血管内皮细胞hCASK-Id1通路对p53表达调控的机制研究
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摘要
研究背景
多脏器功能不全综合征(multiple organ dysfunction syndrome, MODS)是严重烧伤病人死亡的重要原因之一,它的发生和发展与烧伤严重程度、烧伤后休克期渡过是否平稳以及烧伤感染有密切关系,提高烧伤的救治水平,必须重视MODS的防治。组织血液灌流不足及细胞缺血缺氧性损害是严重烧伤后脏器功能损害的病理生理基础。烧伤后多种因素可导致血管内皮细胞的激活和损伤,损伤的血管内皮细胞使毛细血管通透性增加,导致脏器间质水肿,加重脏器的缺血缺氧性损害。同时,激活的血管内皮细胞还可释放大量的细胞因子、炎症介质,直接参与炎症反应、影响血管的收缩/舒张平衡、导致血液的高凝状态、促进中性粒细胞与血管内皮细胞的黏附。黏附的中性粒细胞可释放多种炎症介质加重血管内皮细胞的损伤,使微循环功能障碍加重,进一步减少重要脏器的血供和氧气交换,最终导致多脏器功能不全综合征。可以说,血管内皮细胞活化与损伤是导致烧伤、创伤后器官功能障碍的中心环节之一。
内皮细胞的损伤早期表现为细胞活化,此时细胞释放大量细胞因子和炎症介质参与全身的炎症反应。当损伤因素得不到及时纠正时,内皮细胞损伤加重,自血管壁剥脱、死亡,内皮下胶原纤维暴露引发血管内凝血,进一步加重微循环障碍。微循环功能的恢复依赖血管内皮细胞结构及功能的修复,因此,如何加速损伤内皮细胞的修复是烧伤后脏器功能障碍防治研究的重要内容之一。
损伤修复过程涉及细胞的运动、黏附、通讯、增殖和分化,也包含细胞内外物质代谢基因的启动、调控等一系列生化和分子生物学反应。细胞的增殖反应是其中重要环节之一,许多细胞因子及信号转导通路参与这个复杂的过程,它们相互促进、制约,共同发挥作用。如果能全面揭示细胞增殖调控的信号网络,我们就可以主动参与内皮细胞损伤的修复进程,在治疗大面积烧创伤、防治内脏并发症时就可以掌握主动权。
CASK是膜相关鸟苷酸激酶同源家族(membrane-associated guanylate kinase homologs family, MAGUK)成员,主要功能是作为脚手架蛋白质(scaffold protein),利用其多蛋白结构域,结合功能相关的蛋白分子,形成多蛋白复合物,参与许多重要的细胞生理过程。既往的研究通过酵母双杂交技术、免疫共沉淀和激光共聚焦等方法,在ECV-304细胞中证实了内源性hCASK和Id1蛋白分子的相互作用;发现诱导表达hCASK的ECV-304细胞增殖缓慢,并且能诱导细胞周期相关蛋白p21、p16等表达上调,E蛋白家族成员(如E12、E47及HEB)参与了这一信号调节过程,并由此提出hCASK-Id1通路抑制ECV-304细胞增殖的信号机制:hCASK通过与Id1结合,抑制Id1对bHLH转录因子的负调控作用,促进bHLH转录因子(如E蛋白家族的E12、E47和HEB)与p21启动子E-box的结合,转录活化p21等基因,抑制细胞G1/S期转换,抑制细胞增殖。
本组研究发现,诱导表达hCASK可使p53表达升高,由于p53表达升高可通过调控细胞周期导致细胞增殖功能的抑制,因此hCASK-Id1信号通路是否可以通过调控p53的表达发挥细胞增殖抑制的功能值得进一步研究。
研究目的
本研究的重点是考察hCASK-Id1信号通路调控细胞增殖的机制,探讨hCASK-Id1通路是否可通过影响p53表达发挥其在细胞增殖调节中的调控作用,并初步探讨该通路调节p53表达的机制。
研究方法
1.采用RNA干扰技术(RNAi),应用质粒载体介导的细胞内小干扰RNA(siRNA)表达策略,将针对CASK的RNA干扰质粒pGenesil-1-hCASK转染ECV-304细胞,筛选并建立CASK抑制表达细胞株,采用MTT、流式细胞技术和Western Blot印迹技术研究其对细胞增殖功能及p53表达的影响。
2.构建Id1强制表达载体pIRES2-EGFP-Id1,转染ECV-304细胞,筛选并建立了Id1强制表达细胞株,用MTT、流式细胞技术和Western Blot印迹技术分析其对细胞增殖及p53表达的影响。
3.构建针对p53基因启动子的荧光素酶报告基因载体,并在此基础上构建含p53基因启动子不同截短片段的载体,了解在p53基因启动子上起主要作用的转录因子结合区域,为深入研究hCASK-Id1通路影响p53表达的信号机制提供信息。
结果
1. RNA干扰对于hCASK的抑制率与其作用位点直接相关,本研究中2号靶点的抑制率约70%,而1号靶点几乎没有明显的抑制效果。hCASK抑制表达可以使ECV-304细胞的生长曲线左移,促进细胞增殖,细胞周期分析也发现抑制hCASK表达可导致细胞群中G2M+S期细胞比率升高,是细胞增殖能力增强的表现。
