水通道蛋白1基因对白血病K562细胞红系分化的影响
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摘要
研究背景和目的
白血病是世界范围内常见的血液系统恶性肿瘤,是一种细胞分化障碍性疾病,其核心问题是白血病细胞失去进一步分化为成熟细胞的能力而停滞在细胞发育的不同阶段。人红白血病细胞株K562最初源于一位慢性髓性白血病患者的胸膜渗出液,K562细胞携有Ph染色体t(9；22)(q34；q21),9号染色体abl基因与22号染色体bcr基因由于染色体异常重组形成bcr/abl融合基因,翻译产生P210蛋白,该蛋白是致病的主要原因,其具有增强酪氨酸激酶的活性。K562细胞在体外可被诱导向红系分化,表现为抑制该细胞的恶性增殖能力,以及具有相应的红系成熟标志。以β-珠蛋白是否表达来判断其是否真正的向红系细胞方向分化。研究证明,小鼠红白血病细胞(MEL)、K562细胞、人早幼粒白血病HL-60细胞等造血系统肿瘤细胞,都可以通过诱导分化剂在体外促进其向终末方向分化,并表达终末分化产物,从而使这些肿瘤细胞失去恶性增殖的能力。因此利用诱导分化物使癌细胞的增殖和基因的活动趋于正常,使基因表达亦趋于正常,为肿瘤的治疗领域提供了新策略。
目前诱导分化疗法是肿瘤研究的新领域,尤其是白血病的治疗,使恶性肿瘤细胞能够在体内外分化诱导剂的作用下向正常细胞方向分化逆转。K562细胞是体外研究肿瘤细胞恶性表达调控的极好模型,因为他能够向多种血细胞系方向分化。因此分化策略主要是将K562细胞向红系方向诱导分化。其中,全反式维甲酸诱导分化治疗已成为治疗急性早幼粒细胞白血病(Acute promyelocytic leukemia, APL)的首选手段。随着对白血病诱导分化机制的深入研究和动物实验的开展,诱导分化疗法将成为白血病临床治疗的主攻方向,但目前仍有诸多问题阻碍着其应用：1、诱导分化的具体机制尚未明确；2、诱导分化剂本身毒副作用大。因此白血病诱导分化相关基因或治疗靶点的发现将是解决这些问题的突破点。
水通道蛋白(Aquaporin, AQP)是一组小分子跨膜蛋白家族,参与水的快速跨膜转运。对于水通道蛋白的认识最开始主要是基于AQP1的结构分析,AQP1是第一个被发现的AQPs蛋白,Agre因此获得了2003年的诺贝尔化学奖,之后陆续发现哺乳动物水通道家族有13个成员(AQPO-AQP12),其基因结构、基因表达调控、染色体定位、蛋白结构、组织分布和生理功能均得到了较为深入的研究。AQP1能使红细胞适应环境巾血浆的渗透压变化,通过调节水的运输使红细胞膨胀或皱缩。促红细胞生成素可使红细胞容积瞬间增高,胞膜AQP1密度也大大增高。研究发现氧气除了在红细胞膜上自由扩散,也能够在AQP1运输和扩散。因此AQP1既是红细胞胞内胞外水进出交换的通道,同样也是红细胞运输和交换气体的通道,AQP1不仅是水通道蛋白,还是氧气氧分子进出细胞的通道。
大量研究显示AQP1的功能或表达异常与多种肿瘤的发生关系密切。用丁酸盐诱导HEL细胞向红系分化后,AQP1的转录表达明显增加,在人红白血病K562细胞中也出现了同样的结果。重要的是在AQP1启动子巾包含CACCC元件,该元件具有红细胞特异性,并且AQP1启动子的活性在K562和HEL细胞巾是非红系细胞的24倍。无论是9-顺式维甲酸(9-cis retinoic acid)还是全反式维甲酸(all-transretinoic acid), AQP1启动子中含有的维甲酸(retinoic acid, RA)反应元件可通过与RA结合诱导人红白血病细胞细胞表达AQP1,并且随着RA剂量增加,AQP1的表达明显增加,维甲酸诱导的AQP1蛋白主要在细胞质膜上表达。另外,还有研究发现除了RA,二甲亚砜和皮质类固醇亦可诱导小鼠MEL细胞表达AQP1。以上研究可以看出多种促红系分化的药物都可增强AQP1的表达,说明红白血病细胞的红系分化调控和AQP1的表达可能有着密切联系。
目前对于AQP1基因的功能研究还多局限于与液体吸收或分泌有关的上皮细胞及可能协同水跨细胞转运的内皮细胞中。AQP1高表达在红白血病细胞向红系分化过程的作用如何及其具体机制,有待进一步研究。因此在以前的研究基础上,结合国内外文献资料,首次将AQP1作为靶基因,构建AQP1基因的真核表达重组载体,在K562细胞中稳定表达,同时采用逆转录病毒载体shRNA表达系统对K562细胞中AQP1基因表达进行长效抑制,研究AQP1的高表达和表达抑制对K562细胞向红系分化过程及生理生化各项指标的影响,深入研究AQP1基因在细胞诱导分化中的功能,以期进一步明确AQP1基因与白血病发生机制之间的关系,为临床诱导分化治疗白血病提供新的治疗靶点。
目的
确定AQP1与K562细胞红系分化的相关性。构建稳定表达AQP1基因的K562-AQP1细胞系以及稳定抑制AQP1基因表达的K562-shAQP1细胞系。比较三种不同细胞系K562、K562-AQP1、K562-shAQP1在肿瘤细胞诱导分化、生长增殖等方面的差异,明确AQP1基因的在红系分化中的新功能。应用生物信息学方法比较K562细胞与AQP1表达增高或降低的K562细胞中大量相关基因表达的变化,从而发现一组疾病相关基因在特定条件下的相互作用,为临床诱导分化治疗白血病提供一组新的治疗靶点。
方法
1.使用RA处理K562细胞,收集24h、48h及72h的细胞,同时设立未加RA的K562细胞为对照组。采用RA诱导K562细胞向红系分化之后,通过Real-time PCR法检测红系指标γ-globin的表达变化；分光光度法检测血红蛋白(hemoglobin)含量,研究RA对K562细胞红系分化的影响。为了研究APQ1基因与K562细胞红系分化的关联,使用Real-time PCR法、蛋白质印迹法检测RA诱导后K562细胞AQP1mRNA和蛋白表达水平。
2.以人脑cDNA文库为模板,通过PCR扩增出AQP1基因的编码序列,构建pBABE-puro-AQP1真核表达载体；转染K562细胞,筛选建立稳定过表达AQP1基因的K562细胞株(K562-AQP1); real-time PCR法、细胞免疫荧光染色法及蛋白质印迹法检测AQP1转录和蛋白表达水平。通过MTT法检测细胞生长增殖、real-time PCR法检测红系指标γ-globin表达和分光光度法检测血红蛋白含量,研究AQP1过表达对K562细胞红系分化、增殖的影响。
3.设计特异AQP1shRNA序列,构建pSUPER-retro-puro重组质粒,转染K562细胞,筛选建立稳定抑制AQP1基因表达的K562细胞株(K562-shAQP1); real-time PCR法、细胞免疫荧光染色法及蛋白质印迹法检测AQP1转录和蛋白表达水平。通过Real-time PCR和紫外分光光度计法比较K562-shAQP1细胞株与对照组细胞在RA诱导后红系指标γ-globin和hemoglobin的表达,研究AQP1在RA诱导K562细胞红系分化中的作用,能否阻断RA诱导的K562细胞红系分化。
4.比较三种不同细胞系K562、K562-AQP1、K562-shAQP1在肿瘤细胞诱导分化、生长增殖等方面的差异,明确AQP1基因的在红系分化中的新功能。
5.利用K562细胞和AQP1过表达的K562细胞所生成的全基因组表达谱芯片,获取共同表达差异基因,然后应用GO、Pathway等生物信息学方法比较相关基因表达的变化,从而发现一组白血病相关基因在特定条件下的相互作用,为临床诱导分化治疗白血病提供一组新的治疗靶点。
6.本文统计分析采用SPSS13.0。计量资料的统计描述用均数±标准差的形式给出,两组比较采用两独立样本t检验,多组比较的情况下用one-way ANOVA,重复测量资料采用重复资料的方差分析；两两之间的比较,方差齐时用LSD法,不齐则用Dunnett's T3检验法。检验水准设为P<0.05。
结果
1.RA体外诱导K562细胞红系分化
Real-time PCR检测RA处理K562细胞不同时间后红系分化指标γ-globin mRNA的表达增加,48h时表达增加最显著(可达未加RA处理组的10.05倍),且差异具有统计学意义(F=20.191,P=0.008)。紫外分光光度计法进行K562细胞hemoglobin含量的测定结果显示,RA诱导后的K562细胞hemoglobin含量较对照K562细胞增加,且随RA作用时间延长含量递增,差异具有统计学意义(F=651.197,P<0.001)。
2.AQP1基因表达与K562细胞红系分化的相关性
Real-time PCR检测RA诱导K562细胞红系分化后AQP1基因mRNA的表达较对照组均有所增加(24h、48h、72h与对照组相比分别为8.23、12.81、12.57倍),不同水平间差异具有统计学意义(F=18.834,P=0.009); Western blot结果也显示AQP1蛋白表达量也随RA作用时间延长而递增。
3.AQP1基因稳定过表达与K562细胞红系分化和增殖
成功构建AQP1基因的真核表达载体之后,使用免疫组化法鉴定K562细胞中AQP1的表达。AQP1经一抗孵育后用goat anti-rabbit IgG-R荧光二抗显色为红色,DAPI进行核复染显色为蓝色,将抗原显色部分与核染色部分叠加在一起观察抗原表达的变化。结果显示K562-AQP1细胞中红色荧光较对照组K562细胞增强,表明pBaBb-puro-AQP1逆转录病毒载体感染K562细胞后,AQP1蛋白在细胞中的表达增多。
Real-time PCR和Western blot检测K562-AQP1细胞中AQP1mRNA和蛋白表达情况。Real-time PCR检测结果显示K562-AQP1细胞中AQP1mRNA的表达较对照组增加,较对照细胞增高700倍以上,且差异具有统计学意义(t=24.845,P=0.002)；Western blot结果显示K562-AQP1细胞中AQP1蛋白表达量也有所增加。
Real-time PCR结果显示,在稳定转染AQP1基因真核表达载体的K562-AQP1细胞中γ-globin mRNA的表达较对照K562细胞有所增加(7.369：1),且差异具有统计学意义(t=24.965,P=0.001)。紫外分光光度计法的测定结果显示,AQP1稳定过表达细胞株血红蛋白含量较对照K562细胞有所增加(增加49%),差异具有统计学意义(t=26.561,P<0.001)。
