促凋亡基因PDCD5在人胶质瘤中的表达及其对化疗敏感性的影响
摘要
目的
脑胶质瘤是中枢神经系统中最常见的恶性肿瘤之一,发生率约占所有颅内肿瘤的35.12-61.10%,具有发病率、复发率、死亡率高和治愈率低等“三高一低”的特点。目前临床治疗胶质瘤的方法主要是手术治疗、放疗、化疗,近几十年来,尽管新的诊疗技术不断出现,但由于大多数胶质瘤呈浸润性生长,与脑组织无明显分界,手术即使达到显微镜下和影像学全切除,复发还是在所难免,病人的总体生存率没有明显提高。胶质瘤术后化疗是胶质瘤综合治疗中的一个重要环节,特别是对进一步杀灭残留肿瘤细胞,防止肿瘤复发和延长患者生存时间起着十分重要的作用。然而,临床上相当一部分病人常规化疗疗效欠佳,过去认为是由于受血脑屏障的影响,化疗药物难以到达肿瘤细胞。近年来研究发现,影响脑胶质瘤化疗效果有多方面的原因,其中化疗药物产生耐药性是化疗失败的重要原因之一。而如何提高化疗药物的敏感性则成为胶质瘤治疗的瓶颈。随着分子生物学研究的深入,为神经胶质瘤的基因研究方面提供了广阔的前景,而寻找影响化疗敏感性的基因,对于胶质瘤的治疗具有重要价值,是目前一个亟待解决的问题。
PDCD5是近年来发现的新基因,在凋亡诱导因素存在下能够明显促进细胞凋亡的发生,研究结果显示卵巢癌、胃癌、肾透明细胞癌等肿瘤中PDCD5表达下调,那么PDCD5在人神经胶质瘤组织中的表达状况如何?PDCD5在神经胶质瘤进展中是否发挥作用?能否影响胶质瘤的化疗敏感性并成为胶质瘤基因治疗的靶点之一?尚不清楚。
为了明确PDCD5基因与人神经胶质瘤发生、发展的关系,为进一步以此为靶点进行基因治疗提供实验依据,本论文从以下几个方面进行了探讨:
1.PDCD5在人神经胶质瘤中的表达状态及其临床意义
2.PDCD5表达对胶质瘤细胞生长及化疗敏感性的影响
3.PDCD5影响胶质瘤细胞化疗敏感性的分子机制
4.PDCD5与凋亡相关基因(如PDCD4)的关系
方法
一、PDCD5在人神经胶质瘤组织中的表达及其临床意义
1.病例收集:收集山东大学齐鲁医院神经外科2003～2006年神经胶质瘤患者的新鲜标本共30例包括其中3例患者的相应癌旁组织,以及石蜡包埋的肿瘤标本58例。所有患者术前均未接受任何放、化疗治疗。
2.分别用RT-PCR及Western blot方法检测神经胶质瘤细胞系U251、U87及神经胶质瘤组织中PDCD5mRNA及蛋白质的表达。
3.χ~2检验分析PDCD5在不同胶质瘤病例中的表达与胶质瘤临床病理生理学特征之间的关系,Kaplan-Meier生存曲线分析PDCD5的表达与胶质瘤患者生存率之间的关系。
二、PDCD5过表达或沉默表达对胶质瘤细胞系生长及化疗敏感性的影响
1.用空载体或PDCD5重组表达载体转染胶质瘤细胞系,应用RT-PCR及Western blot及免疫细胞化学方法检测转染后PDCD5的表达,建立低表达及高表达PDCD5的胶质瘤细胞模型。
2.倒置显微镜观察转染PDCD5后神经胶质瘤细胞系形态、生长分化的变化,绘制生长曲线。
3.采用不同的化疗药(顺铂等),设定不同浓度,用MTT法检测稳定转染空载体或PDCD5的胶质瘤细胞系生存率变化。
4.克隆形成实验观察PDCD5转染后胶质瘤细胞系在长期低浓度化疗药处理时细胞集落形成的情况。
5.设计合成PDCD5特异的siRNA(HPLC纯度＞95%),应用siRNA沉默胶质瘤细胞系中PDCD5的表达,转染48小时后在mRNA及蛋白质水平检测PDCD5的表达。
6.倒置显微镜观察和活细胞计数分析siRNA沉默PDCD5的表达后神经胶质瘤细胞形态、增殖的变化,绘制生长曲线。
7.MTT法检测siRNA沉默PDCD5的表达后对胶质瘤细胞系化疗敏感性的影响。
三、PDCD5影响胶质瘤细胞化疗敏感性的机制研究
1.化疗药处理后的PDCD5及空载体稳定转染细胞系的凋亡检测:通过Hoechst染色荧光显微镜下观察细胞凋亡的形态学变化;流式细胞术分析细胞晚期凋亡率变化。
2.凋亡通路蛋白检测:应用Western blot方法检测凋亡相关蛋白caspase-3、caspase-8、caspase-9的活性变化及Bcl-2、Bax蛋白表达变化。
四、PDCD5对凋亡相关基因PDCD4的影响
1.用空载体或PDCD4重组表达载体转染稳定表达外源性PDCD5或空载体的U87胶质瘤细胞系,应用RT-PCR及Western blot方法检测瞬时转染后PDCD4的表达。
2.荧光显微镜观察瞬时转染PDCD4后神经胶质瘤细胞系形态、生长的变化,绘制生长曲线。
3.应用Western blot方法检测凋亡相关蛋白Bcl-2、Bax蛋白表达变化。
结果
一、PDCD5在人神经胶质瘤中的表达及其临床意义
1.PDCD5在人神经胶质瘤细胞系中的表达
为了研究PDCD5在人神经胶质瘤中的作用,首先应用RT-PCR及Western blot的方法检测胶质瘤细胞系U87及U251中PDCD5的表达,结果显示在U251细胞系中PDCD5表达较高,而在U87细胞系中PDCD5 mRNA及蛋白质则均低表达,提示PDCD5有可能参与影响胶质瘤的进展过程。
2.PDCD5在人原发性神经胶质瘤中表达降低
为了更深入研究PDCD5在人神经胶质细胞瘤中的作用,我们进一步收集胶质瘤临床病例,应用RT-PCR及Western blot方法对30例新鲜原发神经胶质肿瘤及3例神经胶质瘤中的PDCD5进行检测,同时用免疫组织化学的方法分析了PDCD5在88例神经胶质细胞瘤中的表达部位及表达状态。
结果显示癌旁组织表达高水平的PDCD5,而30例新鲜胶质瘤组织中有16例组织中PDCD5 mRNA表达降低,低表达率为53.33%(16/30)。Western blot结果显示PDCD5蛋白肿瘤组织中低表达率达83.33%(25/30),高于其mRNA,这提示有的肿瘤组织尽管PDCD5 mRNA的表达水平接近正常组织,但PDCD5蛋白表达仍存在明显降低,从而造成PDCD5的表达缺陷。
为了证实神经胶质瘤组织中PDCD5的降低表达情况,为分析PDCD5的表达与病人临床病理特征和预后关系奠定基础,进一步采用免疫组织化学的方法检测88例胶质瘤组织中PDCD5的表达,表达趋势与Western blot一致,癌旁组织表达较高水平PDCD5,而肿瘤组织PDCD5蛋白则出现明显的降低表达趋势。
3.PDCD5的降低表达与胶质瘤临床病理特征及预后的关系
为了进一步揭示PDCD5在胶质瘤发生、发展中的作用,我们对PDCD5的表达水平与临床病理特征进行了详细分析,结果显示不同性别、年龄、组织学类型的神经胶质瘤标本中PDCD5的表达没有显著差异;然而PDCD5的降低表达与肿瘤病理分级密切相关,在同一组织类型胶质瘤标本即星形胶质瘤的不同病理分级中,低级别(Ⅰ-Ⅱ)胶质瘤标本中,PDCD5降低表达占45.16%(14/31),而高级别(Ⅲ-Ⅳ)组织标本中PDCD5降低表达占86.00%(43/50),不同级别间PDCD5表达有显著差异(p＜0.01);然而,临床随访结果显示PDCD5的表达水平与星形胶质瘤病人总的生存率之间没有明显联系(p＞0.05),在不同级别星形胶质瘤中,PDCD5的表达与病人的生存率之间统计之后也没有明显统计学意义。