2.通过克隆Id1全长编码基因,成功的构建了Id1真核表达载体,转染ECV-304细胞,经G418筛选得到稳定Id1强制表达的细胞亚系。在所得到的ECV-304-Id1(+)细胞株中,转染阳性细胞比率达到90%以上,为今后深入研究hCASK-Id1通路功能奠定了基础。Id1的强制表达可以使细胞增殖曲线左移,细胞群中G2M+S期细胞比率升高,促进细胞增殖。
3. Western Blot印迹实验表明hCASK抑制表达和Id1强制表达均可使p53蛋白的表达下降。
4.成功构建含p53基因启动子的荧光素酶报告基因质粒,同未转染ECV-304细胞相比,hCASK抑制表达细胞株和Id1强制表达细胞株都能使p53基因启动子报告基因质粒活性降低,提示hCASK、Id1对p53表达的调节至少部分是通过影响p53基因转录水平造成的。
5.构建了系列截短的p53基因启动子荧光素酶报告基因质粒。通过将这些质粒转染hCASK抑制表达和Id1强制表达细胞株,发现p53基因启动子3’端约150bp的序列(从-150到-1)是激活p53基因表达必须的,在这段序列中,包含了E-box、Ets、AP-2、HIF-1、HES1、Sum1等潜在的转录因子结合位点。
6.将Id1强制表达和抑制表达质粒与PGL3-p53p-luc共转染已筛选出的CASK抑制表达细胞株,检测Id1表达变化对p53基因启动子活性的影响,结果Id1抑制可使p53基因启动子活性较hCASK抑制表达细胞株升高,而Id1强制表达可使hCASK抑制表达细胞p53基因启动子的活性进一步下降。
结论:
1. hCASK抑制表达及Id1强制表达均可促进细胞增殖、下调p53蛋白表达。
2. hCASK-Id1通路对p53蛋白表达的调节至少部分是在转录水平发挥的, p53基因启动子3’端约150bp的序列(从-150到-1)是激活p53基因表达必须的,这段序列中包含了E-box、Ets、AP-2、HIF-1、HES1、Sum1等潜在的转录因子结合位点。
3. Id1的表达变化可以影响hCASK抑制表达细胞p53基因启动子活性,是Id1直接参与hCASK对p53表达调节的证据。
4. p53在细胞周期调节、细胞增殖、细胞老化及细胞损伤修复等过程中起到十分重要的作用,hCASK-Id1通路可能通过对p53表达的调控发挥广泛的的生物学效应。
Background
Multiple organ dysfunction syndrome (MODS) was one of the most important causes of burn mortality. The hypoperfusion of tissues and ischemia-hypoxia injury were the pathophysiological bases of postburn organ dysfunction. Many factors could activate and harm vascular endothelial cells (VECs), which could elevate vascular permeability and lead to organ edema and aggravate organ dysfunction. The activated vascular endothelial cells could release cytokines and inflammatory mediators contributing to inflammation, influencing vasomotion, causing hypercoagulable state and promoting neutrophil-vascular endothelial cell adhesion. The adhesive neutrophils release large amount of inflammatory mediators and in turn deteriorate vascular injury, worsen microcirculation dysfunction,decrease blood supply of important organs and lead to MODS. Thus, the activation and injury of vascular endothelial cells plays a key role in post-burn MODS.