MTT法检测K562-AQP1细胞的生长曲线结果显示,AQP1过表达组(K562-AQP1)较对照组(K562)细胞的生长速度降低(F=171.36,P=0.001),说明AQP1在促进K562细胞向红系分化的同时对细胞增殖有抑制作用。
4.AQP1基因表达抑制与K562细胞诱导分化
筛选建立稳定抑制AQP1基因表达的K562细胞株(K562-shAQP1)之后,细胞免疫荧光染色法检测AQP1在K562细胞巾的表达改变结果显示,AQP1荧光显色为红色,定位在细胞膜上,K562-shAQP1细胞膜上红色荧光较对照组K562细胞有所减少,表明pSUPERretro-puro-shAQP1逆转录病毒载体感染K562细胞后,AQP1蛋白在细胞中的表达受到抑制。
用Real-time PCR法检测K562-shAQP1细胞中AQP1基因mRNA的表达结果显示, K562-shAQP1细胞中AQP1mRNA的表达较对照组有所下降,表达量约为对照组的27%,差异具有统计学意义(t=-34.138,P=0.001)；Western blot结果显示K562-shAQP1细胞中AQP1蛋白表达量也有所降低,Bandleade软件分析内对照GAPDH与目的蛋白条带灰度比,抑制效率达89.8%。
通过比较K562-shAQP1细胞株与对照组细胞在RA诱导后红系指标γ-globin和hemoglobin的表达,研究AQP1在RA诱导K562细胞红系分化中的作用,能否阻断RA诱导的K562细胞红系分化。用Real-time PCR法检测γ-globin mRNA表达结果显示,RA作用于AQP1表达下调的K562-shAQP1细胞系24h、48h、72h的γ-globin mRNA表达量为2.348、2.926、2.836,与RA处理的对照K562细胞组(6.709、9.561、8.284)相比有所下降,差异具有统计学意义(F=53.062,P<0.001)。紫外分光光度计法的测定结果显示,RA处理的K562-shAQP1细胞系hemoglobin含量与RA处理的对照K562细胞比较也有有所降低,差异具有统计学意义(F=1.736,P=0.177)。当RA诱导K562细胞红系分化时,下调AQP1表达可抑制RA的诱导分化作用。
5.应用全基因组表达谱芯片分析AQP1基因对K562细胞红系诱导分化的作用机制
本研究重点分析K562组与K562-AQP1组之间的差异表达基因,其中363个基因表达上调,421个基因表达下调。这些差异表达基因的GO分类有317个,分析发现差异基因的生物学途径主要与氧气运输、程序性凋亡以及红系分化等相关；分子功能则与氧气运输活性相关,证实AQP1基因的过表达确实能促进红系分化并且在该进程中发生细胞凋亡；差异表达基因的细胞组成多为血红蛋白复合物,证实AQP1过表达能显著增加K562细胞血红蛋白的表达,表现出红系分化的显著特征。
将K562组和K562-AQP1组共同上调和下调的基因经过整理筛选,上传至STRING分析这两组的差异基因所编码蛋白质的相互关系,结果发现有24个基因编码的蛋白质的相互作用主要集中在核心位置。将上述24个基因上传至pSTIING工具,进一步分析这些基因及其转录因子的网络拓扑结构,其中7个差异基因仍然处在重要结点位置,分别是：POLR2L、SOS1、 CDKN1A、PIN1、NCOA3、ATF3、FOS。再根据参与重要分子通路的基因、以及在重要GO term有所富集的基因使用pSTIING工具进行网络图谱的构建,有5个基因位于重要结点位置,分别是：TRIB3、CDKN1A、DDIT3、 ATF3、ALCAM。最终将这12个基因将进行下一步的文献挖掘,以探讨与白血病以及红系分化之间的联系。
FACTA文本挖掘工具对10个差异表达基因进行进一步的分析比较,结果发现,其中大部分都与白血病相关,并且大多数基因存在大量的相关文献报道,为白血病红系分化分子靶标的筛选提供明确有力的支持。相关文献挖掘的结果发现FOS、ATF3在白血病中高表达,促进白血病细胞的增殖；SOS1、NCOA3这2个基因经文献查阅跟白血病没有直接关系,但是通过信号通路或者与相关基因融合而参与白血病的发生,在白血病的治疗以及预后起到负面作用；并且这4个基因在本研究巾均发生了下调。另外,与红系分化相关的血红蛋白HBD、HBE1、HBG2、HBQ1在K562-AQP1细胞中均差异表达上调,其中HBD基因的差异表达最显著,因此推测AQP1与血红蛋白表达关系密切,并且在红系分化过程巾存在着共表达作用。
预计后期再加以实验验证。下一步再加上AQP1表达抑制的表达谱芯片进行共同研究,从巾再筛选出特异性和实用性更强的基因,作为白血病临床诊断的分子靶标候选基因。还要确定AQP1参与的信号通路以及如何发挥诱导血红蛋白表达的功能。
结论
本研究通过基因增加、基因抑制、Real-time PCR、Western Blot等分了生物学方法,采用Y-珠蛋白、血红蛋白表达,细胞增殖曲线作为红系分化检测指标,对水通道蛋白1(AQP1基因)在K562细胞红系分化巾的作用进行了研究,取得以下研究结果：
1.维甲酸(RA)诱导K562细胞红系分化的过程巾,红系分化指标Y-珠蛋白、血红蛋白表达均明显增加：并且AQP1mRNA及蛋白的表达显著增加。确定了AQP1表达与K562细胞红系分化的相关性；
2.通过构建pBABE-puro-AQP1真核表达载体确定了AQP1过表达可以显著促进K562细胞向红系分化,同时抑制细胞增殖。
3.AQP1基因的表达抑制可部分阻断RA诱导的红系分化作用,证实AQP1基因在RA诱导K562细胞红系分化过程中发挥重要作用,提示AQP1基因可能作为治疗红白血病新的靶基因。
4.以K562-AQP1和K562为对象,利用全基因组表达谱芯片获取差异表达基因进行分析。选取8个与AQP1基因相关的基因,分别是：差异表达下调基因FOS、ATF3、SOS1、NCOA3,以及差异表达上调基因HBD、 HBE1、HBG2、HBQ1。推测这些基因与红系分化相关,预计后期再加以实验验证并进行深入研究。
本研究的创新之处
1.研究AQP1的表达变化在红白血病细胞红系诱导分化中的作用及机制,国内外未见报道,有明显创新。
2.本课题从基因增强、基因抑制正反两个方面研究AQP1的作用,采用高特异性的RNA干扰技术,方法先进,可望发现AQP1在红白血病诱导分化中的新机制。
3.应用全基因组表达谱芯片和生物信息学工具筛选到若干红系分化相关基因,下一步拟针对这些基因再加以实验验证,进一步研究AQP1基因对K562细胞红系诱导分化的作用机制,从而发现一组疾病相关基因作为药物筛选靶标。
BACKGROUND
Leukemia is a worldwide malignant tumor in blood system, which is caused by cell differentiation disorder, its core nosogenesis is leukemia cells lose the ability to further differentiate stagnation in the cell growth phase. K562cell is the human erythroleukemia cell which is cultivated from the pleural effusion of chronic myelogenous leukemia patients, its t(9;22)(q34;qll) chromosomal recombinant to be bcr/abl fusion gene, which translates the P210protein increases the tyrosine kinase activity is the main pathogenic factor. K562cell is in the low state of differentiation, rapid rate of proliferation and high degree of malignant. But can be induced to the erythroid by inductive substance in vitro, first performance of malignant proliferation is restrained, and corresponding differentiation mature symbol of blood cells style. Erythroid maturation product β-globin is the key indicator to estimate the direction of erythroid differentiation. Using the differentiation material to change the abnormal state of gene expression, guiding the cancer cell that proliferation is out of control and gene ability is abnormal in the normal direction, it provides a new strategy of cancer therapy. A large number of experiments have proved, use the differentiation inducer most of the hematopoietic system tumor cells can differentiate to the terminal direction and express terminal differentiation product, so that tumor cell lose the ability of maliganant proliferation, such as murine erythroleukemia cell (MEL), human erythroleukemia K562cell, human promyelocytic leukemia HL-60cell.