二、外源性PDCD5的过表达对胶质瘤细胞生长及化疗敏感性的影响
第一部分研究显示PDCD5在大部分神经胶质瘤中表达降低。已有研究报道在胃癌,宫颈癌,肺癌等肿瘤中均存在PDCD5的表达降低,同时,对PDCD5的作用研究显示其是一个促凋亡基因,提示PDCD5可能参与了肿瘤的发生、发展过程。为了进一步确定PDCD5在胶质瘤发生、发展中的作用,我们从临床应用的角度出发,应用基因转染技术使外源性PDCD5在低表达PDCD5的神经胶质瘤细胞系中表达,并分析其对胶质瘤细胞生长的影响。
1.外源性PDCD5在胶质瘤细胞中的稳定表达
通过基因转染分别将空载体及PDCD5重组表达载体瞬时转染U87细胞系,并应用G418进行稳筛,建立稳定表达的细胞系,通过RT-PCR、Western blot及免疫细胞化学方法检测后发现,与空细胞及空载体对照组相比,PDCD5转染组U87细胞高表达PDCD5。
2.外源性PDCD5的过表达对胶质瘤细胞的形态及生长无明显影响
显微镜下观察稳定筛选后过表达PDCD5的U87细胞,其形态与空细胞和空载体相比没有明显改变;通过生长曲线发现转染PDCD5后U87细胞的增殖未受到明显影响。
3.外源性PDCD5过表达可增强胶质瘤细胞化疗敏感性
PDCD5是一个促凋亡因子,在有凋亡因素存在情况下发挥促凋亡作用。因此我们加入不同浓度顺铂48小时后,MTT方法检测细胞的存活率,发现PDCD5高表达组细胞存活率明显低于空载体对照组,当顺铂浓度为12.5μg或25μg时,差异具有显著统计学意义,提示过表达PDCD5后U87胶质瘤细胞对顺铂敏感性增强。
同时应用低浓度顺铂长时间作用于PDCD5过表达的U87细胞及空载体组细胞后,活细胞计数发现PDCD5过表达组细胞明显低于对照组,差异显著(p＜0.05),形态学上细胞出现向正常胶质细胞再分化的趋势。进一步进行克隆形成实验发现在低浓度顺铂维持下,过表达PDCD5的U87细胞其克隆形成能力明显低于对照组,差异显著。
进一步,我们选用其他化疗药物进行上述实验,发现另一种铂类药物卡铂及长春新碱的化疗敏感性也有相应提高,而依托泊甙的化疗敏感性较对照组相比没有显著差异。
三、PDCD5沉默表达对胶质瘤细胞生长及化疗敏感性的影响
1.PDCD5特异性siRNA可有效沉默PDCD5的表达
通过设计合成两条PDCD5 siRNA片段,采用转染的方法将siRNA导入U251细胞和稳定表达外源性PDCD5的U87细胞后,应用RT-PCR及Western blot的方法检测PDCD5的沉默效果。结果显示PDCD5特异性的siRNA-2(siPDCD5-2)沉默作用更为明显,因此选取siPDCD5-2进行后续实验。
2.PDCD5沉默表达对胶质瘤细胞的形态和生长速率无明显影响
倒置显微镜下观察发现PDCD5沉默表达后两种胶质瘤细胞系形态均没有明显改变,通过活细胞计数绘制生长曲线发现在同一时间点细胞增殖同对照组相比没有明显差异(p＞0.05)。
3.PDCD5沉默表达后胶质瘤细胞对顺铂的化疗敏感性降低
采用转染的方法将siPDCD5-2导入胶质瘤细胞系U87,48小时后加入不同浓度顺铂,作用48小时后再进一步应用MTT法检测细胞的存活率,发现PDCD5表达后对顺铂化疗敏感性降低。同时U251细胞经siPDCD5-2作用后对顺铂的化疗敏感性也有相应降低。
四、外源性PDCD5增强化疗敏感性的机制研究
在前述结果中我们发现PDCD5过表达能增强顺铂等对胶质瘤细胞的敏感性,而应用siRNA技术沉默PDCD5的表达后其对药物的敏感性降低。PDCD5作用的具体的机制是什么?是否通过凋亡通路进行?因此我们又做了如下研究
1.外源性PDCD5的过表达对细胞凋亡率的影响
首先通过应用Hoechst方法检测外源性PDCD5表达对细胞凋亡率的影响观察发现在同一浓度的顺铂作用下(25μg/ml或50μg/ml),过表达PDCD5的U87细胞凋亡显著,出现明显的染色质凝集、核固缩,而空载体对照组则没出现明显凋亡。
进一步采用TUNEL方法应用流式细胞仪检测细胞晚期凋亡率。流式结果显示在顺铂为25μg/ml时,PDCD5转染组细胞凋亡率达到了44.22%,而空载体对照组没有出现明显晚期凋亡;同样,在顺铂为50μg/ml时,也得到一致的结果。
2.外源性PDCD5过表达对凋亡通路蛋白活性影响
为了更详细揭示PDCD5促进细胞凋亡的机制,采用Western blot的方法进一步研究凋亡通路的关键性蛋白caspase-3、caspase-8、caspase-9及抗凋亡的Bcl-2蛋白和促凋亡蛋白Bax的表达。结果显示与空载体对照组相比较,PDCD5转染组活性caspase-3、caspase-9蛋白量明显升高,具有显著差异。而caspase-8活性没有明显差别。同时Bcl-2蛋白明显降低,但Bax蛋白没有明显差异,Bcl-2/Bax比值降低,具有显著差异,提示PDCD5主要是通过促进线粒体通路凋亡来增强胶质瘤细胞对顺铂的化疗敏感性。
五、PDCD5对凋亡相关基因PDCD4的促进效应
1.PDCD5与PDCD4共表达对胶质瘤病人预后的影响
PDCD4是近几年新发现的凋亡相关基因,我们课题组已经发现PDCD4在胶质细胞瘤中高频缺失且与病人的不良预后有关,本课题发现PDCD5在胶质瘤中表达明显降低,为了确定促凋亡基因PDCD5与凋亡相关基因PDCD4在神经胶质瘤发生、进展中的关系,我们首先分析了PDCD5与PDCD4的表达与病人预后之间的关系。发现在PDCD4阳性表达组,PDCD5高表达病人其预后率要好于PDCD5低表达组(p＜0.05),而在PDCD4阴性组,PDCD5的表达高低与病人的预后没有明显联系;进一步对PDCD5高表达病人组的生存率进行分析,发现PDCD4阳性表达组其生存率明显高于PDCD4阴性组(p＜0.05),而PDCD5低表达组病人的预后与PDCD4的表达没有显著联系。这些说明PDCD4和PDCD5共表达可提高胶质瘤病人的生存率,二者之间可能具有协同作用。
2.体外PDCD5、PDCD4共表达对胶质瘤细胞生长的影响
PDCD4基因具有诱导细胞凋亡的作用,而PDCD5基因能够促进细胞的凋亡,那么这两个基因是否相互作用,PDCD5是否能促进PDCD4引起的凋亡呢?我们在体外进行了初步研究。通过基因转染将PDCD4或空载体导入我们已建立的稳定表达PDCD5或空载体的U87细胞系,24小时后观察荧光显微镜下观察细胞转染效果,发现空载体组及PDCD4转染组都有明显的绿色荧光。在PDCD5低表达细胞组,转染PDCD4的胶质瘤细胞生长状态差于转染空载体组细胞;而在PDCD5高表达细胞组,转染PDCD4的胶质瘤细胞生长状态与空载体对照组比较,细胞有变圆,变小、皱缩甚至碎裂。进行活细胞计数发现同其他三个对照组相比,高表达PDCD5且共表达PDCD4的细胞明显生长明显减缓,提示PDCD5和PDCD4联合作用时能更好的抑制胶质瘤细胞的生长。
