p38 伪 MAPK) and the proliferation of NB4 cells were observed. The eukaryotic expression plasmid p38 伪 MAPK was constructed by molecular cloning, and the transcriptional activation activity of p38 伪 MAPK was detected by double luciferase assay. Using lentivirus-mediated overexpression of NLS-RAR 伪 gene, the overexpression efficiency of NLS-RAR 伪 and the expression of C/EBP 尾, CD11b and p38 伪 MAPK in NB4 cells were examined by Western blotting assay. At the same time, the expression levels of C/EBP 尾, CD11b and p38 伪 MAPK were detected. CCK-8 proliferation assay was used to detect the effect of NLS-RAR 伪 on the proliferation of NB4 cells. The spatial co-localization of nls-rar 伪 and p38 伪 mapk was analyzed by indirect immunofluorescence assay, and the interaction between nls-rar 伪 and p38 伪 mapk was analyzed by immunoprecipitation assay. Using p38 伪 mapk activity inhibitor pd169316 to study whether atra activates p38 伪 mapk before recruiting nls-rar 伪 or atra recruitment with nls-rar 伪 combined with p38 伪 mapk to activate p38 伪 mapk. Results: in all trans retinoic acid (all-transretinoicacid:atra) treated group, c/ebp 尾, cd11b, phosphorylated mitogen-activated protein kinase p38 伪 (phosphorylated-mitogen-activatedproteinkinase:p-p38 伪 mapk) increased (p0.05). The level of cell proliferation was decreased (p0. 05), but the expression of p38 伪 mapk was not different (p0. 05). The eukaryotic expression plasmid p38 伪 mapk was constructed successfully. The luciferase activity of p38 伪 mapk eukaryotic expression plasmid transfected group was increased (p0. 05). Nls-rar 伪 was successfully expressed in nb4 cells. When the cells were not treated with atra, the expression levels of c/ebp 尾 and cd11b in nb4 differentiation index were decreased (p0.05), and the levels of p38 伪 mapk and p-p38 伪 mapk were not changed (p0.05). When the cells were treated with atra, the nb4 differentiation indexes c/ebp 尾, cd11b and p-p38 伪 mapk decreased (p0.05), the expression of p38 伪 mapk was not different (p0.05), and the cell proliferation was increased (p0.05). The direct interaction between nls-rar 伪 and p38 伪 mapk was confirmed by immunofluorescence and co-immunoprecipitation. The application of p38 伪 mapk inhibitor pd169316 in lv-nls-rar 伪-nb4 cells showed that the quantity of c/ebp 尾, cd11b and p-p38 伪 mapk in lv-nls-rar 伪-nb4 cells treated with atra pd169316 was lower than that in atra treatment group (p0. 05). There was no difference in the expression of nls-rar 伪 and p38 伪 mapk (p0.05), but the level of cell proliferation increased (p0.05). The application of pd169316 in 293t cells showed that p38 伪 MAPK and NLS-RAR 伪 could interact with each other in both the inhibitor group and the untreated group. Conclusion: ATRA recruitment combined with p38 伪 MAPK NLS-RAR 伪 can inhibit the proliferation of NB4 cells by activating p38 伪 MAPK. However, NLS-RAR 伪 can inhibit the effect of ATRA on NB4 cells by down-regulating p-p38 伪 MAPK.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7

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