In early stage, vascular endothelial cells were activated and produced cytokines and mediators which functioned in inflammation. While injury persisted, the VECs would shed off from vascular wall and even proceed to cell death; the exposure of collagens under endothelium could trigger DIC and worsen microcirculation disturbance. Reconstruction of vascular function relys on damage repair of vascular cells, which was very important in the rehabilitation of MODS.
There are many cellular factors and pathways involved in this process. The interfering of vascular repair could only be achieved after the thorough understanding of the complicated networks of cellular proliferation signal pathways.
hCASK (human calcium/calmodulin-dependent serine protein kinase)is a member of the membrane associated guanylate kinase (MAGUK) family. As a scaffolding protein, it participates in many physiological and pathological processes including cytoskeleton assembly, cell junction formation and adhesion, protein sub-cellular localization, signal transduction and gene expression by binding associated protein with its multi-protein domain.
Previous studies, using mammalian two-hybrid protein-protien interaction assays, immunoprecipitation and laser con-focal microscopy techniques, have shown that hCASK could interact with Id1 in vivo, compelling expression of hCASK could restrain cell proliferation and induce the expression of p21WAF1/CIP1 and p16INK4a.The regulation of p21 expression by hCASK attributes to the E-box in p21 gene promoter, which could bind the E protein family members like E12,E47and HEB.Thus a putative signal pathway involving proliferation effects of hCASK on ECV-304 cell might be: the binding of hCASK with Id1 repress the dominant negative effect of Id1 on bHLH protein,which promotes the binding of bHLH transcription factors (E12,E47 and HEB) with the E-box on p21 gene promoter. The activated p21 expression could lead to G1/S arrest and inhibit cell proliferation.
It was also found that compelling expression of hCASK could increase the expression of p53, which may also inhibit cell proliferation and involve in cell cycle and differentiation control. It was speculated that p53 might also be one of the downstream signals of the hCASK-Id1 pathway.
Aim
The study was focused on cell proliferation regulation function of hCASK-Id1 pathway and whether it could promote cell proliferation via regulating p53 expression and try to find out the mechanism of p53 regulation by hCASK-Id1 pathway.
Methods
1. Recombinant siRNA expression plasmids, pGenesil-1-hCASK, were transfected into ECV-304 cells. Stable hCASK knockdown cell strain of ECV-304 cells was obtained under sustained pressure of G418.The influences on cell proliferation and p53 expression were examined in the screened cell strain using MTT, flow cytomytry and Western Blot techniques.
2. Recombinant Id1 compelling expression plasmids were constructed using pIRES2-EGFP vector and were transfected into ECV-304 cells. Stable Id1 compelling expression cell strain of ECV-304 cells were obtained under sustained pressure of G418.The influences on cell proliferation and p53 expression were examined in screened cell strain using MTT,flow cytomytry and Western Blot techniques.
3. p53 gene promoter was subcloned into the luciferase reporter vector, PGL3-basic, as wild type PGL3-p53p-luc vector.To create truncated PGL3-p53p-luc constructs, a series of 5’truncated p53 gene promoter sequences were obtain by PCR using PGL3-p53p-luc as template. The reporter gene constructs were transiently transfected into ECV-304 cells, hCASK knock down and Id1 compelling expression cell lines. Luciferase activity of cell lysate was detected using a dual-luciferase reporter assay kit. The ratio of firefly luciferase activity to renilla luciferase activity was taken as the relative activity of the PGL3 plasmids.
Results
1. The inhibition rate of hCASK interfering was associated with sequences selected in siRNA design, the pGenesil-1-hCASK based on the No.2 target sequence exhibited 70% inhibition rate compared with that of No.1, which had nearly no effect. The inhibition of CASK expression could promote proliferation of ECV-304 cells, which was revealed by the left shift of growth curve and the increase of G2M+S cells in hCASK-knockdown ECV-304 cells.