3.凋亡相关蛋白的改变
Bcl-2家族成员是细胞凋亡基因家族中重要的组成部分,Bcl-2家族成员如Bcl-2,Bax表达在线粒体上以抑制或促进凋亡。因此我们进一步检测凋亡相关蛋白Bcl-2、Bax的变化。通过半定量分析后发现同PDCD5~(low)PDCD4~-、PDCD5~(low)PDCD4~+、PDCD5~(high)PDCD4~-组细胞比较PDCD5~(high)PDCD4~+组细胞即PDCD5与PDCD4共表达时Bcl-2/Bax比值降低的更为明显,表明PDCD5、PDCD4协同作用时可能通过促进细胞凋亡的发生更好地抑制胶质瘤细胞生长。
结论
1.PDCD4在人原发性胶质瘤中表达显著降低;并且这种降低表达与高级别(Ⅲ-Ⅳ级)的神经胶质瘤密切相关。
2.从正反两方面证明PDCD5能增强胶质瘤细胞对化疗药物的敏感性。
3.PDCD5主要是通过线粒体通路来促进顺铂引起的凋亡以增强胶质瘤细胞系对顺铂的化疗敏感性。
4.PDCD5能促进PDCD4对胶质瘤细胞的抑制作用。
创新性和意义
1.在国内外首先发现PDCD5在胶质瘤中表达降低,而且与高级别(Ⅲ-Ⅳ级)的神经胶质瘤密切相关。
2.将PDCD5基础与临床应用联系起来,对PDCD5表达与药物化疗敏感性之间的关系进行了系统的研究,(1)PDCD5能够增强胶质瘤细胞系对化疗药物(顺铂)的敏感性,而沉默PDCD5的表达则降低其化疗敏感性,从正反两方面有力的证明了PDCD5的增敏作用。(2)在分子机制方面进一步研究发现PDCD5主要是通过线粒体通路促进顺铂诱导的凋亡从而增强其化疗敏感性。这为将PDCD5作为化疗增敏剂应用于胶质瘤的临床治疗提供了有力的证据。
3.首次发现PDCD5与PDCD4两个基因共表达时明显提高胶质瘤病人的生存率,进一步体外实验发现PDCD5与PDCD4共表达对胶质瘤细胞的生长具有更好的抑制作用,提示其在胶质瘤进展中具有潜在的协同促进作用,为判断病人的预后及转归提供更好的依据。
研究的局限性
1.PDCD5促进顺铂化疗敏感性的体内效应有待进一步研究。
2.导致PDCD5在神经胶质瘤中出现降低表达的可能原因尚有待进一步证明。
3.PDCD5、PDCD4在胶质瘤进展中的协同作用机制还有待进一步研究。
Objective
Glioma is one of the common tumors in central nervous system,which accounts for approximately 35.12%～61.10%of all intracranial tumor.It has such characters as high morbidity,high relapse rate,high mortality and low cure rate.Although the main medical technology such as operation,radiotherapy and chemotherapy has been used for cancer treatments,the prognosis of patients with glioma has remained dismal.As nearly half of gliomas are histologically malignant and most of gliomas are infiltrative growth,patients still cannot avoid relapse even when the fully excision of tumor under the microscope and imaging is performed.Therefore,new diagnostic and therapeutic strategies are required in order to control this devastating disease. Chemotherapy is still the main protocols for glioma other than surgery and radiation.But there are still some barriers for this therapy,mainly because of the adverse side effects of the anticancer agents and the development of chemoresistance. So it requires sequential use of a number of therapeutic agents in an attempt to increase efficacy while maintaining anticancer drugs tolerable toxicity.Studies of molecular biology for tumor are key issue to get insight into the mechanism underlying the pathogenesis of glioma and thus,lead to more effective methods.It is might be of important value to seek for target genes which could enhance chemosensitivity of glioma.
Programmed cell death 5(PDCD5),also designated as TF-1 cell apoptosis-related gene-19(TFAR19),is a new identified gene which facilitates apoptosis triggered by certain stimuli.Recent evidences indicate that the expression of PDCD5 protein is down-regulated in some human tumors such as ovarian cancer, gastric cancer,and renal clear cell carcinoma.How about the status of PDCD5 in gliomas and what an effect of PDCD5 is on the progression of glioma? Whether PDCD5 could be one of valuable target genes for glioma chemosensitivity? All these problems remain to be investigated.