At present, induction differentiation therapy is the new field of tumor therapeutics, especially leukemia. K.562is an erythroleukemia cell line, which is situated in the common stage of erythroid lineages of the hematopoietic stem cell differentiation and its normally following differentiation is blockaded. But K562cells can be induced to erythroid differentiation by agents such as all-trans retinoic acid. As further studies of leukemia cell differentiation mechanism and carry out of animal experiments, induction differentiation therapy become the main attack direction of the clinical treatment of leukemia, but there are still some questions hamper its application:①The concrete mechanism of differentiation is not clear;②The side effects of differentiation inducer. Therefore the breakthrough point to solve these problems is to find out key genes or therapeutic targets related to leukemia differentiation.
Aquaporins (AQPs) are water channel proteins that facilitate transcellular water movements. For the understanding of the water channel protein is began to the structure analysis of AQP1, Agre won the2003Nobel Prize in chemistry because of AQP1that was isolated initially from human erythrocytes, and from then13members of AQPs (AQP0-AQP12) were identified one after another. We made a thorough study about the gene structure, gene expression regulation, chromosome location, protein structure, tissue distribution and physiological function of AQPs. AQP1making red blood cells accommodate to plasma osmotic pressure by adjusting the transport of water to make red blood cells to expand or shrink. Erythropoietin can instantly increase red blood cell volume, AQP1density in cell membrane also increased greatly. Study found that oxygen not only free spread on the red cell membrane, but also can transport and diffusion in AQP1. So AQP1is not only a water channel protein, but also channel for oxygen or oxygen molecules in and out of the cell.
A lot of studies have shown that the function of AQP1is close related to abnormal expression in a variety of cancers. The regulation of AQP1gene especially has been addressed in the erythrocyte. Some scientist's study has demonstrated that the AQP1expression in human erythroleukemia HEL and K562cells was induced by sodium butyrate that is a strong agent for erythroid differentiation. In addition, the AQP1promoter contained CACCC element, the research found that AQP1promoter activity was more than24-fold higher in HEL and K562cells than in nonerythroid (Hela) cells. Retinoic acid can specially induce expression of AQP1in HEL cell by combined with RA reaction component. In the present study we demonstrate that RAs, ATRA, and9-cis-RA, strongly induce the AQP1mRNA and protein expression in human erythroleukemia in dose dependent manner. AQP1protein induced by retinoic acid is mainly expressed in cytoplasm membrane. In addition, other studies have found that dimethyl sulfoxide and corticosteroids can induce expression of AQP1protein in murine erythroleukemia MEL cell. The researches above demonstrate that many kinds of drugs promote erythroid differentiation can significantly enhance the expression of AQP1, illustrate that AQP1play an important role in the regulation of erythroleukemia cells.
Function research of AQP1gene is limited to epithelial cells related to fluid absorption or secretion and possible endothelial cells that synergy water transport across a cell membrane. The role and specific mechanism of high expression of AQP1in the procedure of erythroid differentiation in leukemia cells need further study. Therefore on the basis of previous studies, and combined with domestic and foreign literature, for the first time make AQP1as target gene to build AQP1gene eukaryotic expression recombinant vectors, and then make it stabled express in K562cells, on the other hand using a retro virus vector shRNA expression system to build long-term restrain AQP1gene expression in K562cells, So can illustrate the high expression and inhibition of AQP1expression in K562cells influence the physiological and biochemical indicators in the process of erythroid differentiation, Further research on the function of AQP1gene in erythroid differentiation. In order to make clear the mechanism of the relationship between gene AQP1and leukemia, provide new therapeutic targets for clinical differentiation treatment of leukemia.
OBJECTIVE
Confirm the correlation between AQP1and erythroid differentiation of K562cells. Construction stable cell line K562-AQP1over expression of AQP1gene and establish the K562-shAQP1cell line with stable down-regulation of AQP1. Compare the difference of three different cell lines K562, K562-AQP1, K562-shAQP1in tumor induced differentiation, growth and proliferation, clarify new features of AQP1in erythroid differentiation.
METHODS
1. Collected24h,48h and72h cells after retinoic acid (RA) treatment, and at the same time collected K562cells without RA treatment as control group. After using RA induced K562to erythroid differentiation, detect expression changes of red blood indicators y-globin by real-time PCR method; detect content of hemoglobin by spectrophotometric method to study the impact of RA to erythroid differentiation. In order to illustrate the relationship between AQPlgene and erythroid differentiation of K562, examined the AQP1mRNA and protein before and after RA treatment detected by real-time PCR and western blot.
2. Constructed pBABE-puro-AQP1eukaryotic expression vector by AQP1gene with the human brain cDNA library as a template, and then transfection k562cell established stable cell line K562-AQP1over expression of AQP1gene; AQP1mRNA and protein expression were detected by real-time PCR, Cell immunofluorescence staining method, and western blot. The cell growth and proliferation was determined by MTT method, red blood indicators γ-globin detected by real-time PCR method; and content of hemoglobin detect by spectrophotometric method, these studies could illustrate the correlation of AQP1expression and erythrocytic differentiation and proliferation in K562cells.
3. The retroviral expression vector of AQP1small interfering RNA (pSUPER-retro-puro-shAQP1) was constructed and transfected into K562cells to establish the K562cells (K562-shAQPl) with stable down-regulation of AQP1; AQP1mRNA and protein expression were detected by real-time PCR, Cell immunofluorescence staining method, and western blot. Compared to the control K562cells, y-globin and hemoglobin expression in K562-shAQPl cells induced by RA were detected by real-time PCR and spectrophotometric method, to study the role of down-regulated expression of AQP1in erythrocytic differentiation of K562cells.
4. Compare the difference of three different cell lines K562, K562-AQP1, K562-shAQPl in tumor induced differentiation, growth and proliferation, clarify new features of AQP1in erythroid differentiation.
5. Use genome-wide expression profile chip of K562and K562-AQP1cells, abtain common differential genes, and compared the changes of relevant genes by GO, Pathway methods in order to discorved interaction of these genes, provides a set of new therapeutic targets for clinical leukemia treatment.
6. The statistical method in this article using SPSS13.0software for data analysis. Test samples by real-time PCR, ultraviolet spectrophotometry, and test cell growth curve determined by MTT method, the statistical method of all these experiment use ANOVA for repeated measurements. Firstly conduct homogeneity test for variance, secondly conduct independent-samples T test between two treatment groups or one way ANOVA between multiple treatment groups, at last, using LSD method between multiple comparisons if the differences are statistically significant; when there is constant in control group, use single sample t-test, if more than two groups, use independent-samples T test or ANOVA method after eliminate the control group, finally compare with control group respectively. All data is expressed by x±s, With P<0.05for the difference is statistically significant.
