NFκB上调USP16基因的转录及其分子机制研究
发布时间：2018-08-14 15:28
【摘要】：目的:泛素特异性蛋白酶USP16在基因表达、细胞周期、细胞自我更新和/或衰老过程中起重要作用,且其在唐氏综合症中的过度表达是神经功能缺损的主要促进者,与神经干细胞缺陷密切相关。但USP16基因的转录调控基本上是未知的。本研究主要探讨核转录因子NFκB对人USP16基因的转录调控及其分子机制。方法:引物设计、质粒构建、细胞培养与转染、荧光素酶活性检测及分析、核蛋白和Ch IP DNA提取、电泳迁移率分析、染色质免疫共沉淀、细胞干预、实时定量PCR。结果:1.成功扩增USP16基因启动子区-1856至+468共2324bp DNA序列,插入载体p GL4.10构建重组荧光素酶报告基因质粒p USP16-A,在此基础上构建一系列删切质粒。并通过荧光素酶活性检测得到了一系列正性和负性调控区域。2.转录因子预测分析发现人USP16基因启动子区-1856至+468含有若干核转录因子结合位点,如:NFκB、HIF、SP1。3.根据转录因子预测结果显示的结合位点,依次构建4个重组荧光素酶报告基因质粒p USP16-N1~p USP16-N4。在HEK293细胞中,与空载PMTF相比,共转PMTF-p65质粒后荧光素酶活性分别增加2.69,2.51,2.24和2.04倍(p0.001),并且对N1 vs.N3,N1 vs.N4以及N2vs.N4统计发现,结合位点2和3的缺失对NFkB在上调USP16基因启动子中的作用有很大的影响。在SH-SY5Y细胞中,得到类似的结果(p0.001)。4.电泳迁移率分析,我们发现预测到的结合位点2,3和4在体外可与NFκB p65结合;而染色质免疫共沉淀实验表明,预测到的结合位点2和3可与NFkBp65结合,而位点1和4不能结合。6.LPS干预导致SH-SY5Y细胞内源性USP16 m RNA水平显着增加(1.58倍)(p0.05),但在HEK293细胞中无显着影响(数据未显示)。然而,在HEK293细胞和SH-SY5Y细胞中,TNFa干预分别使内源性USP16 m RNA的水平增加了1.90倍(p0.01)和1.85倍(p0.05)。结论:人USP16基因的5'侧翼区,从-1856至+468,具有显著启动子活性;过表达NF?B p65可以上调人USP16启动子活性;无论在体内或体外,有两个(-826~-815和-510~-501)NF?B p65结合位点与均可与USP16相互作用;过表达NF?Bp65或LPS和TNFα的刺激活化NF?B均能上调人类USP16基因的转录。总之,NF?B可通过两个真正的顺式作用元件上调人USP16基因的转录。目的:分析棉鼠肺表面活性相关蛋白A(SP-A)基因序列结构和生物信息学特点,观察棉鼠肺损伤模型中SP-A m RNA和蛋白表达水平,初探SP-A表达规律与肺损伤的相关性。方法:将32只棉鼠随机均分为4组:3个实验组棉鼠分别腹腔注射2 mg/kg LPS处理24、48和96 h,对照组腹腔注射等量生理盐水。建模后提取肺组织总RNA,经RT-PCR扩增SP-A基因,并对其进行生物信息学分析;组织切片观察LPS作用不同时期肺组织病理学变化;q RT-PCR分析SP-A m RNA水平;蛋白印迹法检测SP-A蛋白表达。结果:棉鼠SP-A基因编码区长744bp,编码248个氨基酸,具有多个半胱氨酸保守位点、α螺旋结构和糖基化位点,与其他物种相比其核苷酸(75.4%~90.1%)和氨基酸(70.6%~87.1%)序列均具有较高同源性;在LPS诱导肺损伤模型中发现,与对照组相比,实验组肺组织病理学改变随LPS刺激时间延长而加重,具有时间依赖性;SP-A m RNA和蛋白表达水平在LPS处理24h后开始迅速增加,差异有统计学意义(P0.001和P0.01),48h时仍持续上升(P0.001和P0.01),96h时略有下降,但仍保持在较高水平,与对照组相比差异有统计学意义(P0.05和P0.01)。结论:棉鼠SP-A基因具有高度保守性;棉鼠SP-A m RNA和蛋白表达水平与肺损伤严重程度密切相关,可反应肺损伤的不同时程。
[Abstract]:OBJECTIVE: Ubiquitin-specific protease USP16 plays an important role in gene expression, cell cycle, cell self-renewal and/or senescence, and its overexpression in Down syndrome is a major promoter of neurological deficits and is closely related to neural stem cell deficits. However, the transcriptional regulation of USP16 gene is largely unknown. Methods: Primer design, plasmid construction, cell culture and transfection, detection and analysis of luciferase activity, extraction of nucleoprotein and CHIP DNA, electrophoretic mobility analysis, chromatin immunoprecipitation, cell intervention, real-time quantitative PCR were successfully amplified. A recombinant luciferase reporter gene plasmid P USP16-A was constructed by inserting the vector p GL4.10 into the 2324 BP DNA sequence of the promoter region of USP16 gene - 1856 to +468. A series of deletion plasmids were constructed on the basis of the recombinant luciferase reporter gene plasmid P USP16-A. Promoter regions - 1856 to + 468 contain several binding sites for nuclear transcription factors, such as NF-kappa B, HIF, and SP1.3. According to the binding sites predicted by transcription factors, four recombinant luciferase reporter plasmids P USP16-N1~p