In this study,we analyzed the relationship between PDCD5 expression and glioma progression according to four main points:
1.The status of PDCD5 expression in human primary gliomas and its clinical significance
2.The effect of PDCD5 expression on the growth and chemosensitivity of glioma derived cell lines
3.The mechanism for PDCD5 influencing chemosensitivity
4.The relationship between PDCD5 and other apoptosis related genes(such as PDCD4)
Method
1.The expression status and clinical significance of PDCD5 in human primary gliomas
(1) Case collection:a total of 88 glioma specimens including 30 frozen and 58 paraffin-embedded tissues obtained from patients aged between 30 and 60 years who underwent surgery at the department of Neurosurgery in Qilu Hospital of Shandong University during year 2003-2006.None of the patient had received adjuvant treatments such as radiotherapy or chemotherapy treatment prior to surgery in order to eliminate their effects on gene expression.Also 3 normal nervous tissues were excised from tumor-adjacent sites.
(2) The expression of PDCD5 mRNA and protein in 88 glioma tissues and glioma derived cell lines U251 and U87 were detected by RT-PCR and western blot.
(3) The correlation between PDCD5 expression and the pathological characteristics was analyzed byχ~2 test.Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test.P<0.05 was considered statistically significant.The calculation was performed using the SPSS statistical software.
2.The effect of exogenous PDCD5 overexpression or silenced expression on the growth and chemosensitivity of glioma cell lines
1.To determine the effect of PDCD5 on the growth and survival of glioma cells, a recombinant plasmid carrying the full-length PDCD5 cDNA transfected into U87 glioma cells which with low expression of PDCD5.Then transfected cells were selected by G418.The analysis of PDCD5 expression after transfection was by RT-PCR,Western blot and immunocytochemistry.
2.The morphology detection of the glioma cell line after PDCD5 transfection by microscope.The growth curve was detected by viable cell counts.
3.Analysis of the chemosensitivity of PDCD5 transfected cells and controlled cells at different concentration of chemotherapeutic agents(such as cisplatin) by MTT assay.
4.The morphology detection for the growth of transfected cells treated with long-term low concentration cisplatin by microscope.
5.Synthesis of siRNA targeted PDCD5(HPLC purity >95%) and non-silencing control siRNA.Transfected U251 cells and U87 cells with siRNA,then examined the expression of PDCD5 by RT-PCR and Western blot after 48 hours.
6.The morphology detection for the growth of cells transfected with siRNA,and viable cell counts for the proliferation of glioma cells.
7.Analysis of the chemosensitivity of U87 cells and U251 cells with silenced expression of PDCD5 by MTT assay.
3.The mechanism for PDCD5 enhancing CDDP chemosensitivity on glioma derived cell line
(1) The morphology apoptosis was analyzed by Hoechst and late apoptosis was detected by TUNEL assay.
(2) The apoptosis pathway was tested by Western blot of cleaved caspase-3, cleaved caspase-8,cleaved caspase-9,Bcl-2 and Bax.
4.The facilitating effect of PDCD5 on apoptosis related gene PDCD4
(1) Transfected glioma cell line(with high expression of PDCD5 or low expression of PDCD5) with PDCD4 plasmid or empty vector,and then examined PDCD4 expression by RT-PCR or Western blot.
(2) The morphology detection for the growth of glioma cells transfected with PDCD4 or empty vector by fluorescence microscope.
(3) Detection of changes of apoptosis related protein Bcl-2,Bax.
Result
1.The expression of PDCD5 in human primary gliomas and its clinical significance
(1) Expression of PDCD5 at mRNA and protein levels in glioma cell lines
We first examined the expression of PDCD5 in glioma derived cell lines U87 and U251 by semi-quantitative RT-PCR and Western blot.The result showed that U87 cells with high expression of PDCD5 while U251 cells with low expression of PDCD5,indicating that PDCD5 may involve in the development of glioma.
(2) The expression of PDCD5 in human primary gliomas
In order to explore the role of PDCD5,we then collected samples of human primary gliomas.PDCD5 expression at mRNA and protein levels in primary gliomas was also detected by semi-quantitative RT-PCR,Western blot and immunohistochemi -stry.
It showed that adjacent tissues of gliomas expressed high levels of PDCD5 both at mRNA and protein levels.Decreased expression of PDCD5 at mRNA level was detected in 16 fresh samples,the decreased rated was 53.33%(16/30);The expression of PDCD5 at protein level also decreased,about 83.33%(25/30),which consistent with the result of mRNA.
In order to validate the decreased expression of PDCD5 in gliomas,we explored the status and location of PDCD5 expression by imunohistochemistry in all 88 samples.The result also showed the consistency with mRNA and protein result: adjacent tissues expressed high levels of PDCD5,while tumor tissues expressed low levels of PDCD5.
(3) Relationship between PDCD5 expression and pathological characteristics for patients with gliomas
To examine the prognostic value of PDCD5 expression in primary gliomas,we studied a large cohort of patients with this disease.No significant correlation between PDCD5 expression and age,gender and histological type.However,the expression of PDCD5 correlated significantly with the pathological grade(p<0.001).Within the same histological glioma(astrocytoma) type,the rate of reduced expression of PDCD5 was higher in high-grade gliomas(Ⅲ-Ⅳ) than that in low-grade gliomas(Ⅰ-Ⅱ) (86.00%,43/50 vs.45.16%,14/31).Furthermore,we analyzed the association of the overall survival of the astrocytoma patients with low or high PDCD5 expression. However,as judged by the Kaplan-Meier analysis,PDCD5 expression had no statistically significant impact on the prognosis of astrocytoma patients(p>0.1). Similarly,no prognostic value of PDCD5 expression can be detected for patients with same grades of gliomas.
2.The effect of exogenous PDCD5 expression on growth and chemosensitivity of glioma derived cell line
To further determine effect of PDCD5 expression on glioma progress,we overexpressed PDCD5 in glioma cell line U87 by transfection with exogenous PDCD5 and analyzed its effect on tumor growth.
(1) The expression of PDCD5 in glioma cells stably transfected with PDCD5 recombinant plasmid
High level expression of PDCD5 was detected in U87 cells transfected with PDCD5 compared with untreated control or mock-transfected groups by RT-PCR, Western blot and immunocytochemistry.
(2) The exogenous expression of PDCD5 has no influence on morphology and growth of glioma cells
The morphology of U87 cells transfected with PDCD5 showed little variation compared with untreated control or empty vector transfected groups,also we found no significance for the growth curve among the U87 cells transfected with PDCD5, untreated control or empty vector transfected groups(p>0.05).