RESULTS
1. RA induction erythroid differentiation of K562cells in vitro
After using RA induced K.562to erythroid differentiation, expression of red blood indicator γ-globin was significantly increased detect by real-time PCR method, especially at48h (can be up to10.05times than that without RA treatment group), the difference is statistically significant (F=20.191, P=0.008). The result of hemoglobin level in K562cells which detected by spectrophotometric was showed, Hemoglobin level in K562cells induced by RA significantly increased compared with the K562-control group, And the content increasing with the duration of RA treatment, the difference is statistically significant (F=651.197, P<0.001)
2. The correlation between AQP1and erythroid differentiation of K562cells
After using RA induced K562to erythroid differentiation, the expression of AQP1mRNA detected by real-time PCR was significantly increased compared to the control group (The expression of AQP1mRNA at24h,48h and72h was8.23、12.81、12.57times than control group, respectively), the difference is statistically significant(F=18.834, P=0.009); Expression of AQP1protein also increased with the duration of RA treatment detected by Western Blot.
3. AQP1gene over-expressed compacted differentiation of K562cells.
After constructed eukaryotic expression vector of AQP1gene successfully, evaluated the expression of AQP1in K562cells by immunohistochemistry staining. Made AQP1incubated by primary antibody and then fluorescent color in red after use goat anti-rabbit IgG-R second antibody, nuclear color for blue counterstained by DAPI, eventually antigen color parts and nuclear staining are stacked up together then observe the changes of antigen expression. The result of immunohistochemistry staining was observed under a fluorescence microscope (X400), AQP1was located with red fluorescence which is stronger than control group, indicated that AQP1protein expression in K562-AQP1was obviously increased than that of control.
Expression of AQP1mRNA and protein in K562-AQP1cells detected by real-time PCR and western blot. The result of real-time PCR showed that expression of AQP1mRNA in K562-AQP1cells increased more than700times compared with the K562-control group; Western Blot showed that expression of AQP1protein in K562-AQP1cells also increased compared with the K562-control group, the difference is statistically significant (t=24.845, P=0.002).
The result of real-time PCR displayed, expression of γ globulin mRNA in K562-AQP1cells significantly increased compared with the K562-control group (increased7.369times), the difference is statistically significant (^=24.965, P=0.001); The result of ultraviolet spectrophotometry displayed, hemoglobin level in K562-AQP1cells also increased compared with the K562-control group, the difference is statistically significant (t=26.561, P<0.001). So the content of γ-globin and haemoglobin were significantly increased in AQP1over-expression cells line (K562-AQP1).
While growth curves of K562-AQP1cells detected by MTT method revealed, the cell growth rate of K562-AQP1group was markedly reduced compared to K562control cells (F=171.36, P=0.001), indicated the over expression of AQP1promoted erythroid differentiation of K562cells and at the same time inhibited the cell proliferation significantly.
4. AQP1gene down-regulated compacted differentiation of K562cells.
After established the K562cells (K562-shAQP1) with stable down-regulation of AQP1, change of AQP1expression in K562cell was detected by immunohistochemistry staining, AQP1located on the cell membrane and color in red fluorescence; the nuclei of K562cells were visualised by DAPI counter-staining, the figure showed red fluorescence in cell membrane of K562-shAQP1was less than K.562control group, indicated that expression of AQP1protein was suppressed.
Expression of AQP1mRNA in K562-shAQP1cells detected by real-time PCR showed that expression of AQP1mRNA in K562-shAQP1cells descended to27%of K562-control group, the difference is statistically significant (f=-34.138, P=0.001); Expression of AQP1protein in K562-shAQP1cells detected by Western Blot showed that expression of AQP1protein in K562-AQP1cells also descended compared with the K562-control group, use bandleade software analysis band gray ratio between housekeeping gene GAPDH and purpose protein, we concluded that inhibition efficiency was up to89.8%.
Whether down-regulated AQP1can blocking erythroid differentiation through compared the expression of Y globulin and hemoglobin in K562-sh562before and after induced by RA. The result of real-time PCR displayed, expression of Y globulin mRNA in K562-shAQP1cells was2.348,2.926,2.836after induced by RA at24h,48h,72h, respectively, which is significantly decreased compared with the K562-RA group (6.709、9.561、8.284), the difference is statistically significant (F=53.062, P<0.001). The result of ultraviolet spectrophotometry displayed, hemoglobin level in K562-shAQPl cells also significantly decreased compared with the K562-control group, the difference is statistically significant (F=1.736, P=0.177). Down-regulated expression of AQP1can partially blocked the induction effect of RA in the procedure of erythroid differentiation.
5. Application of expression profile chip analysis the mechanism of erythroid differentiation of K562cells influenced by AQPl
This study focused on the differential expressed genes between K562and K562-AQP1groups, there are363genes up-regulate, and421genes down-regulate. And317GO Terms of the differential expressed genes, the biological process mainly concern about oxygen transport, apoptosis, erythroid differentiation, et al; molecular function is relevant to oxygen transport activity, confirmed that the over-expression of AQP1induce erythroid differentiation and also lead apoptosis during this process; the cellular component of differential expressed genes mainly is hemoglobin complex, confirmed that the over-expression of AQP1reduce the expression of hemoglobin, and characteristic of erythroid differentiation.
Screened the up-regulated and down-regulated differential expressed genes, and then uploaded to the STRING tool to analysis the relationship of proteins encoded by these genes, there are24genes concentrate in the core position. Following uploaded to pSTIING tool to further analysis the network topology of24genes and their transcription factors, screened7genes. Uploaded the genes participate in the important singnal pathways and enrichment in fatal GO terms to pSTIING tool, screened5genes. Eventually use the literature mining of these genes to discuss the relationship with leukemia and erythroid differentiation.
This research find that FOS and ATF3over-expressed in leukemia and promote the proliferation; SOS1and NCOA3have no direct relationship with leukemia, but influenced leukemia through signaling pathways or fused with related genes, have an negative effect on the treatment and prognosis of leukemia,and all down-regulated in this research. Furthermore, up-regulated differential genes:HBD, HBE1, HBG2, HBQ1are related to erythroid differentian. Speculate that AQP1closely relate to the expression of hemoglobin, and they co-express in the process of erythroid differentiation.
CONCLUSION
This study investigated the gene function of Aquaporin-1(AQP1) in erythroid differentiation of leukemia K562cells in vitro using various molecular biology meanings, such as gene augment, gene suppression, Real-time PCR, Western Blot etc. We first found:
1. K562cells with erythroid indicators γ-globin and hemoglobin expression increased and proliferation speed reduced markedly in the process of the erythroid differentiation. AQP1mRNA and protein expression were significantly increased with the duration of RA treatment. The correlation of AQP1expression and erythrocytic differentiation in K.562cells was confirmed.
2. The retroviral expression vector of AQP1(pBABE-puro-AQP1) was constructed and transfected into K562cells to establish the stable K562cells (K562-AQP1) over-expressing AQP1. Results of experiment confirmed that over-expression of AQP1can obviously promote K562cells erythroid differentiation and in the same time inhibit cell proliferation.
3. The retroviral expression vector of AQP1small interfering RNA (pSUPER-retro-puro-shAQP1) was constructed and transfected into K.562cells to establish the K562cells (K562-shAQP1) with stable down-regulation of AQP1. Compared to the control K562cells, γ-globin and hemoglobin expression in K562-shAQP1cells induced by RA were markedly reduced. The results suggest that down-regulated expression of AQP1can partially blocked the induction effect of RA.
4. Use the expression profile chip and bioformatics tools analysis K562and K562-AQP1, obtain down-regulated genes:FOS, ATF3, SOS1, NCOA3, and up-regulated genes:HBD, HBE1, HBG2, HBQ1.
The above results verified AQP1play an important role in regulation to erythrocytic differentiation of K.562cells, and provided a new drug target for induced differentiation treatment of leukemia.
INNOVATIONS
1. There is no report domestic and overseas about the roles and mechanism of AQP1expression changes in erythroid differentiation of erythroleukemia cells.
2. This study investigated the gene function of AQP1in erythroid differentiation of leukemia K562cells in vitro using gene augment and gene suppression. The method is advanced, we expect to find a new mechanism of AQP1in erythroleukemia differentiation.
3. Use the expression profile chip and bioformatics tools screen several genes relate to erythroid differentiation, next step for these genes is expereiment, further study the mechanism of AQP1to erythroid differentiation of K562cells, in order to find disease related genes as drug targets.
