OAZ基因在K562细胞分化中的功能研究
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摘要
多聚胺包括腐胺、亚精胺和精胺,几乎是所有活细胞的必要组分。它们可以结合在RNA和染色体DNA上,在细胞周期、基因表达、细胞增殖与分化上起到重要的作用。多胺的缺失将导致细胞生长减慢甚至死亡,但是当它们的含量升高时也会有致癌作用并且可以诱导细胞凋亡。鸟氨酸脱羧酶(ODC)是多聚胺生物合成中的限速酶,催化多聚胺生物合成途径中的第一步:鸟氨酸向腐胺的转变。ODC的半衰期仅仅几分钟,是已知所有酶中半衰期最短的。在多胺生成的过程中扮演着重要的调控酶角色,其活性可在转录、翻译和翻译后水平被精确调控。鸟氨酸脱羧酶抗酶(OAZ)可以连接到ODC蛋白上,抑制其催化活性并靶向地促使26S蛋白酶降解ODC,在翻译后水平影响ODC活性。此外,OAZ还能抑制细胞对多胺的吸收,促进细胞对多胺的排泄,从而降低细胞内多胺的浓度。研究表明细胞内该抗酶的过度表达除了能降低细胞内多胺水平,还能抑制细胞的生长。抗酶成员目前发现至少有三种,最主要的就是由227个氨基酸组成的抗酶1。抗酶1 C末端的部分是与ODC结合的区域,N末端的部分则促进ODC的降解。抗酶2未发现有通过蛋白酶体降解ODC的功能,但却有抑制多胺转运的能力。抗酶3,只存在于睾丸组织中,其表达特定的出现于精子细胞发生过程中,与生育有关。OAZ的表达需要有一个独特的核糖体阅读框的移位过程。所有的OAZ成员有两个重叠的ORF框,一个带有翻译起始密码子和在移位处的终止密码子的ORF1,ORF2则编码了大部分的OAZ多肽序列却缺乏起始密码子。因此翻译全长的有功能的抗酶蛋白需要有+1的核糖体移位,移位处的核苷酸序列高度保守,有助于从其它组织中对抗酶成员的鉴别。+1的移位过程受到精胺的诱导调控,机制尚不明晰。当多胺水平上升时,可促进核糖体移位,OAZ表达增多,于是反馈抑制细胞内多胺浓度的上升。这样的机制有助于防止细胞内多胺浓度的起伏而造成的细胞毒性。许多实验已证实OAZ对ODC的降解作用与其对细胞的增殖抑制明显相关,使其体现出抗肿瘤恶性增殖的特性。Ruchi M等发现在实体肿瘤细胞如肝癌和前列腺癌中,OAZ基因的表达升高与细胞生长抑制明显相关,通过与细胞周期素D1(cyclinD1)结合并使之降解,使细胞停滞于G1期,减缓细胞生长。在鼠皮肤癌还有胃上皮癌的模型中,OAZ的过度表达均导致了癌细胞的生长抑制,提示OAZ基因在肿瘤细胞的治疗上能发挥作用。我们的目的是通过定点突变技术,得到相应的去除frameshift位点后的突变OAZ基因,并构建至真核表达载体pEGFP-N1质粒中,转染至K562细胞中,通过检测K562细胞生长分化及cyclinD1基因变化情况,来研究OAZ基因对白血病肿瘤细胞的生物学效应。
     K562细胞诱导分化:K562细胞是一株恶性程度较高的人红白血病细胞系,建株于慢性淋巴细胞性白血病的患者。第九号与第22号染色体相互易位形成的Ph染色体是CML的肿瘤恶性克隆标志。这种染色体平衡易位导致9q34上的c-abl原癌基因与22q11上的Bcr基因断裂后重组,形成新的融合基因bcr-abl,后者转录翻译的P210蛋白具有异常增高的蛋白酪氨酸激酶(PTK)活性。K562细胞具有多项分化潜能并能被多种诱导物诱导分化,因此K562细胞成为一个重要的关于白血病研究的细胞模型。K562细胞能被诱导表达胚胎型和胎儿型的血红蛋白,对这种机制的阐明有助于临床上鎌型红细胞性贫血和β地贫的治疗研究。当前,对白血病治疗的观念正逐步从杀死白血病细胞向使其往良性方向分化转变。许多化学物质对K562细胞诱导分化的同时,会出现诱导细胞凋亡的副作用,如维甲酸、佛波酯、羟基脲均在诱导细胞分化同时出现促凋亡的现象。有研究发现OAZ在抑制细胞生长的同时,被抑制细胞未出现凋亡的迹象,这给K562细胞的诱导分化研究提供了新的思路。
     研究方法:通过荧光定量PCR技术检测OAZ基因在Hemin诱导K562细胞红系分化过程中的表达变化,结果说明OAZ的表达升高与K562红系分化呈正相关。根据Genebank的数据,查找OAZ基因序列,并选用Primer primier5.0设计扩增ORF2与OAZ全长的引物。引物的5’端分别修饰上相应的酶切位点,以利于扩增产物重组至表达载体pET32a和pEGFP-N1。测序鉴定后命名为pET32a-OAZ-ORF2和pEGFP-N1-OAZ。将pET32a-OAZ-ORF2转化至BL21菌,IPTG诱导下表达出OAZ的ORF2部分蛋白。收集并纯化蛋白后,免疫新西兰兔,制备多克隆抗体。对测序鉴定后的pEGFP-N1-OAZ重组体,采用定点碱基突变技术,将框移位点处TCCTGATG的T碱基缺失,使OAZ基因的两段开放阅读框没有重叠,无需精胺的诱导即可表达出全长OAZ蛋白,减少实验过程中外源物质的添加对实验结果的干扰。测序后命名为pEGFP-N1-OAZ-muration,并采用脂质体法,转染至K562细胞,G418筛选得到稳定表达OAZ的细胞株,命名为K562_(OAZ)。同时针对OAZ基因序列,设计针对性的27mer siRNA,转染K562细胞和K562_(OAZ)细胞,抑制OAZ基因表达。实验中,我们采用荧光定量PCR技术检测siRNA干扰OAZ基因表达的效率。当OAZ在K562细胞中过表达及抑制表达时,通过细胞生长检测、分化检测、细胞周期分析和CyclinD1基因变化检测,初步探讨OAZ在K562细胞生长分化中的功能。
     实验结果:采用50mmol/L Hemin诱导K526细胞,细胞生长明显减慢,联苯胺阳性细胞增加,血红蛋白数量增加,说明K562细胞被诱导向红系方向分化。采用荧光定量PCR技术检测OAZ基因表达变化,发现OAZ mRNA水平随诱导时间的延长表达量逐渐增多,OAZ在Hemin诱导24、48和72h后,其mRNA表达分别上升了18.67%、51.12%和92.11%。为深入研究OAZ对K562的生物学效应,我们构建了OAZ突变基因重组质粒,测序鉴定后,转染至K562细胞。发现随时间的增加,细胞生长明显减慢,联苯胺阳性细胞增多。进一步的实验发现,生长受到抑制的K562细胞在G1期被阻滞,与cyclinD1的表达下调相一致。为验证实验的可靠性,我们设计了针对OAZ基因的27mere siRNA,转染K562_(OAZ)抑制OAZ基因的表达。当转染siRNA 24h后,处理组OAZ基因较对照组的抑制率为46.04%,48h为55.41%,72h为50.07%,说明siRNA的干扰效率可信。随OAZ基因表达的减少,细胞的联苯胺阳性率逐渐减少,由开始时的6.6%±1.0%分别下降4.3%±0.5%(24h)、4.1%±0.7%(48h)和4.6%±0.9%(72h),细胞的增殖速度亦随之增加,有逐步恢复恶性的趋势。同时,RT-PCR检测cyclinD1的表达也随之增多,提示细胞周期的抑制也随之解除。