(3) The exogenous overexpression of PDCD5 enhances the chemosensitivity of glioma derived cell line
We tested the cell viability of U87 cells transfected with PDCD5 or MOCK control treated with different concentration of CDDP by MTT assay.The cell viability of PDCD5 overexpressed U87 cells was lower than that of mock control.The deference had statistics significance when the concentration of CDDP was 12.5μg/mlor 25μg/ml.
We also examined effect of PDCD5 on chemosensitivity of other chemotherapeutic drugs.Consistent with CDDP,the chemosensitivity of U87 cells transfected with PDCD5 for CBDCA and VCR was higher than mock control,having statistics significance,while no significant changes was detected in U87 cells transfected with PDCD5 when treated by VP-16.
We then treated the U87 cells with low concentration of CDDP,lower viable cell counts was detected in PDCD5 overexpressed compared with mock control.Also the morphology had the differentiation tendency toward normal glial cells. Remarkably,PDCD5 transfection significantly diminished the capacity of these cells to form colonies treated with low concentration CDDP.
3.The effect of PDCD5 silenced expression on growth and chemosensitivity of glioma derived cell line
(1) Silenced expression of PDCD5 in glioma cell lines both at mRNA and protein levels
Non-silencing siRNA or the two candidate siRNAs targeted PDCD5 (siPDCD5-1,-2) were transfected into PDCD5 transfected U87 cells or U251 cells.48 hours after transfection,both PDCD5 mRNA and protein levels were significantly decreased in those cells that were transfected with siPDCD5-1 or siPDCD5-2,as assessed by RT-PCR and Western blot.Effect of siPDCD5-2 was more obvious,so we selected it for future experiment.
(2) Silenced expression of PDCD5 has no influence on the growth of glioma derived cell line
The morphology of glioma cells transfected with siPDCD5 showed little variation compared with non-silencing or untreated groups,also we found no significance for the proliferation among the glioma cells transfected with siPDCD5 (p>0.05).
(3) Silenced expression of PDCD5 decreases the chemosensitivity of glioma cell lines to CDDP
Transfected U87 cells with exogenous PDCD5 expression with siPDCD5 or non-silencing siRNA and then subjected to CDDP and analyzed chemosensitivity by MTT assay.The viable cell rates of U87 cells transfected with PDCD5 were greatly restored at the concentration of CDDP at 10μg/ml or 12.5μg/ml.The viable cell rates of U251 cells also increased when transfected with siRNA targeted PDCD5,which means the chemosensitivity to CDDP decreased.
4.The mechanism for PDCD5 enhancing chemosensitivity of glioma cells on CDDP
(1) The effect of exogenous PDCD5 overexpression on apoptosis
To furthermore determine the mechanism of PDCD5 enhancing chemosensitivity of CDDP,we tested the apoptosis of U87 cells transfected with PDCD5 or mock control.Hoechst assay revealed that PDCD5-transfected glioma cells appeared conspicuous nuclear condense and underwent more apoptosis than the mock control.
Furthermore,TUNEL analysis also clearly showed that PDCD5-transfected glioma cells appeared underwent more apoptosis(44.22%) than the mock control (0.16%) at 25μg/ml of CDDP.Similar results were achieved when at 50μg/ml of CDDP.
(2) The influence of exogenous PDCD5 overexpression on the activity of apoptosis related protein
In order to further expatiate the mechanism for enhanced apoptosis by combination with PDCD5 and CDDP,we further detected the cleavage of caspase-3 caspase-8 and caspase-9,also protein of Bcl-2 and Bax was determined by Western blot.Compared to mock control,Western blot analysis showed that the cleavage of caspase-3,caspase-9 increased greatly in PDCD5 transfected cells compared with controlled groups,whereas caspase-8 showed little difference among the three groups. Expression of Bcl-2 protein also showed obvious decreased in PDCD5 transfected cells,while Bax had no remarkable difference,the ratio of Bcl-2/Bax decreased obviously in PDCD5 stably transfected group.
5.The facilitating effect of PDCD5 on apoptosis related gene PDCD4
(1) The effect of PDCD5 and PDCD4 expression on the prognosis of glioma patients
We further studied the role of PDCD5,PDCD4 in the development of glioma in order to explore their relationship.We found that in PDCD4~(positive) group,glioma patients with high PDCD5 expression had better prognosis than that with low expression of PDCD5(p<0.05),while in the PDCD4~(negative) groups,glioma patients with high PDCD5 had no better prognosis than that with low expression of PDCD5. Further in the PDCD5~(high) groups,glioma patients with PDCD4 expression had better prognosis than that with no expression of PDCD4(p<0.05),meanwhile in the PDCD5~(low) Wgroups,glioma patients with PDCD4 expression had no better prognosis than that with no expression of PDCD4,these result suggested that PDCD5 may facilitate the role of PDCD4.
(2) The effect of PDCD5 and PDCD4 expression on the growth of glioma cells in vitro
PDCD5 is a pro-apoptosis factor while PDCD4 could induce the apoptosis,but whether they had potential interaction remained unknown.Preliminary study was done in vitro.PDCD4 was transfected into U87 cells with high or low expression of PDCD5,and then the morphology was observed by fluorescence microscope.In PDCD5~(low) group,the cells with co-expression of PDCD4 had poor status than the cells with no expression of PDCD4;In PDCD5~(high)group,the cells with co-expression of PDCD4 had poor status than the cells with no expression of PDCD4,with cells turning round,shrinkage.Viable cell counts showed that the group with co-expression of PDCD4 and high PDCD5 had slower growth than another three groups,which suggested that PDCD5 and PDCD4 coeffect could enhance the inhibition of glioma cells.
(3) The impact of expression of PDCD5 and PDCD4 on the apoptosis related protein
The balance of proapoptotic and antiapoptotic Bcl-2 family members regulates the susceptibility of cells to apoptosis.The change of the ratio of Bcl-2/Bax may mean the mitochondrial dysfunction with alterations in the permeability transition pore,the release of cytochrome c and apoptosis taken place.So we further detected the expression of Bcl-2 and Bax.By semi-quantitative analysis,the value of Bcl-2/Bax decreased more obviously in PDCD5~(high)PDCD4 p~(sitive)group of cells compared with another three groups.
Conclusion
1.Expression of PDCD5 was decreased in primary human glioma tissues and the decreased expression of PDCD5 correlated significantly with the high grade (Ⅲ-Ⅳ) of astrocytoma.
2.We demonstrated that the expression of PDCD5 could enhance the chemosensitivity of CDDP on glioma cell lines from two aspects.
3.Exogenous PDCD5 expression enhanced the chemosensitivity of glioma cell line on CDDP mainly by promoting apoptosis of mitochondrial pathway.