白血病是世界范围内常见的血液系统恶性肿瘤,是一种细胞分化障碍性疾病,其核心问题是白血病细胞失去进一步分化为成熟细胞的能力而停滞在细胞发育的不同阶段。人红白血病细胞株K562最初源于一位慢性髓性白血病患者的胸膜渗出液,K562细胞携有Ph染色体t(9；22)(q34；q21),9号染色体abl基因与22号染色体bcr基因由于染色体异常重组形成bcr/abl融合基因,翻译产生P210蛋白,该蛋白是致病的主要原因,其具有增强酪氨酸激酶的活性。K562细胞在体外可被诱导向红系分化,表现为抑制该细胞的恶性增殖能力,以及具有相应的红系成熟标志。以β-珠蛋白是否表达来判断其是否真正的向红系细胞方向分化。研究证明,小鼠红白血病细胞(MEL)、K562细胞、人早幼粒白血病HL-60细胞等造血系统肿瘤细胞,都可以通过诱导分化剂在体外促进其向终末方向分化,并表达终末分化产物,从而使这些肿瘤细胞失去恶性增殖的能力。因此利用诱导分化物使癌细胞的增殖和基因的活动趋于正常,使基因表达亦趋于正常,为肿瘤的治疗领域提供了新策略。
目前诱导分化疗法是肿瘤研究的新领域,尤其是白血病的治疗,使恶性肿瘤细胞能够在体内外分化诱导剂的作用下向正常细胞方向分化逆转。K562细胞是体外研究肿瘤细胞恶性表达调控的极好模型,因为他能够向多种血细胞系方向分化。因此分化策略主要是将K562细胞向红系方向诱导分化。其中,全反式维甲酸诱导分化治疗已成为治疗急性早幼粒细胞白血病(Acute promyelocytic leukemia, APL)的首选手段。随着对白血病诱导分化机制的深入研究和动物实验的开展,诱导分化疗法将成为白血病临床治疗的主攻方向,但目前仍有诸多问题阻碍着其应用：1、诱导分化的具体机制尚未明确；2、诱导分化剂本身毒副作用大。因此白血病诱导分化相关基因或治疗靶点的发现将是解决这些问题的突破点。
水通道蛋白(Aquaporin, AQP)是一组小分子跨膜蛋白家族,参与水的快速跨膜转运。对于水通道蛋白的认识最开始主要是基于AQP1的结构分析,AQP1是第一个被发现的AQPs蛋白,Agre因此获得了2003年的诺贝尔化学奖,之后陆续发现哺乳动物水通道家族有13个成员(AQPO-AQP12),其基因结构、基因表达调控、染色体定位、蛋白结构、组织分布和生理功能均得到了较为深入的研究。AQP1能使红细胞适应环境巾血浆的渗透压变化,通过调节水的运输使红细胞膨胀或皱缩。促红细胞生成素可使红细胞容积瞬间增高,胞膜AQP1密度也大大增高。研究发现氧气除了在红细胞膜上自由扩散,也能够在AQP1运输和扩散。因此AQP1既是红细胞胞内胞外水进出交换的通道,同样也是红细胞运输和交换气体的通道,AQP1不仅是水通道蛋白,还是氧气氧分子进出细胞的通道。
大量研究显示AQP1的功能或表达异常与多种肿瘤的发生关系密切。用丁酸盐诱导HEL细胞向红系分化后,AQP1的转录表达明显增加,在人红白血病K562细胞中也出现了同样的结果。重要的是在AQP1启动子巾包含CACCC元件,该元件具有红细胞特异性,并且AQP1启动子的活性在K562和HEL细胞巾是非红系细胞的24倍。无论是9-顺式维甲酸(9-cis retinoic acid)还是全反式维甲酸(all-transretinoic acid), AQP1启动子中含有的维甲酸(retinoic acid, RA)反应元件可通过与RA结合诱导人红白血病细胞细胞表达AQP1,并且随着RA剂量增加,AQP1的表达明显增加,维甲酸诱导的AQP1蛋白主要在细胞质膜上表达。另外,还有研究发现除了RA,二甲亚砜和皮质类固醇亦可诱导小鼠MEL细胞表达AQP1。以上研究可以看出多种促红系分化的药物都可增强AQP1的表达,说明红白血病细胞的红系分化调控和AQP1的表达可能有着密切联系。
目前对于AQP1基因的功能研究还多局限于与液体吸收或分泌有关的上皮细胞及可能协同水跨细胞转运的内皮细胞中。AQP1高表达在红白血病细胞向红系分化过程的作用如何及其具体机制,有待进一步研究。因此在以前的研究基础上,结合国内外文献资料,首次将AQP1作为靶基因,构建AQP1基因的真核表达重组载体,在K562细胞中稳定表达,同时采用逆转录病毒载体shRNA表达系统对K562细胞中AQP1基因表达进行长效抑制,研究AQP1的高表达和表达抑制对K562细胞向红系分化过程及生理生化各项指标的影响,深入研究AQP1基因在细胞诱导分化中的功能,以期进一步明确AQP1基因与白血病发生机制之间的关系,为临床诱导分化治疗白血病提供新的治疗靶点。
目的
确定AQP1与K562细胞红系分化的相关性。构建稳定表达AQP1基因的K562-AQP1细胞系以及稳定抑制AQP1基因表达的K562-shAQP1细胞系。比较三种不同细胞系K562、K562-AQP1、K562-shAQP1在肿瘤细胞诱导分化、生长增殖等方面的差异,明确AQP1基因的在红系分化中的新功能。应用生物信息学方法比较K562细胞与AQP1表达增高或降低的K562细胞中大量相关基因表达的变化,从而发现一组疾病相关基因在特定条件下的相互作用,为临床诱导分化治疗白血病提供一组新的治疗靶点。
方法
1.使用RA处理K562细胞,收集24h、48h及72h的细胞,同时设立未加RA的K562细胞为对照组。采用RA诱导K562细胞向红系分化之后,通过Real-time PCR法检测红系指标γ-globin的表达变化；分光光度法检测血红蛋白(hemoglobin)含量,研究RA对K562细胞红系分化的影响。为了研究APQ1基因与K562细胞红系分化的关联,使用Real-time PCR法、蛋白质印迹法检测RA诱导后K562细胞AQP1mRNA和蛋白表达水平。
2.以人脑cDNA文库为模板,通过PCR扩增出AQP1基因的编码序列,构建pBABE-puro-AQP1真核表达载体；转染K562细胞,筛选建立稳定过表达AQP1基因的K562细胞株(K562-AQP1); real-time PCR法、细胞免疫荧光染色法及蛋白质印迹法检测AQP1转录和蛋白表达水平。通过MTT法检测细胞生长增殖、real-time PCR法检测红系指标γ-globin表达和分光光度法检测血红蛋白含量,研究AQP1过表达对K562细胞红系分化、增殖的影响。
3.设计特异AQP1shRNA序列,构建pSUPER-retro-puro重组质粒,转染K562细胞,筛选建立稳定抑制AQP1基因表达的K562细胞株(K562-shAQP1); real-time PCR法、细胞免疫荧光染色法及蛋白质印迹法检测AQP1转录和蛋白表达水平。通过Real-time PCR和紫外分光光度计法比较K562-shAQP1细胞株与对照组细胞在RA诱导后红系指标γ-globin和hemoglobin的表达,研究AQP1在RA诱导K562细胞红系分化中的作用,能否阻断RA诱导的K562细胞红系分化。
4.比较三种不同细胞系K562、K562-AQP1、K562-shAQP1在肿瘤细胞诱导分化、生长增殖等方面的差异,明确AQP1基因的在红系分化中的新功能。
5.利用K562细胞和AQP1过表达的K562细胞所生成的全基因组表达谱芯片,获取共同表达差异基因,然后应用GO、Pathway等生物信息学方法比较相关基因表达的变化,从而发现一组白血病相关基因在特定条件下的相互作用,为临床诱导分化治疗白血病提供一组新的治疗靶点。
6.本文统计分析采用SPSS13.0。计量资料的统计描述用均数±标准差的形式给出,两组比较采用两独立样本t检验,多组比较的情况下用one-way ANOVA,重复测量资料采用重复资料的方差分析；两两之间的比较,方差齐时用LSD法,不齐则用Dunnett's T3检验法。检验水准设为P<0.05。
结果
1.RA体外诱导K562细胞红系分化
Real-time PCR检测RA处理K562细胞不同时间后红系分化指标γ-globin mRNA的表达增加,48h时表达增加最显著(可达未加RA处理组的10.05倍),且差异具有统计学意义(F=20.191,P=0.008)。紫外分光光度计法进行K562细胞hemoglobin含量的测定结果显示,RA诱导后的K562细胞hemoglobin含量较对照K562细胞增加,且随RA作用时间延长含量递增,差异具有统计学意义(F=651.197,P<0.001)。
2.AQP1基因表达与K562细胞红系分化的相关性
Real-time PCR检测RA诱导K562细胞红系分化后AQP1基因mRNA的表达较对照组均有所增加(24h、48h、72h与对照组相比分别为8.23、12.81、12.57倍),不同水平间差异具有统计学意义(F=18.834,P=0.009); Western blot结果也显示AQP1蛋白表达量也随RA作用时间延长而递增。
3.AQP1基因稳定过表达与K562细胞红系分化和增殖
成功构建AQP1基因的真核表达载体之后,使用免疫组化法鉴定K562细胞中AQP1的表达。AQP1经一抗孵育后用goat anti-rabbit IgG-R荧光二抗显色为红色,DAPI进行核复染显色为蓝色,将抗原显色部分与核染色部分叠加在一起观察抗原表达的变化。结果显示K562-AQP1细胞中红色荧光较对照组K562细胞增强,表明pBaBb-puro-AQP1逆转录病毒载体感染K562细胞后,AQP1蛋白在细胞中的表达增多。
Real-time PCR和Western blot检测K562-AQP1细胞中AQP1mRNA和蛋白表达情况。Real-time PCR检测结果显示K562-AQP1细胞中AQP1mRNA的表达较对照组增加,较对照细胞增高700倍以上,且差异具有统计学意义(t=24.845,P=0.002)；Western blot结果显示K562-AQP1细胞中AQP1蛋白表达量也有所增加。
Real-time PCR结果显示,在稳定转染AQP1基因真核表达载体的K562-AQP1细胞中γ-globin mRNA的表达较对照K562细胞有所增加(7.369：1),且差异具有统计学意义(t=24.965,P=0.001)。紫外分光光度计法的测定结果显示,AQP1稳定过表达细胞株血红蛋白含量较对照K562细胞有所增加(增加49%),差异具有统计学意义(t=26.561,P<0.001)。