USP16-N4 were constructed in turn. In HEK293 cells, the luciferase activity of PMTF-p65 plasmids increased respectively after co-transfection with PMTF-p65 plasmid. Adding 2.69, 2.51, 2.24 and 2.04 times (p0.001), and statistic analysis of N1 vs. N3, N1 vs. N4 and N2vs. N4 showed that the deletion of binding sites 2 and 3 had a great influence on the role of NFkB in the up-regulation of USP16 promoter. Similar results were obtained in SH-SY5Y cells (p0.001). 4. Electrophoretic mobility analysis showed that predicted binding sites 2, 3 and 4 were found. The predicted binding sites 2 and 3 could bind to NFkBp65 in vitro, whereas sites 1 and 4 could not bind to NFkBp65. 6. LPS intervention resulted in a significant increase (1.58 times) (p0.05) in endogenous USP16 m RNA levels in SH-SY5Y cells, but no significant effect (data not shown) in HEK293 cells. In 293 cells and SH-SY5Y cells, TNFa intervention increased the level of endogenous USP16 m RNA by 1.90 times (p0.01) and 1.85 times (p0.05), respectively. Conclusion: The 5'flanking region of human USP16 gene, from -1856 to +468, has prominent promoter activity; Overexpression of NF? B p65 can up-regulate the activity of human USP16 promoter; in vivo and in vitro, there are two (-826~-815 and-468) promoters. 510~-501) NF? B p65 binding sites interact with USP16; stimulating activation of NF? B by over-expression of NF? B p65 or LPS and TNFa can up-regulate the transcription of human USP16 gene. In short, NF? B can up-regulate the transcription of human USP16 gene through two true cis-acting elements. Methods: Thirty-two cotton rats were randomly divided into four groups: three experimental groups were treated with 2 mg/kg LPS intraperitoneally for 24, 48 and 96 hours, and the control group was treated with the same amount of normal saline. SP-A gene was amplified by RT-PCR and analyzed by bioinformatics. The pathological changes of lung tissues were observed in different periods of LPS treatment. SP-A m RNA level was analyzed by Q RT-PCR. SP-A protein expression was detected by Western blotting. Results: SP-A gene coding region of cotton mouse was 744 BP long and encoded 248 amino acids. The nucleotide (75.4%~90.1%) and amino acid (70.6%~87.1%) sequences of the acid conservative sites, alpha helix structure and glycosylation sites were homologous to those of other species. In the LPS-induced lung injury model, the pathological changes of lung tissue in the experimental group were aggravated and time-dependent compared with those in the control group. The levels of AMRNA and protein expression increased rapidly after LPS treatment for 24 hours (P 0.001 and P 0.01), and continued to rise at 48 hours (P 0.001 and P 0.01), decreased slightly at 96 hours, but remained at a higher level, with significant difference compared with the control group (P 0.05 and P 0.01). The expression of SP-AMRNA and protein was closely related to the severity of lung injury and could reflect the different duration of lung injury.