4.PDCD5 could enhance the inhibition of PDCD4 to glioma cells in vitro.
Originality
1.We demonstrate,for the first time,that decreased expression of PDCD5 is a frequent event in human glioma and that the expression of PDCD5 significantly correlated with the grade of astrocytomas,which suggested that PDCD5 may involve in the pathological process of glioma.
2.We studied the expression of PDCD5 and chemosensitivity in gliomas for the first time.(1) PDCD5 overexpression enhanced the chemosensitivity of glioma cell line to CDDP,while silenced expression of PDCD5 decreased the chemosensitivity,which indicated the potential value of PDCD5 as a sensitizer in glioma treatment. (2)Exogenous PDCD5 overexpression mainly by promoting apoptosis of mitochondrial pathway to exert its role on chemosensitivity,which provide first hand data for the gene therapy targeted PDCD5 for glioma.
3.For the first time we found that PDCD5 and PDCD4 co-expression was valuable to the prognosis of glioma patients and better inhibited the glioma cells growth in vitro,which predict the potential role of PDCD5 and PDCD4 for prognosis and outcome of glioma patients.
Limitations of this study
1.The promoting effect of PDCD5 on CDDP chemosensitivity in vivo and potential clinical application value needs further to be demonstrated.
2.The mechanism of decreased PDCD5 expression in gliomas needs further study.
脑胶质瘤是中枢神经系统中最常见的恶性肿瘤之一,发生率约占所有颅内肿瘤的35.12-61.10%,具有发病率、复发率、死亡率高和治愈率低等“三高一低”的特点。目前临床治疗胶质瘤的方法主要是手术治疗、放疗、化疗,近几十年来,尽管新的诊疗技术不断出现,但由于大多数胶质瘤呈浸润性生长,与脑组织无明显分界,手术即使达到显微镜下和影像学全切除,复发还是在所难免,病人的总体生存率没有明显提高。胶质瘤术后化疗是胶质瘤综合治疗中的一个重要环节,特别是对进一步杀灭残留肿瘤细胞,防止肿瘤复发和延长患者生存时间起着十分重要的作用。然而,临床上相当一部分病人常规化疗疗效欠佳,过去认为是由于受血脑屏障的影响,化疗药物难以到达肿瘤细胞。近年来研究发现,影响脑胶质瘤化疗效果有多方面的原因,其中化疗药物产生耐药性是化疗失败的重要原因之一。而如何提高化疗药物的敏感性则成为胶质瘤治疗的瓶颈。随着分子生物学研究的深入,为神经胶质瘤的基因研究方面提供了广阔的前景,而寻找影响化疗敏感性的基因,对于胶质瘤的治疗具有重要价值,是目前一个亟待解决的问题。
PDCD5是近年来发现的新基因,在凋亡诱导因素存在下能够明显促进细胞凋亡的发生,研究结果显示卵巢癌、胃癌、肾透明细胞癌等肿瘤中PDCD5表达下调,那么PDCD5在人神经胶质瘤组织中的表达状况如何?PDCD5在神经胶质瘤进展中是否发挥作用?能否影响胶质瘤的化疗敏感性并成为胶质瘤基因治疗的靶点之一?尚不清楚。
为了明确PDCD5基因与人神经胶质瘤发生、发展的关系,为进一步以此为靶点进行基因治疗提供实验依据,本论文从以下几个方面进行了探讨:
1.PDCD5在人神经胶质瘤中的表达状态及其临床意义
2.PDCD5表达对胶质瘤细胞生长及化疗敏感性的影响
3.PDCD5影响胶质瘤细胞化疗敏感性的分子机制
4.PDCD5与凋亡相关基因(如PDCD4)的关系
方法
一、PDCD5在人神经胶质瘤组织中的表达及其临床意义
1.病例收集:收集山东大学齐鲁医院神经外科2003～2006年神经胶质瘤患者的新鲜标本共30例包括其中3例患者的相应癌旁组织,以及石蜡包埋的肿瘤标本58例。所有患者术前均未接受任何放、化疗治疗。
2.分别用RT-PCR及Western blot方法检测神经胶质瘤细胞系U251、U87及神经胶质瘤组织中PDCD5mRNA及蛋白质的表达。
3.χ~2检验分析PDCD5在不同胶质瘤病例中的表达与胶质瘤临床病理生理学特征之间的关系,Kaplan-Meier生存曲线分析PDCD5的表达与胶质瘤患者生存率之间的关系。
二、PDCD5过表达或沉默表达对胶质瘤细胞系生长及化疗敏感性的影响
1.用空载体或PDCD5重组表达载体转染胶质瘤细胞系,应用RT-PCR及Western blot及免疫细胞化学方法检测转染后PDCD5的表达,建立低表达及高表达PDCD5的胶质瘤细胞模型。
2.倒置显微镜观察转染PDCD5后神经胶质瘤细胞系形态、生长分化的变化,绘制生长曲线。
3.采用不同的化疗药(顺铂等),设定不同浓度,用MTT法检测稳定转染空载体或PDCD5的胶质瘤细胞系生存率变化。
4.克隆形成实验观察PDCD5转染后胶质瘤细胞系在长期低浓度化疗药处理时细胞集落形成的情况。
5.设计合成PDCD5特异的siRNA(HPLC纯度＞95%),应用siRNA沉默胶质瘤细胞系中PDCD5的表达,转染48小时后在mRNA及蛋白质水平检测PDCD5的表达。