MTT法检测K562-AQP1细胞的生长曲线结果显示,AQP1过表达组(K562-AQP1)较对照组(K562)细胞的生长速度降低(F=171.36,P=0.001),说明AQP1在促进K562细胞向红系分化的同时对细胞增殖有抑制作用。
4.AQP1基因表达抑制与K562细胞诱导分化
筛选建立稳定抑制AQP1基因表达的K562细胞株(K562-shAQP1)之后,细胞免疫荧光染色法检测AQP1在K562细胞巾的表达改变结果显示,AQP1荧光显色为红色,定位在细胞膜上,K562-shAQP1细胞膜上红色荧光较对照组K562细胞有所减少,表明pSUPERretro-puro-shAQP1逆转录病毒载体感染K562细胞后,AQP1蛋白在细胞中的表达受到抑制。
用Real-time PCR法检测K562-shAQP1细胞中AQP1基因mRNA的表达结果显示, K562-shAQP1细胞中AQP1mRNA的表达较对照组有所下降,表达量约为对照组的27%,差异具有统计学意义(t=-34.138,P=0.001)；Western blot结果显示K562-shAQP1细胞中AQP1蛋白表达量也有所降低,Bandleade软件分析内对照GAPDH与目的蛋白条带灰度比,抑制效率达89.8%。
通过比较K562-shAQP1细胞株与对照组细胞在RA诱导后红系指标γ-globin和hemoglobin的表达,研究AQP1在RA诱导K562细胞红系分化中的作用,能否阻断RA诱导的K562细胞红系分化。用Real-time PCR法检测γ-globin mRNA表达结果显示,RA作用于AQP1表达下调的K562-shAQP1细胞系24h、48h、72h的γ-globin mRNA表达量为2.348、2.926、2.836,与RA处理的对照K562细胞组(6.709、9.561、8.284)相比有所下降,差异具有统计学意义(F=53.062,P<0.001)。紫外分光光度计法的测定结果显示,RA处理的K562-shAQP1细胞系hemoglobin含量与RA处理的对照K562细胞比较也有有所降低,差异具有统计学意义(F=1.736,P=0.177)。当RA诱导K562细胞红系分化时,下调AQP1表达可抑制RA的诱导分化作用。
5.应用全基因组表达谱芯片分析AQP1基因对K562细胞红系诱导分化的作用机制
本研究重点分析K562组与K562-AQP1组之间的差异表达基因,其中363个基因表达上调,421个基因表达下调。这些差异表达基因的GO分类有317个,分析发现差异基因的生物学途径主要与氧气运输、程序性凋亡以及红系分化等相关；分子功能则与氧气运输活性相关,证实AQP1基因的过表达确实能促进红系分化并且在该进程中发生细胞凋亡；差异表达基因的细胞组成多为血红蛋白复合物,证实AQP1过表达能显著增加K562细胞血红蛋白的表达,表现出红系分化的显著特征。
将K562组和K562-AQP1组共同上调和下调的基因经过整理筛选,上传至STRING分析这两组的差异基因所编码蛋白质的相互关系,结果发现有24个基因编码的蛋白质的相互作用主要集中在核心位置。将上述24个基因上传至pSTIING工具,进一步分析这些基因及其转录因子的网络拓扑结构,其中7个差异基因仍然处在重要结点位置,分别是：POLR2L、SOS1、 CDKN1A、PIN1、NCOA3、ATF3、FOS。再根据参与重要分子通路的基因、以及在重要GO term有所富集的基因使用pSTIING工具进行网络图谱的构建,有5个基因位于重要结点位置,分别是：TRIB3、CDKN1A、DDIT3、 ATF3、ALCAM。最终将这12个基因将进行下一步的文献挖掘,以探讨与白血病以及红系分化之间的联系。
FACTA文本挖掘工具对10个差异表达基因进行进一步的分析比较,结果发现,其中大部分都与白血病相关,并且大多数基因存在大量的相关文献报道,为白血病红系分化分子靶标的筛选提供明确有力的支持。相关文献挖掘的结果发现FOS、ATF3在白血病中高表达,促进白血病细胞的增殖；SOS1、NCOA3这2个基因经文献查阅跟白血病没有直接关系,但是通过信号通路或者与相关基因融合而参与白血病的发生,在白血病的治疗以及预后起到负面作用；并且这4个基因在本研究巾均发生了下调。另外,与红系分化相关的血红蛋白HBD、HBE1、HBG2、HBQ1在K562-AQP1细胞中均差异表达上调,其中HBD基因的差异表达最显著,因此推测AQP1与血红蛋白表达关系密切,并且在红系分化过程巾存在着共表达作用。
预计后期再加以实验验证。下一步再加上AQP1表达抑制的表达谱芯片进行共同研究,从巾再筛选出特异性和实用性更强的基因,作为白血病临床诊断的分子靶标候选基因。还要确定AQP1参与的信号通路以及如何发挥诱导血红蛋白表达的功能。
结论
本研究通过基因增加、基因抑制、Real-time PCR、Western Blot等分了生物学方法,采用Y-珠蛋白、血红蛋白表达,细胞增殖曲线作为红系分化检测指标,对水通道蛋白1(AQP1基因)在K562细胞红系分化巾的作用进行了研究,取得以下研究结果：
1.维甲酸(RA)诱导K562细胞红系分化的过程巾,红系分化指标Y-珠蛋白、血红蛋白表达均明显增加：并且AQP1mRNA及蛋白的表达显著增加。确定了AQP1表达与K562细胞红系分化的相关性；
2.通过构建pBABE-puro-AQP1真核表达载体确定了AQP1过表达可以显著促进K562细胞向红系分化,同时抑制细胞增殖。
3.AQP1基因的表达抑制可部分阻断RA诱导的红系分化作用,证实AQP1基因在RA诱导K562细胞红系分化过程中发挥重要作用,提示AQP1基因可能作为治疗红白血病新的靶基因。
4.以K562-AQP1和K562为对象,利用全基因组表达谱芯片获取差异表达基因进行分析。选取8个与AQP1基因相关的基因,分别是：差异表达下调基因FOS、ATF3、SOS1、NCOA3,以及差异表达上调基因HBD、 HBE1、HBG2、HBQ1。推测这些基因与红系分化相关,预计后期再加以实验验证并进行深入研究。
本研究的创新之处
1.研究AQP1的表达变化在红白血病细胞红系诱导分化中的作用及机制,国内外未见报道,有明显创新。
2.本课题从基因增强、基因抑制正反两个方面研究AQP1的作用,采用高特异性的RNA干扰技术,方法先进,可望发现AQP1在红白血病诱导分化中的新机制。
3.应用全基因组表达谱芯片和生物信息学工具筛选到若干红系分化相关基因,下一步拟针对这些基因再加以实验验证,进一步研究AQP1基因对K562细胞红系诱导分化的作用机制,从而发现一组疾病相关基因作为药物筛选靶标。
BACKGROUND
Leukemia is a worldwide malignant tumor in blood system, which is caused by cell differentiation disorder, its core nosogenesis is leukemia cells lose the ability to further differentiate stagnation in the cell growth phase. K562cell is the human erythroleukemia cell which is cultivated from the pleural effusion of chronic myelogenous leukemia patients, its t(9;22)(q34;qll) chromosomal recombinant to be bcr/abl fusion gene, which translates the P210protein increases the tyrosine kinase activity is the main pathogenic factor. K562cell is in the low state of differentiation, rapid rate of proliferation and high degree of malignant. But can be induced to the erythroid by inductive substance in vitro, first performance of malignant proliferation is restrained, and corresponding differentiation mature symbol of blood cells style. Erythroid maturation product β-globin is the key indicator to estimate the direction of erythroid differentiation. Using the differentiation material to change the abnormal state of gene expression, guiding the cancer cell that proliferation is out of control and gene ability is abnormal in the normal direction, it provides a new strategy of cancer therapy. A large number of experiments have proved, use the differentiation inducer most of the hematopoietic system tumor cells can differentiate to the terminal direction and express terminal differentiation product, so that tumor cell lose the ability of maliganant proliferation, such as murine erythroleukemia cell (MEL), human erythroleukemia K562cell, human promyelocytic leukemia HL-60cell.