     结论:(1)首次采用荧光定量PCR的方法检测了OAZ基因在Hemin诱导K562细胞红系分化时的表达变化,结果提示OAZ的表达增多与K562细胞的红系分化呈正相关。(2)构建了原核表达重组载体pET32a-OAZ-ORF2和真核表达重组载体pEGFP-N1-OAZ、pEGFP-N1-OAZ-mutation,并成功在BL21菌和K562细胞中表达。这对深入研究OAZ基因的功能提供了良好的研究基础。(3)首次采用27mer的siRNA对OAZ基因的表达进行抑制实验,并通过荧光定量PCR证实了基因抑制效果,给OAZ基因的功能研究提供了新的选择。(4)实验结果证明OAZ基因与K562的分化存在相关性,在抑制肿瘤细胞恶性增殖的基础上,还具有使K562细胞向红系方向分化趋势。
The polyamines (putrescine,spermidine,and spermine) are essential for allliving cells. They are largely bound to negatively charged molecules such as DNAand RNA and play an important role in cell proliferation, gene expression, cellproliferation and differentiation. The absence of polyamines will lead cellproliferation stepping down, while rising of the polyamines level results incancerogenesis and apoptosis.Ornithine decarboxylase(ODC)is the rate-limitingenzyme in polyamine biosynthesis and catalyzes ornithine to putrescine. ODC whichhas a short half-life of several minutes to 1 hour(the shortest half-life of any knownenzyme) acts an important key enzyme in polyamine biosynthesis and its activity canbe regulated at level of transtription, translation and posttranslation. Ornithinedecarboxylase antizyme inhibits ODC and then directs its degradation by the 26Sproteasome. Overexpression of OAZ in cells not only decreases the level ofpolyamines, but also coincides with growth inhibition. Furthermore, OAZ candecrease the concentration of polyamine inside cell by inhibiting polyamine uptakeand stimulating polyamine excretion. There are at least three isoforms in OAZ family,in which the important isoform is OAZ1 composed of 227 amino acids. TheC-terminal domain of OAZ1 is sufficient for ODC binding whereas the N-terminal region is important to promote degradation of ODC. Isoform-2 of antizyme (OAZ-2)does not appear to stimulate ODC degradation at the proteaome but does maintain theability to negatively regulate polyamine transport. A third isoform,OAZ-3,is limitedto only one cell type, testis germ cells, where its expression occurs at a particularstage of spermatogenesis, with implications fertility. A unique ribosomal frameshiftmechanism is needed for the expression of functional antizymes. All antizymes havetwo overlapping open reading frames, a short ORF1 with a translational start codonand a stop codon at the frameshifl site and another ORF2, which encodes most of theprotein but lacks a discrete start codon. Therefore, translation of a functional,full-length antizyme requires a +1 ribosomal frameshift. The nucleotide sequencesurrounding the frameshif site