6.倒置显微镜观察和活细胞计数分析siRNA沉默PDCD5的表达后神经胶质瘤细胞形态、增殖的变化,绘制生长曲线。
7.MTT法检测siRNA沉默PDCD5的表达后对胶质瘤细胞系化疗敏感性的影响。
三、PDCD5影响胶质瘤细胞化疗敏感性的机制研究
1.化疗药处理后的PDCD5及空载体稳定转染细胞系的凋亡检测:通过Hoechst染色荧光显微镜下观察细胞凋亡的形态学变化;流式细胞术分析细胞晚期凋亡率变化。
2.凋亡通路蛋白检测:应用Western blot方法检测凋亡相关蛋白caspase-3、caspase-8、caspase-9的活性变化及Bcl-2、Bax蛋白表达变化。
四、PDCD5对凋亡相关基因PDCD4的影响
1.用空载体或PDCD4重组表达载体转染稳定表达外源性PDCD5或空载体的U87胶质瘤细胞系,应用RT-PCR及Western blot方法检测瞬时转染后PDCD4的表达。
2.荧光显微镜观察瞬时转染PDCD4后神经胶质瘤细胞系形态、生长的变化,绘制生长曲线。
3.应用Western blot方法检测凋亡相关蛋白Bcl-2、Bax蛋白表达变化。
结果
一、PDCD5在人神经胶质瘤中的表达及其临床意义
1.PDCD5在人神经胶质瘤细胞系中的表达
为了研究PDCD5在人神经胶质瘤中的作用,首先应用RT-PCR及Western blot的方法检测胶质瘤细胞系U87及U251中PDCD5的表达,结果显示在U251细胞系中PDCD5表达较高,而在U87细胞系中PDCD5 mRNA及蛋白质则均低表达,提示PDCD5有可能参与影响胶质瘤的进展过程。
2.PDCD5在人原发性神经胶质瘤中表达降低
为了更深入研究PDCD5在人神经胶质细胞瘤中的作用,我们进一步收集胶质瘤临床病例,应用RT-PCR及Western blot方法对30例新鲜原发神经胶质肿瘤及3例神经胶质瘤中的PDCD5进行检测,同时用免疫组织化学的方法分析了PDCD5在88例神经胶质细胞瘤中的表达部位及表达状态。
结果显示癌旁组织表达高水平的PDCD5,而30例新鲜胶质瘤组织中有16例组织中PDCD5 mRNA表达降低,低表达率为53.33%(16/30)。Western blot结果显示PDCD5蛋白肿瘤组织中低表达率达83.33%(25/30),高于其mRNA,这提示有的肿瘤组织尽管PDCD5 mRNA的表达水平接近正常组织,但PDCD5蛋白表达仍存在明显降低,从而造成PDCD5的表达缺陷。
为了证实神经胶质瘤组织中PDCD5的降低表达情况,为分析PDCD5的表达与病人临床病理特征和预后关系奠定基础,进一步采用免疫组织化学的方法检测88例胶质瘤组织中PDCD5的表达,表达趋势与Western blot一致,癌旁组织表达较高水平PDCD5,而肿瘤组织PDCD5蛋白则出现明显的降低表达趋势。
3.PDCD5的降低表达与胶质瘤临床病理特征及预后的关系
为了进一步揭示PDCD5在胶质瘤发生、发展中的作用,我们对PDCD5的表达水平与临床病理特征进行了详细分析,结果显示不同性别、年龄、组织学类型的神经胶质瘤标本中PDCD5的表达没有显著差异;然而PDCD5的降低表达与肿瘤病理分级密切相关,在同一组织类型胶质瘤标本即星形胶质瘤的不同病理分级中,低级别(Ⅰ-Ⅱ)胶质瘤标本中,PDCD5降低表达占45.16%(14/31),而高级别(Ⅲ-Ⅳ)组织标本中PDCD5降低表达占86.00%(43/50),不同级别间PDCD5表达有显著差异(p＜0.01);然而,临床随访结果显示PDCD5的表达水平与星形胶质瘤病人总的生存率之间没有明显联系(p＞0.05),在不同级别星形胶质瘤中,PDCD5的表达与病人的生存率之间统计之后也没有明显统计学意义。
二、外源性PDCD5的过表达对胶质瘤细胞生长及化疗敏感性的影响
第一部分研究显示PDCD5在大部分神经胶质瘤中表达降低。已有研究报道在胃癌,宫颈癌,肺癌等肿瘤中均存在PDCD5的表达降低,同时,对PDCD5的作用研究显示其是一个促凋亡基因,提示PDCD5可能参与了肿瘤的发生、发展过程。为了进一步确定PDCD5在胶质瘤发生、发展中的作用,我们从临床应用的角度出发,应用基因转染技术使外源性PDCD5在低表达PDCD5的神经胶质瘤细胞系中表达,并分析其对胶质瘤细胞生长的影响。
1.外源性PDCD5在胶质瘤细胞中的稳定表达
通过基因转染分别将空载体及PDCD5重组表达载体瞬时转染U87细胞系,并应用G418进行稳筛,建立稳定表达的细胞系,通过RT-PCR、Western blot及免疫细胞化学方法检测后发现,与空细胞及空载体对照组相比,PDCD5转染组U87细胞高表达PDCD5。
2.外源性PDCD5的过表达对胶质瘤细胞的形态及生长无明显影响
显微镜下观察稳定筛选后过表达PDCD5的U87细胞,其形态与空细胞和空载体相比没有明显改变;通过生长曲线发现转染PDCD5后U87细胞的增殖未受到明显影响。
3.外源性PDCD5过表达可增强胶质瘤细胞化疗敏感性
PDCD5是一个促凋亡因子,在有凋亡因素存在情况下发挥促凋亡作用。因此我们加入不同浓度顺铂48小时后,MTT方法检测细胞的存活率,发现PDCD5高表达组细胞存活率明显低于空载体对照组,当顺铂浓度为12.5μg或25μg时,差异具有显著统计学意义,提示过表达PDCD5后U87胶质瘤细胞对顺铂敏感性增强。
同时应用低浓度顺铂长时间作用于PDCD5过表达的U87细胞及空载体组细胞后,活细胞计数发现PDCD5过表达组细胞明显低于对照组,差异显著(p＜0.05),形态学上细胞出现向正常胶质细胞再分化的趋势。进一步进行克隆形成实验发现在低浓度顺铂维持下,过表达PDCD5的U87细胞其克隆形成能力明显低于对照组,差异显著。
进一步,我们选用其他化疗药物进行上述实验,发现另一种铂类药物卡铂及长春新碱的化疗敏感性也有相应提高,而依托泊甙的化疗敏感性较对照组相比没有显著差异。
三、PDCD5沉默表达对胶质瘤细胞生长及化疗敏感性的影响
1.PDCD5特异性siRNA可有效沉默PDCD5的表达
通过设计合成两条PDCD5 siRNA片段,采用转染的方法将siRNA导入U251细胞和稳定表达外源性PDCD5的U87细胞后,应用RT-PCR及Western blot的方法检测PDCD5的沉默效果。结果显示PDCD5特异性的siRNA-2(siPDCD5-2)沉默作用更为明显,因此选取siPDCD5-2进行后续实验。
2.PDCD5沉默表达对胶质瘤细胞的形态和生长速率无明显影响
倒置显微镜下观察发现PDCD5沉默表达后两种胶质瘤细胞系形态均没有明显改变,通过活细胞计数绘制生长曲线发现在同一时间点细胞增殖同对照组相比没有明显差异(p＞0.05)。
3.PDCD5沉默表达后胶质瘤细胞对顺铂的化疗敏感性降低
