NFκB上調(diào)USP16基因的轉(zhuǎn)錄及其分子機(jī)制研究
發(fā)布時(shí)間：2018-08-14 15:28
【摘要】：目的:泛素特異性蛋白酶USP16在基因表達(dá)、細(xì)胞周期、細(xì)胞自我更新和/或衰老過程中起重要作用,且其在唐氏綜合癥中的過度表達(dá)是神經(jīng)功能缺損的主要促進(jìn)者,與神經(jīng)干細(xì)胞缺陷密切相關(guān)。但USP16基因的轉(zhuǎn)錄調(diào)控基本上是未知的。本研究主要探討核轉(zhuǎn)錄因子NFκB對(duì)人USP16基因的轉(zhuǎn)錄調(diào)控及其分子機(jī)制。方法:引物設(shè)計(jì)、質(zhì)粒構(gòu)建、細(xì)胞培養(yǎng)與轉(zhuǎn)染、熒光素酶活性檢測及分析、核蛋白和Ch IP DNA提取、電泳遷移率分析、染色質(zhì)免疫共沉淀、細(xì)胞干預(yù)、實(shí)時(shí)定量PCR。結(jié)果:1.成功擴(kuò)增USP16基因啟動(dòng)子區(qū)-1856至+468共2324bp DNA序列,插入載體p GL4.10構(gòu)建重組熒光素酶報(bào)告基因質(zhì)粒p USP16-A,在此基礎(chǔ)上構(gòu)建一系列刪切質(zhì)粒。并通過熒光素酶活性檢測得到了一系列正性和負(fù)性調(diào)控區(qū)域。2.轉(zhuǎn)錄因子預(yù)測分析發(fā)現(xiàn)人USP16基因啟動(dòng)子區(qū)-1856至+468含有若干核轉(zhuǎn)錄因子結(jié)合位點(diǎn),如:NFκB、HIF、SP1。3.根據(jù)轉(zhuǎn)錄因子預(yù)測結(jié)果顯示的結(jié)合位點(diǎn),依次構(gòu)建4個(gè)重組熒光素酶報(bào)告基因質(zhì)粒p USP16-N1~p USP16-N4。在HEK293細(xì)胞中,與空載PMTF相比,共轉(zhuǎn)PMTF-p65質(zhì)粒后熒光素酶活性分別增加2.69,2.51,2.24和2.04倍(p0.001),并且對(duì)N1 vs.N3,N1 vs.N4以及N2vs.N4統(tǒng)計(jì)發(fā)現(xiàn),結(jié)合位點(diǎn)2和3的缺失對(duì)NFkB在上調(diào)USP16基因啟動(dòng)子中的作用有很大的影響。在SH-SY5Y細(xì)胞中,得到類似的結(jié)果(p0.001)。4.電泳遷移率分析,我們發(fā)現(xiàn)預(yù)測到的結(jié)合位點(diǎn)2,3和4在體外可與NFκB p65結(jié)合;而染色質(zhì)免疫共沉淀實(shí)驗(yàn)表明,預(yù)測到的結(jié)合位點(diǎn)2和3可與NFkBp65結(jié)合,而位點(diǎn)1和4不能結(jié)合。6.LPS干預(yù)導(dǎo)致SH-SY5Y細(xì)胞內(nèi)源性USP16 m RNA水平顯著增加(1.58倍)(p0.05),但在HEK293細(xì)胞中無顯著影響(數(shù)據(jù)未顯示)。然而,在HEK293細(xì)胞和SH-SY5Y細(xì)胞中,TNFa干預(yù)分別使內(nèi)源性USP16 m RNA的水平增加了1.90倍(p0.01)和1.85倍(p0.05)。結(jié)論:人USP16基因的5'側(cè)翼區(qū),從-1856至+468,具有顯著啟動(dòng)子活性;過表達(dá)NF?B p65可以上調(diào)人USP16啟動(dòng)子活性;無論在體內(nèi)或體外,有兩個(gè)(-826~-815和-510~-501)NF?B p65結(jié)合位點(diǎn)與均可與USP16相互作用;過表達(dá)NF?Bp65或LPS和TNFα的刺激活化NF?B均能上調(diào)人類USP16基因的轉(zhuǎn)錄�？傊�,NF?B可通過兩個(gè)真正的順式作用元件上調(diào)人USP16基因的轉(zhuǎn)錄。目的:分析棉鼠肺表面活性相關(guān)蛋白A(SP-A)基因序列結(jié)構(gòu)和生物信息學(xué)特點(diǎn),觀察棉鼠肺損傷模型中SP-A m RNA和蛋白表達(dá)水平,初探SP-A表達(dá)規(guī)律與肺損傷的相關(guān)性。方法:將32只棉鼠隨機(jī)均分為4組:3個(gè)實(shí)驗(yàn)組棉鼠分別腹腔注射2 mg/kg LPS處理24、48和96 h,對(duì)照組腹腔注射等量生理鹽水。建模后提取肺組織總RNA,經(jīng)RT-PCR擴(kuò)增SP-A基因,并對(duì)其進(jìn)行生物信息學(xué)分析;組織切片觀察LPS作用不同時(shí)期肺組織病理學(xué)變化;q RT-PCR分析SP-A m RNA水平;蛋白印跡法檢測SP-A蛋白表達(dá)。結(jié)果:棉鼠SP-A基因編碼區(qū)長744bp,編碼248個(gè)氨基酸,具有多個(gè)半胱氨酸保守位點(diǎn)、α螺旋結(jié)構(gòu)和糖基化位點(diǎn),與其他物種相比其核苷酸(75.4%~90.1%)和氨基酸(70.6%~87.1%)序列均具有較高同源性;在LPS誘導(dǎo)肺損傷模型中發(fā)現(xiàn),與對(duì)照組相比,實(shí)驗(yàn)組肺組織病理學(xué)改變隨LPS刺激時(shí)間延長而加重,具有時(shí)間依賴性;SP-A m RNA和蛋白表達(dá)水平在LPS處理24h后開始迅速增加,差異有統(tǒng)計(jì)學(xué)意義(P0.001和P0.01),48h時(shí)仍持續(xù)上升(P0.001和P0.01),96h時(shí)略有下降,但仍保持在較高水平,與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05和P0.01)。結(jié)論:棉鼠SP-A基因具有高度保守性;棉鼠SP-A m RNA和蛋白表達(dá)水平與肺損傷嚴(yán)重程度密切相關(guān),可反應(yīng)肺損傷的不同時(shí)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R725.9
本文編號(hào)：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R725.9
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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2 王謙,宋勇;肺表面活性蛋白A在急性肺損傷發(fā)病機(jī)制中作用的研究進(jìn)展[J];中國急救醫(yī)學(xué);2005年08期
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