2. Id1 compelling expression plasmid (pIRES2-EGFP-Id1)was successfully constructed and was transfected into ECV-304 cells. The stable cell strain of Id1 compelling expression , with EGFP positive rate over 90%, was achieved by G418 screening. Id1 compelling expression could lead to a left shift of cell growth curve and increase of G2M+S ratio and promote the proliferation of ECV-304 cells.
3. hCASK inhibition and Id1 compelling expression could decrease the expression of p53 protein.
4. The luciferase reporter gene vector containing p53 gene promoter was constructed successfully. The activation of p53 gene promoter was decreased under CASK inhibition and Id1 compelling expression, which implied that hCASK and Id1 transactivates the expression of p53 by stimulating the promoter activity of p53 and regulate its expression at transcription level.
5. Deletion analysis of the p53 gene promoter revealed that a 150bp sequence in the 3’end of p53 gene promoter, containing potent transcription factor binding sequences of E-box, Ets, AP-2, HIF-1, HES1 and Sum1, was required for the activation of p53 gene expression by hCASK and Id1.
6. The expression of Id1 could influence p53 gene promoter activity in hCASK knock down cells. The inhibited activation of p53 gene promoter by hCASK down-regulation was elevated at Id1 inhibition and even lower at Id1 compelling expression.
Conclusion
1. Id1 compelling expression and hCASK inhibition could promote cell proliferation and inhibit p53 expression.
2. hCASK and Id1 regulated p53 expression at transcriptional level, and a 150bp sequence in the 3’end of the promoter , containing binding sequences of E-box, Ets, AP-2, HIF-1, HES1 and Sum1, was required for its trans-activation by hCASK and Id1 stimulation.
3. Influence of Id1 expression on p53 gene promoter activity in hCASK-knockdown cells proved a direct effect of Id1 in the p53 regulation by hCASK.
4. p53 plays very important rolls in cell proliferation, cell cycle regulation and repair, as a downstream molecule of hCASK-Id1 pathway, it might endow this pathway with more physiological and pathological significances and shine a new light on the hCASK gene function .
多脏器功能不全综合征(multiple organ dysfunction syndrome, MODS)是严重烧伤病人死亡的重要原因之一,它的发生和发展与烧伤严重程度、烧伤后休克期渡过是否平稳以及烧伤感染有密切关系,提高烧伤的救治水平,必须重视MODS的防治。组织血液灌流不足及细胞缺血缺氧性损害是严重烧伤后脏器功能损害的病理生理基础。烧伤后多种因素可导致血管内皮细胞的激活和损伤,损伤的血管内皮细胞使毛细血管通透性增加,导致脏器间质水肿,加重脏器的缺血缺氧性损害。同时,激活的血管内皮细胞还可释放大量的细胞因子、炎症介质,直接参与炎症反应、影响血管的收缩/舒张平衡、导致血液的高凝状态、促进中性粒细胞与血管内皮细胞的黏附。黏附的中性粒细胞可释放多种炎症介质加重血管内皮细胞的损伤,使微循环功能障碍加重,进一步减少重要脏器的血供和氧气交换,最终导致多脏器功能不全综合征。可以说,血管内皮细胞活化与损伤是导致烧伤、创伤后器官功能障碍的中心环节之一。
内皮细胞的损伤早期表现为细胞活化,此时细胞释放大量细胞因子和炎症介质参与全身的炎症反应。当损伤因素得不到及时纠正时,内皮细胞损伤加重,自血管壁剥脱、死亡,内皮下胶原纤维暴露引发血管内凝血,进一步加重微循环障碍。微循环功能的恢复依赖血管内皮细胞结构及功能的修复,因此,如何加速损伤内皮细胞的修复是烧伤后脏器功能障碍防治研究的重要内容之一。