is highly conserved which was particularly helpful inidentifying antizyme orthologues from various organisms. The +1 translationalframeshift is induced by polyamines via a mechanism that is not completelyunderstood. When polyamine level rises, it can promote the frameshift and induce theexpression of OAZ, which can reduce the polyamines in a feedback mechanism. Thismechanism serves to prevent extreme fluctuation in polyamine levels, which arethought to be toxic. Many experiments prove that OAZ has the effect of anti-tumorcharacteristic while the degradation of ODC is associated with its ability of growthinhibition of cells. Overproduction of OAZ in a variety of cell types, includingmalignant oral keratinocytes, heaptoma cell lines, and prostate cancer cells, coincideswith growth inhibition and cell cycle arrest in the G1 phase.
     Furthermore, overexpression of antizyme in mouse skin cancer models hasbeen shown to result in tumor suppression. Our goal is to gain the mutation OAZgene without the site of frame-shift using site-directed mutagenesis method. Then wetransfect the pEGFP-N1-OAZ-mutation into K562 cells after construction of therecombinant between mutation OAZ gene and pEGFP-N1 vector. Study of the biology effect of OAZ gene upon leukemia cells undergoes through the assay ofgrowth and differentiation of K562 as well as the expression of cyclin D1.
     K562 cell is highly malignant human immortalized erythroleukemia cellderived from a patient with chronic myelogenous leukemia(CML).Philadelphiachromosome(Ph) is the result of a t(9:22)reciprocal chromosomal translocation and isthe malignant clonal marker of CML. At the molecular level,Ph translocation leads aprotein tyrosine kinase(PTK),to a new position downstream of the gene bcr gene on22q11 and forms a new bcr-abl fusion gene which encodes a chimeric protein P210.The k562 cells are bipotential and can be induced differentiation by many inducers,which represent an important in vitro model for basic studies of leukemogenesis.K562 can be induced express embryonic and fetal hemoglobins, so elucidation of themechanism will do well to therapy sickle cell disease andβ-thalassemia syndromes.The concept of leukermia therapy has changed from the traditional method of killingtumor cells to induce leukermia cells differentiation. Side effect of apoptosis willoccur while the K562 cells are induced differentiation by many chemistry materials,such as retinoic acid, Phorbol ester, hydrea, which promote cells apoptosis when theyinduce K562 cells differentiation. Some experiments show that there is no connectionbetween inhibitions of cell growth by OAZ with apoptosis phenomenon. This gives anew thinking about induction differentiation of K562 cells.