At present, induction differentiation therapy is the new field of tumor therapeutics, especially leukemia. K.562is an erythroleukemia cell line, which is situated in the common stage of erythroid lineages of the hematopoietic stem cell differentiation and its normally following differentiation is blockaded. But K562cells can be induced to erythroid differentiation by agents such as all-trans retinoic acid. As further studies of leukemia cell differentiation mechanism and carry out of animal experiments, induction differentiation therapy become the main attack direction of the clinical treatment of leukemia, but there are still some questions hamper its application:①The concrete mechanism of differentiation is not clear;②The side effects of differentiation inducer. Therefore the breakthrough point to solve these problems is to find out key genes or therapeutic targets related to leukemia differentiation.
Aquaporins (AQPs) are water channel proteins that facilitate transcellular water movements. For the understanding of the water channel protein is began to the structure analysis of AQP1, Agre won the2003Nobel Prize in chemistry because of AQP1that was isolated initially from human erythrocytes, and from then13members of AQPs (AQP0-AQP12) were identified one after another. We made a thorough study about the gene structure, gene expression regulation, chromosome location, protein structure, tissue distribution and physiological function of AQPs. AQP1making red blood cells accommodate to plasma osmotic pressure by adjusting the transport of water to make red blood cells to expand or shrink. Erythropoietin can instantly increase red blood cell volume, AQP1density in cell membrane also increased greatly. Study found that oxygen not only free spread on the red cell membrane, but also can transport and diffusion in AQP1. So AQP1is not only a water channel protein, but also channel for oxygen or oxygen molecules in and out of the cell.
A lot of studies have shown that the function of AQP1is close related to abnormal expression in a variety of cancers. The regulation of AQP1gene especially has been addressed in the erythrocyte. Some scientist's study has demonstrated that the AQP1expression in human erythroleukemia HEL and K562cells was induced by sodium butyrate that is a strong agent for erythroid differentiation. In addition, the AQP1promoter contained CACCC element, the research found that AQP1promoter activity was more than24-fold higher in HEL and K562cells than in nonerythroid (Hela) cells. Retinoic acid can specially induce expression of AQP1in HEL cell by combined with RA reaction component. In the present study we demonstrate that RAs, ATRA, and9-cis-RA, strongly induce the AQP1mRNA and protein expression in human erythroleukemia in dose dependent manner. AQP1protein induced by retinoic acid is mainly expressed in cytoplasm membrane. In addition, other studies have found that dimethyl sulfoxide and corticosteroids can induce expression of AQP1protein in murine erythroleukemia MEL cell. The researches above demonstrate that many kinds of drugs promote erythroid differentiation can significantly enhance the expression of AQP1, illustrate that AQP1play an important role in the regulation of erythroleukemia cells.
Function research of AQP1gene is limited to epithelial cells related to fluid absorption or secretion and possible endothelial cells that synergy water transport across a cell membrane. The role and specific mechanism of high expression of AQP1in the procedure of erythroid differentiation in leukemia cells need further study. Therefore on the basis of previous studies, and combined with domestic and foreign literature, for the first time make AQP1as target gene to build AQP1gene eukaryotic expression recombinant vectors, and then make it stabled express in K562cells, on the other hand using a retro virus vector shRNA expression system to build long-term restrain AQP1gene expression in K562cells, So can illustrate the high expression and inhibition of AQP1expression in K562cells influence the physiological and biochemical indicators in the process of erythroid differentiation, Further research on the function of AQP1gene in erythroid differentiation. In order to make clear the mechanism of the relationship between gene AQP1and leukemia, provide new therapeutic targets for clinical differentiation treatment of leukemia.
OBJECTIVE
Confirm the correlation between AQP1and erythroid differentiation of K562cells. Construction stable cell line K562-AQP1over expression of AQP1gene and establish the K562-shAQP1cell line with stable down-regulation of AQP1. Compare the difference of three different cell lines K562, K562-AQP1, K562-shAQP1in tumor induced differentiation, growth and proliferation, clarify new features of AQP1in erythroid differentiation.
METHODS
1. Collected24h,48h and72h cells after retinoic acid (RA) treatment, and at the same time collected K562cells without RA treatment as control group. After using RA induced K562to erythroid differentiation, detect expression changes of red blood indicators y-globin by real-time PCR method; detect content of hemoglobin by spectrophotometric method to study the impact of RA to erythroid differentiation. In order to illustrate the relationship between AQPlgene and erythroid differentiation of K562, examined the AQP1mRNA and protein before and after RA treatment detected by real-time PCR and western blot.
2. Constructed pBABE-puro-AQP1eukaryotic expression vector by AQP1gene with the human brain cDNA library as a template, and then transfection k562cell established stable cell line K562-AQP1over expression of AQP1gene; AQP1mRNA and protein expression were detected by real-time PCR, Cell immunofluorescence staining method, and western blot. The cell growth and proliferation was determined by MTT method, red blood indicators γ-globin detected by real-time PCR method; and content of hemoglobin detect by spectrophotometric method, these studies could illustrate the correlation of AQP1expression and erythrocytic differentiation and proliferation in K562cells.
3. The retroviral expression vector of AQP1small interfering RNA (pSUPER-retro-puro-shAQP1) was constructed and transfected into K562cells to establish the K562cells (K562-shAQPl) with stable down-regulation of AQP1; AQP1mRNA and protein expression were detected by real-time PCR, Cell immunofluorescence staining method, and western blot. Compared to the control K562cells, y-globin and hemoglobin expression in K562-shAQPl cells induced by RA were detected by real-time PCR and spectrophotometric method, to study the role of down-regulated expression of AQP1in erythrocytic differentiation of K562cells.
4. Compare the difference of three different cell lines K562, K562-AQP1, K562-shAQPl in tumor induced differentiation, growth and proliferation, clarify new features of AQP1in erythroid differentiation.
5. Use genome-wide expression profile chip of K562and K562-AQP1cells, abtain common differential genes, and compared the changes of relevant genes by GO, Pathway methods in order to discorved interaction of these genes, provides a set of new therapeutic targets for clinical leukemia treatment.
6. The statistical method in this article using SPSS13.0software for data analysis. Test samples by real-time PCR, ultraviolet spectrophotometry, and test cell growth curve determined by MTT method, the statistical method of all these experiment use ANOVA for repeated measurements. Firstly conduct homogeneity test for variance, secondly conduct independent-samples T test between two treatment groups or one way ANOVA between multiple treatment groups, at last, using LSD method between multiple comparisons if the differences are statistically significant; when there is constant in control group, use single sample t-test, if more than two groups, use independent-samples T test or ANOVA method after eliminate the control group, finally compare with control group respectively. All data is expressed by x±s, With P<0.05for the difference is statistically significant.