损伤修复过程涉及细胞的运动、黏附、通讯、增殖和分化,也包含细胞内外物质代谢基因的启动、调控等一系列生化和分子生物学反应。细胞的增殖反应是其中重要环节之一,许多细胞因子及信号转导通路参与这个复杂的过程,它们相互促进、制约,共同发挥作用。如果能全面揭示细胞增殖调控的信号网络,我们就可以主动参与内皮细胞损伤的修复进程,在治疗大面积烧创伤、防治内脏并发症时就可以掌握主动权。
CASK是膜相关鸟苷酸激酶同源家族(membrane-associated guanylate kinase homologs family, MAGUK)成员,主要功能是作为脚手架蛋白质(scaffold protein),利用其多蛋白结构域,结合功能相关的蛋白分子,形成多蛋白复合物,参与许多重要的细胞生理过程。既往的研究通过酵母双杂交技术、免疫共沉淀和激光共聚焦等方法,在ECV-304细胞中证实了内源性hCASK和Id1蛋白分子的相互作用;发现诱导表达hCASK的ECV-304细胞增殖缓慢,并且能诱导细胞周期相关蛋白p21、p16等表达上调,E蛋白家族成员(如E12、E47及HEB)参与了这一信号调节过程,并由此提出hCASK-Id1通路抑制ECV-304细胞增殖的信号机制:hCASK通过与Id1结合,抑制Id1对bHLH转录因子的负调控作用,促进bHLH转录因子(如E蛋白家族的E12、E47和HEB)与p21启动子E-box的结合,转录活化p21等基因,抑制细胞G1/S期转换,抑制细胞增殖。
本组研究发现,诱导表达hCASK可使p53表达升高,由于p53表达升高可通过调控细胞周期导致细胞增殖功能的抑制,因此hCASK-Id1信号通路是否可以通过调控p53的表达发挥细胞增殖抑制的功能值得进一步研究。
研究目的
本研究的重点是考察hCASK-Id1信号通路调控细胞增殖的机制,探讨hCASK-Id1通路是否可通过影响p53表达发挥其在细胞增殖调节中的调控作用,并初步探讨该通路调节p53表达的机制。
研究方法
1.采用RNA干扰技术(RNAi),应用质粒载体介导的细胞内小干扰RNA(siRNA)表达策略,将针对CASK的RNA干扰质粒pGenesil-1-hCASK转染ECV-304细胞,筛选并建立CASK抑制表达细胞株,采用MTT、流式细胞技术和Western Blot印迹技术研究其对细胞增殖功能及p53表达的影响。
2.构建Id1强制表达载体pIRES2-EGFP-Id1,转染ECV-304细胞,筛选并建立了Id1强制表达细胞株,用MTT、流式细胞技术和Western Blot印迹技术分析其对细胞增殖及p53表达的影响。
3.构建针对p53基因启动子的荧光素酶报告基因载体,并在此基础上构建含p53基因启动子不同截短片段的载体,了解在p53基因启动子上起主要作用的转录因子结合区域,为深入研究hCASK-Id1通路影响p53表达的信号机制提供信息。
结果
1. RNA干扰对于hCASK的抑制率与其作用位点直接相关,本研究中2号靶点的抑制率约70%,而1号靶点几乎没有明显的抑制效果。hCASK抑制表达可以使ECV-304细胞的生长曲线左移,促进细胞增殖,细胞周期分析也发现抑制hCASK表达可导致细胞群中G2M+S期细胞比率升高,是细胞增殖能力增强的表现。
2.通过克隆Id1全长编码基因,成功的构建了Id1真核表达载体,转染ECV-304细胞,经G418筛选得到稳定Id1强制表达的细胞亚系。在所得到的ECV-304-Id1(+)细胞株中,转染阳性细胞比率达到90%以上,为今后深入研究hCASK-Id1通路功能奠定了基础。Id1的强制表达可以使细胞增殖曲线左移,细胞群中G2M+S期细胞比率升高,促进细胞增殖。
3. Western Blot印迹实验表明hCASK抑制表达和Id1强制表达均可使p53蛋白的表达下降。
4.成功构建含p53基因启动子的荧光素酶报告基因质粒,同未转染ECV-304细胞相比,hCASK抑制表达细胞株和Id1强制表达细胞株都能使p53基因启动子报告基因质粒活性降低,提示hCASK、Id1对p53表达的调节至少部分是通过影响p53基因转录水平造成的。
5.构建了系列截短的p53基因启动子荧光素酶报告基因质粒。通过将这些质粒转染hCASK抑制表达和Id1强制表达细胞株,发现p53基因启动子3’端约150bp的序列(从-150到-1)是激活p53基因表达必须的,在这段序列中,包含了E-box、Ets、AP-2、HIF-1、HES1、Sum1等潜在的转录因子结合位点。
6.将Id1强制表达和抑制表达质粒与PGL3-p53p-luc共转染已筛选出的CASK抑制表达细胞株,检测Id1表达变化对p53基因启动子活性的影响,结果Id1抑制可使p53基因启动子活性较hCASK抑制表达细胞株升高,而Id1强制表达可使hCASK抑制表达细胞p53基因启动子的活性进一步下降。
结论:
1. hCASK抑制表达及Id1强制表达均可促进细胞增殖、下调p53蛋白表达。
2. hCASK-Id1通路对p53蛋白表达的调节至少部分是在转录水平发挥的, p53基因启动子3’端约150bp的序列(从-150到-1)是激活p53基因表达必须的,这段序列中包含了E-box、Ets、AP-2、HIF-1、HES1、Sum1等潜在的转录因子结合位点。
3. Id1的表达变化可以影响hCASK抑制表达细胞p53基因启动子活性,是Id1直接参与hCASK对p53表达调节的证据。
4. p53在细胞周期调节、细胞增殖、细胞老化及细胞损伤修复等过程中起到十分重要的作用,hCASK-Id1通路可能通过对p53表达的调控发挥广泛的的生物学效应。
Background