     Study methods: Up-regulation of OAZ has been identified in erythroiddifferentiation of K562 cells induced by Hemin through the method of fluorescencequantify PCR. The result indicates up-expression of OAZ is associated with K562erythroid differentiation. According to the dates in Genebank, we find out the wholecDNA sequence of OAZ, and design the primers for amplifying the fragments ofORF2 and whole sequence of OAZ. The corresponding restriction site has beenmodified on the 5'end of the primers so that we can use the restriction endonuclease to cut the PCR products and recombine them to expression vector of pET32a andpEGFP-N1. The recombination gene of OAZ and vectors were named ofpET32a-OAZ-ORF2 and pEGFP-N1-OAZ after sequencing. Ecoli BL21 wastransformed with pET32a-OAZ-ORF2, and the OR2 protein was expressed afterIPTG induced. After purification of the combination protein, we further immunizerabbit to prepare polyclonal antibody. Then the T base at the site of frameshiftTCCTGATG was deleted by site base mutation method so that there was no overlappingbetween the two ORFs within OAZ fragment. The OAZ protein can occur withoutany spermine induced which can decrease the interfere of experiment result byxenobiotic added, pEGFP-N1-OAZ-mutation was named after sequencing andtransfected into K562 cells by lipfectin2000. K562 cell line later named K562_(OAZ)which expresses OAZ protein stably was gained by filtration of G418. At the sametime, we designed 27 mer siRNA which was to be transfected into K562 andK562_(OAZ) to inhibit the expression of OAZ. In our experiment we use the method offluorescent quantitation PCR to detect the efficiency of interference of siRNA. Weforced the target OAZ gene up-regulation and down-regulation in K562 cells, growthassay, differentiation assay, cell cycle detection and expression of Cyclin D1 analysiswere performed to discuss the function of OAZ in K562 cell growth anddifferentiation.
     Results: When treated the K562 cells with 50mmol/L Hemin for 24, 48 and72 hours, we detect stepping down of the cell growth, increasement of BZ(+) cellsand hemoglobin. These dates show that K562 cell has been undergone erythroiddifferentiation. The expression of OAZ mRNA rises after hemin treatment using themethod of fluorescent quantification PCR. The expression of OAZ rises of 18.67%、51.12%and 92.11%respectively when treated the K562 cells with hemin for 24,48and 72 hours. To further study the biology effect of OAZ on K562, we construct recombination DNA of mutation OAZ and eukaryotic expression vector pEGFP-N1.The recombination DNA was transfected into K562 cells after sequencing. Whentreated with OAZ, we detect stepping down of the cell growth, increase of BZ(+)cells. And we find that these k562 cells were arrested in G1 phage, coincidence withthe low expression of cyclin D1. To test the confidence of experiment, we designed27met siRNA of target gene OAZ and transfect the siRNA into K562_(oaz) to inhibit theexpression of OAZ. The inhibition of OAZ expression in K562_(OAZ) treated withsiRNA for 24,48 and 72 hours was 46.04%、55.41%和50.07%respectively, whichindicates the efficiency of siRNA was credible. Positive rate of benzidine staining inK562_(OAZ) decreased to 4.3%±0.5%(24h)、4.1%±0.7%(48h) and 4.6%±0.9%(72h) from6.6%±1.0%(0h) following with the down expression of OAZ. Furthermore, thegrowth of K562 cells rose, which indicated K562 cells had the tendency of recoveringits malignant characteristic. The same time we detected the up-expression of cyclinD1 by RT-PCR, which indicated the inhibition of cell cycle was dismissed. K562 celltreated with siRNA had the tendency of recovering its malignant proliferation,coincidence with the decrease of OAZ and the increase of cyclin D1.These dateshowed that OAZ had the ability to influence K562 cells.
     Conclusions: (1)we first use the method of fluorescent quantification PCR todetect the expression of OAZ in K562 cells erythroid-induced by hemin.The dateshows that the up-expression of OAZ is coincident with the erythroid differentiationof K562 cells.(2)Prokaryotic expression vector pET32a-OAZ-ORF2,eukaryoticexpression vectors pEGFP-N1-OAZ and pEGFP-N1-OAZ-mutation are constructedand the ORF2 protein was expressed in Ecoli BL21,while the whole OAZ proteinexpressed in K562 cells.(3)we first use the 27 mer siRNA to interfere the expressionof OAZ and confirmed the inhibition of interferce by fluorescence quantification PCR,which gives us a new choice to study the function of OAZ. (4)The results show that there is a dependability about OAZ gene and differentiation of K562 cells. Based onthe ability of inhibition of tumour cells'malignacy growth, OAZ has the tendency topromote the K562 cells undergoing erythroid differentiation.
引文
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