【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】：R725.9
本文编号：2183304
[Abstract]:OBJECTIVE: Ubiquitin-specific protease USP16 plays an important role in gene expression, cell cycle, cell self-renewal and/or senescence, and its overexpression in Down syndrome is a major promoter of neurological deficits and is closely related to neural stem cell deficits. However, the transcriptional regulation of USP16 gene is largely unknown. Methods: Primer design, plasmid construction, cell culture and transfection, detection and analysis of luciferase activity, extraction of nucleoprotein and CHIP DNA, electrophoretic mobility analysis, chromatin immunoprecipitation, cell intervention, real-time quantitative PCR were successfully amplified. A recombinant luciferase reporter gene plasmid P USP16-A was constructed by inserting the vector p GL4.10 into the 2324 BP DNA sequence of the promoter region of USP16 gene - 1856 to +468. A series of deletion plasmids were constructed on the basis of the recombinant luciferase reporter gene plasmid P USP16-A. Promoter regions - 1856 to + 468 contain several binding sites for nuclear transcription factors, such as NF-kappa B, HIF, and SP1.3. According to the binding sites predicted by transcription factors, four recombinant luciferase reporter plasmids P USP16-N1~p USP16-N4 were constructed in turn. In HEK293 cells, the luciferase activity of PMTF-p65 plasmids increased respectively after co-transfection with PMTF-p65 plasmid. Adding 2.69, 2.51, 2.24 and 2.04 times (p0.001), and statistic analysis of N1 vs. N3, N1 vs. N4 and N2vs. N4 showed that the deletion of binding sites 2 and 3 had a great influence on the role of NFkB in the up-regulation of USP16 promoter. Similar results were obtained in SH-SY5Y cells (p0.001). 4. Electrophoretic mobility analysis showed that predicted binding sites 2, 3 and 4 were found. The predicted binding sites 2 and 3 could bind to NFkBp65 in vitro, whereas sites 1 and 4 could not bind to NFkBp65. 6. LPS intervention resulted in a significant increase (1.58 times) (p0.05) in endogenous USP16 m RNA levels in SH-SY5Y cells, but no significant effect (data not shown) in HEK293 cells. In 293 cells and SH-SY5Y cells, TNFa intervention increased the level of endogenous USP16 m RNA by 1.90 times (p0.01) and 1.85 times (p0.05), respectively. Conclusion: The 5'flanking region of human USP16 gene, from -1856 to +468, has prominent promoter activity; Overexpression of NF? B p65 can up-regulate the activity of human USP16 promoter; in vivo and in vitro, there are two (-826~-815 and-468) promoters. 510~-501) NF? B p65 binding sites interact with USP16; stimulating activation of NF? B by over-expression of NF? B p65 or LPS and TNFa can up-regulate the transcription of human USP16 gene. In short, NF? B can up-regulate the transcription of human USP16 gene through two true cis-acting elements. Methods: Thirty-two cotton rats were randomly divided into four groups: three experimental groups were treated with 2 mg/kg LPS intraperitoneally for 24, 48 and 96 hours, and the control group was treated with the same amount of normal saline. SP-A gene was amplified by RT-PCR and analyzed by bioinformatics. The pathological changes of lung tissues were observed in different periods of LPS treatment. SP-A m RNA level was analyzed by Q RT-PCR. SP-A protein expression was detected by Western blotting. Results: SP-A gene coding region of cotton mouse was 744 BP long and encoded 248 amino acids. The nucleotide (75.4%~90.1%) and amino acid (70.6%~87.1%) sequences of the acid conservative sites, alpha helix structure and glycosylation sites were homologous to those of other species. In the LPS-induced lung injury model, the pathological changes of lung tissue in the experimental group were aggravated and time-dependent compared with those in the control group. The levels of AMRNA and protein expression increased rapidly after LPS treatment for 24 hours (P 0.001 and P 0.01), and continued to rise at 48 hours (P 0.001 and P 0.01), decreased slightly at 96 hours, but remained at a higher level, with significant difference compared with the control group (P 0.05 and P 0.01). The expression of SP-AMRNA and protein was closely related to the severity of lung injury and could reflect the different duration of lung injury.
【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】：R725.9
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2 王谦,宋勇;肺表面活性蛋白A在急性肺损伤发病机制中作用的研究进展[J];中国急救医学;2005年08期
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