Multiple organ dysfunction syndrome (MODS) was one of the most important causes of burn mortality. The hypoperfusion of tissues and ischemia-hypoxia injury were the pathophysiological bases of postburn organ dysfunction. Many factors could activate and harm vascular endothelial cells (VECs), which could elevate vascular permeability and lead to organ edema and aggravate organ dysfunction. The activated vascular endothelial cells could release cytokines and inflammatory mediators contributing to inflammation, influencing vasomotion, causing hypercoagulable state and promoting neutrophil-vascular endothelial cell adhesion. The adhesive neutrophils release large amount of inflammatory mediators and in turn deteriorate vascular injury, worsen microcirculation dysfunction,decrease blood supply of important organs and lead to MODS. Thus, the activation and injury of vascular endothelial cells plays a key role in post-burn MODS.
In early stage, vascular endothelial cells were activated and produced cytokines and mediators which functioned in inflammation. While injury persisted, the VECs would shed off from vascular wall and even proceed to cell death; the exposure of collagens under endothelium could trigger DIC and worsen microcirculation disturbance. Reconstruction of vascular function relys on damage repair of vascular cells, which was very important in the rehabilitation of MODS.
There are many cellular factors and pathways involved in this process. The interfering of vascular repair could only be achieved after the thorough understanding of the complicated networks of cellular proliferation signal pathways.
hCASK (human calcium/calmodulin-dependent serine protein kinase)is a member of the membrane associated guanylate kinase (MAGUK) family. As a scaffolding protein, it participates in many physiological and pathological processes including cytoskeleton assembly, cell junction formation and adhesion, protein sub-cellular localization, signal transduction and gene expression by binding associated protein with its multi-protein domain.
Previous studies, using mammalian two-hybrid protein-protien interaction assays, immunoprecipitation and laser con-focal microscopy techniques, have shown that hCASK could interact with Id1 in vivo, compelling expression of hCASK could restrain cell proliferation and induce the expression of p21WAF1/CIP1 and p16INK4a.The regulation of p21 expression by hCASK attributes to the E-box in p21 gene promoter, which could bind the E protein family members like E12,E47and HEB.Thus a putative signal pathway involving proliferation effects of hCASK on ECV-304 cell might be: the binding of hCASK with Id1 repress the dominant negative effect of Id1 on bHLH protein,which promotes the binding of bHLH transcription factors (E12,E47 and HEB) with the E-box on p21 gene promoter. The activated p21 expression could lead to G1/S arrest and inhibit cell proliferation.