RESULTS
1. RA induction erythroid differentiation of K562cells in vitro
After using RA induced K.562to erythroid differentiation, expression of red blood indicator γ-globin was significantly increased detect by real-time PCR method, especially at48h (can be up to10.05times than that without RA treatment group), the difference is statistically significant (F=20.191, P=0.008). The result of hemoglobin level in K562cells which detected by spectrophotometric was showed, Hemoglobin level in K562cells induced by RA significantly increased compared with the K562-control group, And the content increasing with the duration of RA treatment, the difference is statistically significant (F=651.197, P<0.001)
2. The correlation between AQP1and erythroid differentiation of K562cells
After using RA induced K562to erythroid differentiation, the expression of AQP1mRNA detected by real-time PCR was significantly increased compared to the control group (The expression of AQP1mRNA at24h,48h and72h was8.23、12.81、12.57times than control group, respectively), the difference is statistically significant(F=18.834, P=0.009); Expression of AQP1protein also increased with the duration of RA treatment detected by Western Blot.
3. AQP1gene over-expressed compacted differentiation of K562cells.
After constructed eukaryotic expression vector of AQP1gene successfully, evaluated the expression of AQP1in K562cells by immunohistochemistry staining. Made AQP1incubated by primary antibody and then fluorescent color in red after use goat anti-rabbit IgG-R second antibody, nuclear color for blue counterstained by DAPI, eventually antigen color parts and nuclear staining are stacked up together then observe the changes of antigen expression. The result of immunohistochemistry staining was observed under a fluorescence microscope (X400), AQP1was located with red fluorescence which is stronger than control group, indicated that AQP1protein expression in K562-AQP1was obviously increased than that of control.
Expression of AQP1mRNA and protein in K562-AQP1cells detected by real-time PCR and western blot. The result of real-time PCR showed that expression of AQP1mRNA in K562-AQP1cells increased more than700times compared with the K562-control group; Western Blot showed that expression of AQP1protein in K562-AQP1cells also increased compared with the K562-control group, the difference is statistically significant (t=24.845, P=0.002).
The result of real-time PCR displayed, expression of γ globulin mRNA in K562-AQP1cells significantly increased compared with the K562-control group (increased7.369times), the difference is statistically significant (^=24.965, P=0.001); The result of ultraviolet spectrophotometry displayed, hemoglobin level in K562-AQP1cells also increased compared with the K562-control group, the difference is statistically significant (t=26.561, P<0.001). So the content of γ-globin and haemoglobin were significantly increased in AQP1over-expression cells line (K562-AQP1).
While growth curves of K562-AQP1cells detected by MTT method revealed, the cell growth rate of K562-AQP1group was markedly reduced compared to K562control cells (F=171.36, P=0.001), indicated the over expression of AQP1promoted erythroid differentiation of K562cells and at the same time inhibited the cell proliferation significantly.
4. AQP1gene down-regulated compacted differentiation of K562cells.
After established the K562cells (K562-shAQP1) with stable down-regulation of AQP1, change of AQP1expression in K562cell was detected by immunohistochemistry staining, AQP1located on the cell membrane and color in red fluorescence; the nuclei of K562cells were visualised by DAPI counter-staining, the figure showed red fluorescence in cell membrane of K562-shAQP1was less than K.562control group, indicated that expression of AQP1protein was suppressed.
Expression of AQP1mRNA in K562-shAQP1cells detected by real-time PCR showed that expression of AQP1mRNA in K562-shAQP1cells descended to27%of K562-control group, the difference is statistically significant (f=-34.138, P=0.001); Expression of AQP1protein in K562-shAQP1cells detected by Western Blot showed that expression of AQP1protein in K562-AQP1cells also descended compared with the K562-control group, use bandleade software analysis band gray ratio between housekeeping gene GAPDH and purpose protein, we concluded that inhibition efficiency was up to89.8%.
Whether down-regulated AQP1can blocking erythroid differentiation through compared the expression of Y globulin and hemoglobin in K562-sh562before and after induced by RA. The result of real-time PCR displayed, expression of Y globulin mRNA in K562-shAQP1cells was2.348,2.926,2.836after induced by RA at24h,48h,72h, respectively, which is significantly decreased compared with the K562-RA group (6.709、9.561、8.284), the difference is statistically significant (F=53.062, P<0.001). The result of ultraviolet spectrophotometry displayed, hemoglobin level in K562-shAQPl cells also significantly decreased compared with the K562-control group, the difference is statistically significant (F=1.736, P=0.177). Down-regulated expression of AQP1can partially blocked the induction effect of RA in the procedure of erythroid differentiation.
5. Application of expression profile chip analysis the mechanism of erythroid differentiation of K562cells influenced by AQPl
This study focused on the differential expressed genes between K562and K562-AQP1groups, there are363genes up-regulate, and421genes down-regulate. And317GO Terms of the differential expressed genes, the biological process mainly concern about oxygen transport, apoptosis, erythroid differentiation, et al; molecular function is relevant to oxygen transport activity, confirmed that the over-expression of AQP1induce erythroid differentiation and also lead apoptosis during this process; the cellular component of differential expressed genes mainly is hemoglobin complex, confirmed that the over-expression of AQP1reduce the expression of hemoglobin, and characteristic of erythroid differentiation.
Screened the up-regulated and down-regulated differential expressed genes, and then uploaded to the STRING tool to analysis the relationship of proteins encoded by these genes, there are24genes concentrate in the core position. Following uploaded to pSTIING tool to further analysis the network topology of24genes and their transcription factors, screened7genes. Uploaded the genes participate in the important singnal pathways and enrichment in fatal GO terms to pSTIING tool, screened5genes. Eventually use the literature mining of these genes to discuss the relationship with leukemia and erythroid differentiation.
This research find that FOS and ATF3over-expressed in leukemia and promote the proliferation; SOS1and NCOA3have no direct relationship with leukemia, but influenced leukemia through signaling pathways or fused with related genes, have an negative effect on the treatment and prognosis of leukemia,and all down-regulated in this research. Furthermore, up-regulated differential genes:HBD, HBE1, HBG2, HBQ1are related to erythroid differentian. Speculate that AQP1closely relate to the expression of hemoglobin, and they co-express in the process of erythroid differentiation.
CONCLUSION
This study investigated the gene function of Aquaporin-1(AQP1) in erythroid differentiation of leukemia K562cells in vitro using various molecular biology meanings, such as gene augment, gene suppression, Real-time PCR, Western Blot etc. We first found:
1. K562cells with erythroid indicators γ-globin and hemoglobin expression increased and proliferation speed reduced markedly in the process of the erythroid differentiation. AQP1mRNA and protein expression were significantly increased with the duration of RA treatment. The correlation of AQP1expression and erythrocytic differentiation in K.562cells was confirmed.
2. The retroviral expression vector of AQP1(pBABE-puro-AQP1) was constructed and transfected into K562cells to establish the stable K562cells (K562-AQP1) over-expressing AQP1. Results of experiment confirmed that over-expression of AQP1can obviously promote K562cells erythroid differentiation and in the same time inhibit cell proliferation.
3. The retroviral expression vector of AQP1small interfering RNA (pSUPER-retro-puro-shAQP1) was constructed and transfected into K.562cells to establish the K562cells (K562-shAQP1) with stable down-regulation of AQP1. Compared to the control K562cells, γ-globin and hemoglobin expression in K562-shAQP1cells induced by RA were markedly reduced. The results suggest that down-regulated expression of AQP1can partially blocked the induction effect of RA.
4. Use the expression profile chip and bioformatics tools analysis K562and K562-AQP1, obtain down-regulated genes:FOS, ATF3, SOS1, NCOA3, and up-regulated genes:HBD, HBE1, HBG2, HBQ1.
The above results verified AQP1play an important role in regulation to erythrocytic differentiation of K.562cells, and provided a new drug target for induced differentiation treatment of leukemia.
INNOVATIONS
1. There is no report domestic and overseas about the roles and mechanism of AQP1expression changes in erythroid differentiation of erythroleukemia cells.
2. This study investigated the gene function of AQP1in erythroid differentiation of leukemia K562cells in vitro using gene augment and gene suppression. The method is advanced, we expect to find a new mechanism of AQP1in erythroleukemia differentiation.
3. Use the expression profile chip and bioformatics tools screen several genes relate to erythroid differentiation, next step for these genes is expereiment, further study the mechanism of AQP1to erythroid differentiation of K562cells, in order to find disease related genes as drug targets.
引文
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