LRP16调控NF-κB信号通路的研究
发布时间：2018-12-07 16:32
【摘要】：目前的研究认为,NF-κB介导的信号通路可广泛参与细胞存活、发育和肿瘤进展等过程。尽管决定NF-κB从细胞浆到细胞核转位的分子机制已被广泛报道,对于细胞核内NF-κB活性的调控机制尚不十分清楚。既往研究表明,LRP16作为macro domain蛋白家族的一个特殊成员,属于雌激素受体和雄激素受体的共激活因子；与NF-κB的共激活因子UXT存在相互作用。在本课题中,我们主要研究了LRP16对NF-κB活性的调控作用。方法：首先,通过GST pull -down和免疫共沉淀实验验证LRP16与p65之间的相互作用。然后通过荧光素酶活性分析、电泳迁移率检测、Real-time PCR、染色质免疫共沉淀等方法检测了LRP16对NF-κB的报告基因活性、NF-κB与靶DNA的结合能力、NF-кB下游靶基因的表达水平及NF-κB与靶基因启动子区域结合能力的影响。并通过Annexin V标记与流式细胞计数的方法检测了LRP16对TNF-α诱导细胞凋亡的影响。最终通过免疫组化染色和酶联免疫吸附实验研究了人类原位胃癌样本中胞核分布的LRP16对NF-κB活性的影响。结果：LRP16通过与p65相互作用参与NF-κB转录复合体的形成;通过RNA干扰技术沉默细胞中内源性LRP16的表达可以降低NF-кB活性并明显下调NF-κB下游靶基因的表达。机制研究显示沉默LRP16表达并不影响TNF-α诱导的NF-κB核转位,但能影响细胞核中NF-KB/p300/CREB功能转录复合体的形成和稳定。此外,干扰LRP16的表达后也能增加细胞对TNF-α诱导凋亡的敏感性。最后,在人类原位胃癌标本中初步确立核内LRP16表达阴性组的NF-κB活性整体低于LRP16表达阳性组的NF-κB活性。结论：本研究表明LRP16不仅是细胞核内NF-κB;舌性的一个关键调控因子,对肿瘤中NF-κB的异常激活也有很重要的作用。
[Abstract]:It has been suggested that NF- 魏 B mediated signaling pathway is involved in cell survival, development and tumor progression. Although the molecular mechanism that determines the translocation of NF- 魏 B from cytoplasm to nucleus has been widely reported, the regulatory mechanism of NF- 魏 B activity in the nucleus is not well understood. Previous studies have shown that LRP16, as a special member of the macro domain protein family, is a coactivator of estrogen receptor and androgen receptor, and interacts with NF- 魏 B coactivator UXT. In this study, we studied the regulation of NF- 魏 B activity by LRP16. Methods: firstly, the interaction between LRP16 and p65 was verified by GST pull-down and immunoprecipitation. Then the reporter gene activity of LRP16 to NF- 魏 B and the binding ability of NF- 魏 B to target DNA were detected by luciferase activity analysis, electrophoretic mobility assay and Real-time PCR, chromatin immunoprecipitation. The expression level of target gene downstream of NF- B and the binding ability of NF- 魏 B to the promoter region of target gene. The effect of LRP16 on apoptosis induced by TNF- 伪 was detected by Annexin V labeling and flow cytometry. Finally, the effect of nuclear distribution of LRP16 on the activity of NF- 魏 B was studied by immunohistochemical staining and enzyme-linked immunosorbent assay (Elisa). Results: LRP16 was involved in the formation of NF- 魏 B transcriptional complex by interacting with p65, and the expression of endogenous LRP16 in cells was inhibited by RNA interference technique, and the expression of downstream target gene of NF- 魏 B was significantly down-regulated by RNA interference. The mechanism study showed that the silencing of LRP16 expression did not affect the nuclear translocation of NF- 魏 B induced by TNF- 伪, but affected the formation and stability of NF-KB/p300/CREB functional transcriptional complex in the nucleus. In addition, interfering with the expression of LRP16 increased the sensitivity of cells to apoptosis induced by TNF- 伪. Finally, it was preliminarily established that the activity of NF- 魏 B in nuclear LRP16 negative group was lower than that in LRP16 positive group. Conclusion: LRP16 is not only a key regulatory factor of NF- 魏 B in the nucleus, but also plays an important role in the abnormal activation of NF- 魏 B in tumor.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
本文编号：2367466
[Abstract]:It has been suggested that NF- 魏 B mediated signaling pathway is involved in cell survival, development and tumor progression. Although the molecular mechanism that determines the translocation of NF- 魏 B from cytoplasm to nucleus has been widely reported, the regulatory mechanism of NF- 魏 B activity in the nucleus is not well understood. Previous studies have shown that LRP16, as a special member of the macro domain protein family, is a coactivator of estrogen receptor and androgen receptor, and interacts with NF- 魏 B coactivator UXT. In this study, we studied the regulation of NF- 魏 B activity by LRP16. Methods: firstly, the interaction between LRP16 and p65 was verified by GST pull-down and immunoprecipitation. Then the reporter gene activity of LRP16 to NF- 魏 B and the binding ability of NF- 魏 B to target DNA were detected by luciferase activity analysis, electrophoretic mobility assay and Real-time PCR, chromatin immunoprecipitation. The expression level of target gene downstream of NF- B and the binding ability of NF- 魏 B to the promoter region of target gene. The effect of LRP16 on apoptosis induced by TNF- 伪 was detected by Annexin V labeling and flow cytometry. Finally, the effect of nuclear distribution of LRP16 on the activity of NF- 魏 B was studied by immunohistochemical staining and enzyme-linked immunosorbent assay (Elisa). Results: LRP16 was involved in the formation of NF- 魏 B transcriptional complex by interacting with p65, and the expression of endogenous LRP16 in cells was inhibited by RNA interference technique, and the expression of downstream target gene of NF- 魏 B was significantly down-regulated by RNA interference. The mechanism study showed that the silencing of LRP16 expression did not affect the nuclear translocation of NF- 魏 B induced by TNF- 伪, but affected the formation and stability of NF-KB/p300/CREB functional transcriptional complex in the nucleus. In addition, interfering with the expression of LRP16 increased the sensitivity of cells to apoptosis induced by TNF- 伪. Finally, it was preliminarily established that the activity of NF- 魏 B in nuclear LRP16 negative group was lower than that in LRP16 positive group. Conclusion: LRP16 is not only a key regulatory factor of NF- 魏 B in the nucleus, but also plays an important role in the abnormal activation of NF- 魏 B in tumor.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
【引证文献】
相关硕士学位论文 前1条
1 陈锦汝;冠心合剂主要活性成分对TNF-α损伤人脐静脉内皮细胞的保护作用及其机制的研究[D];浙江中医药大学;2013年
，本文编号：2367466
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