It was also found that compelling expression of hCASK could increase the expression of p53, which may also inhibit cell proliferation and involve in cell cycle and differentiation control. It was speculated that p53 might also be one of the downstream signals of the hCASK-Id1 pathway.
Aim
The study was focused on cell proliferation regulation function of hCASK-Id1 pathway and whether it could promote cell proliferation via regulating p53 expression and try to find out the mechanism of p53 regulation by hCASK-Id1 pathway.
Methods
1. Recombinant siRNA expression plasmids, pGenesil-1-hCASK, were transfected into ECV-304 cells. Stable hCASK knockdown cell strain of ECV-304 cells was obtained under sustained pressure of G418.The influences on cell proliferation and p53 expression were examined in the screened cell strain using MTT, flow cytomytry and Western Blot techniques.
2. Recombinant Id1 compelling expression plasmids were constructed using pIRES2-EGFP vector and were transfected into ECV-304 cells. Stable Id1 compelling expression cell strain of ECV-304 cells were obtained under sustained pressure of G418.The influences on cell proliferation and p53 expression were examined in screened cell strain using MTT,flow cytomytry and Western Blot techniques.
3. p53 gene promoter was subcloned into the luciferase reporter vector, PGL3-basic, as wild type PGL3-p53p-luc vector.To create truncated PGL3-p53p-luc constructs, a series of 5’truncated p53 gene promoter sequences were obtain by PCR using PGL3-p53p-luc as template. The reporter gene constructs were transiently transfected into ECV-304 cells, hCASK knock down and Id1 compelling expression cell lines. Luciferase activity of cell lysate was detected using a dual-luciferase reporter assay kit. The ratio of firefly luciferase activity to renilla luciferase activity was taken as the relative activity of the PGL3 plasmids.
Results
1. The inhibition rate of hCASK interfering was associated with sequences selected in siRNA design, the pGenesil-1-hCASK based on the No.2 target sequence exhibited 70% inhibition rate compared with that of No.1, which had nearly no effect. The inhibition of CASK expression could promote proliferation of ECV-304 cells, which was revealed by the left shift of growth curve and the increase of G2M+S cells in hCASK-knockdown ECV-304 cells.
2. Id1 compelling expression plasmid (pIRES2-EGFP-Id1)was successfully constructed and was transfected into ECV-304 cells. The stable cell strain of Id1 compelling expression , with EGFP positive rate over 90%, was achieved by G418 screening. Id1 compelling expression could lead to a left shift of cell growth curve and increase of G2M+S ratio and promote the proliferation of ECV-304 cells.
3. hCASK inhibition and Id1 compelling expression could decrease the expression of p53 protein.
4. The luciferase reporter gene vector containing p53 gene promoter was constructed successfully. The activation of p53 gene promoter was decreased under CASK inhibition and Id1 compelling expression, which implied that hCASK and Id1 transactivates the expression of p53 by stimulating the promoter activity of p53 and regulate its expression at transcription level.
5. Deletion analysis of the p53 gene promoter revealed that a 150bp sequence in the 3’end of p53 gene promoter, containing potent transcription factor binding sequences of E-box, Ets, AP-2, HIF-1, HES1 and Sum1, was required for the activation of p53 gene expression by hCASK and Id1.
6. The expression of Id1 could influence p53 gene promoter activity in hCASK knock down cells. The inhibited activation of p53 gene promoter by hCASK down-regulation was elevated at Id1 inhibition and even lower at Id1 compelling expression.
Conclusion
1. Id1 compelling expression and hCASK inhibition could promote cell proliferation and inhibit p53 expression.
2. hCASK and Id1 regulated p53 expression at transcriptional level, and a 150bp sequence in the 3’end of the promoter , containing binding sequences of E-box, Ets, AP-2, HIF-1, HES1 and Sum1, was required for its trans-activation by hCASK and Id1 stimulation.
3. Influence of Id1 expression on p53 gene promoter activity in hCASK-knockdown cells proved a direct effect of Id1 in the p53 regulation by hCASK.
4. p53 plays very important rolls in cell proliferation, cell cycle regulation and repair, as a downstream molecule of hCASK-Id1 pathway, it might endow this pathway with more physiological and pathological significances and shine a new light on the hCASK gene function .
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