采用转染的方法将siPDCD5-2导入胶质瘤细胞系U87,48小时后加入不同浓度顺铂,作用48小时后再进一步应用MTT法检测细胞的存活率,发现PDCD5表达后对顺铂化疗敏感性降低。同时U251细胞经siPDCD5-2作用后对顺铂的化疗敏感性也有相应降低。
四、外源性PDCD5增强化疗敏感性的机制研究
在前述结果中我们发现PDCD5过表达能增强顺铂等对胶质瘤细胞的敏感性,而应用siRNA技术沉默PDCD5的表达后其对药物的敏感性降低。PDCD5作用的具体的机制是什么?是否通过凋亡通路进行?因此我们又做了如下研究
1.外源性PDCD5的过表达对细胞凋亡率的影响
首先通过应用Hoechst方法检测外源性PDCD5表达对细胞凋亡率的影响观察发现在同一浓度的顺铂作用下(25μg/ml或50μg/ml),过表达PDCD5的U87细胞凋亡显著,出现明显的染色质凝集、核固缩,而空载体对照组则没出现明显凋亡。
进一步采用TUNEL方法应用流式细胞仪检测细胞晚期凋亡率。流式结果显示在顺铂为25μg/ml时,PDCD5转染组细胞凋亡率达到了44.22%,而空载体对照组没有出现明显晚期凋亡;同样,在顺铂为50μg/ml时,也得到一致的结果。
2.外源性PDCD5过表达对凋亡通路蛋白活性影响
为了更详细揭示PDCD5促进细胞凋亡的机制,采用Western blot的方法进一步研究凋亡通路的关键性蛋白caspase-3、caspase-8、caspase-9及抗凋亡的Bcl-2蛋白和促凋亡蛋白Bax的表达。结果显示与空载体对照组相比较,PDCD5转染组活性caspase-3、caspase-9蛋白量明显升高,具有显著差异。而caspase-8活性没有明显差别。同时Bcl-2蛋白明显降低,但Bax蛋白没有明显差异,Bcl-2/Bax比值降低,具有显著差异,提示PDCD5主要是通过促进线粒体通路凋亡来增强胶质瘤细胞对顺铂的化疗敏感性。
五、PDCD5对凋亡相关基因PDCD4的促进效应
1.PDCD5与PDCD4共表达对胶质瘤病人预后的影响
PDCD4是近几年新发现的凋亡相关基因,我们课题组已经发现PDCD4在胶质细胞瘤中高频缺失且与病人的不良预后有关,本课题发现PDCD5在胶质瘤中表达明显降低,为了确定促凋亡基因PDCD5与凋亡相关基因PDCD4在神经胶质瘤发生、进展中的关系,我们首先分析了PDCD5与PDCD4的表达与病人预后之间的关系。发现在PDCD4阳性表达组,PDCD5高表达病人其预后率要好于PDCD5低表达组(p＜0.05),而在PDCD4阴性组,PDCD5的表达高低与病人的预后没有明显联系;进一步对PDCD5高表达病人组的生存率进行分析,发现PDCD4阳性表达组其生存率明显高于PDCD4阴性组(p＜0.05),而PDCD5低表达组病人的预后与PDCD4的表达没有显著联系。这些说明PDCD4和PDCD5共表达可提高胶质瘤病人的生存率,二者之间可能具有协同作用。
2.体外PDCD5、PDCD4共表达对胶质瘤细胞生长的影响
PDCD4基因具有诱导细胞凋亡的作用,而PDCD5基因能够促进细胞的凋亡,那么这两个基因是否相互作用,PDCD5是否能促进PDCD4引起的凋亡呢?我们在体外进行了初步研究。通过基因转染将PDCD4或空载体导入我们已建立的稳定表达PDCD5或空载体的U87细胞系,24小时后观察荧光显微镜下观察细胞转染效果,发现空载体组及PDCD4转染组都有明显的绿色荧光。在PDCD5低表达细胞组,转染PDCD4的胶质瘤细胞生长状态差于转染空载体组细胞;而在PDCD5高表达细胞组,转染PDCD4的胶质瘤细胞生长状态与空载体对照组比较,细胞有变圆,变小、皱缩甚至碎裂。进行活细胞计数发现同其他三个对照组相比,高表达PDCD5且共表达PDCD4的细胞明显生长明显减缓,提示PDCD5和PDCD4联合作用时能更好的抑制胶质瘤细胞的生长。
3.凋亡相关蛋白的改变
Bcl-2家族成员是细胞凋亡基因家族中重要的组成部分,Bcl-2家族成员如Bcl-2,Bax表达在线粒体上以抑制或促进凋亡。因此我们进一步检测凋亡相关蛋白Bcl-2、Bax的变化。通过半定量分析后发现同PDCD5~(low)PDCD4~-、PDCD5~(low)PDCD4~+、PDCD5~(high)PDCD4~-组细胞比较PDCD5~(high)PDCD4~+组细胞即PDCD5与PDCD4共表达时Bcl-2/Bax比值降低的更为明显,表明PDCD5、PDCD4协同作用时可能通过促进细胞凋亡的发生更好地抑制胶质瘤细胞生长。
结论
1.PDCD4在人原发性胶质瘤中表达显著降低;并且这种降低表达与高级别(Ⅲ-Ⅳ级)的神经胶质瘤密切相关。
2.从正反两方面证明PDCD5能增强胶质瘤细胞对化疗药物的敏感性。
3.PDCD5主要是通过线粒体通路来促进顺铂引起的凋亡以增强胶质瘤细胞系对顺铂的化疗敏感性。
4.PDCD5能促进PDCD4对胶质瘤细胞的抑制作用。
创新性和意义
1.在国内外首先发现PDCD5在胶质瘤中表达降低,而且与高级别(Ⅲ-Ⅳ级)的神经胶质瘤密切相关。
2.将PDCD5基础与临床应用联系起来,对PDCD5表达与药物化疗敏感性之间的关系进行了系统的研究,(1)PDCD5能够增强胶质瘤细胞系对化疗药物(顺铂)的敏感性,而沉默PDCD5的表达则降低其化疗敏感性,从正反两方面有力的证明了PDCD5的增敏作用。(2)在分子机制方面进一步研究发现PDCD5主要是通过线粒体通路促进顺铂诱导的凋亡从而增强其化疗敏感性。这为将PDCD5作为化疗增敏剂应用于胶质瘤的临床治疗提供了有力的证据。
3.首次发现PDCD5与PDCD4两个基因共表达时明显提高胶质瘤病人的生存率,进一步体外实验发现PDCD5与PDCD4共表达对胶质瘤细胞的生长具有更好的抑制作用,提示其在胶质瘤进展中具有潜在的协同促进作用,为判断病人的预后及转归提供更好的依据。
研究的局限性
1.PDCD5促进顺铂化疗敏感性的体内效应有待进一步研究。
2.导致PDCD5在神经胶质瘤中出现降低表达的可能原因尚有待进一步证明。
3.PDCD5、PDCD4在胶质瘤进展中的协同作用机制还有待进一步研究。
Objective
Glioma is one of the common tumors in central nervous system,which accounts for approximately 35.12%～61.10%of all intracranial tumor.It has such characters as high morbidity,high relapse rate,high mortality and low cure rate.Although the main medical technology such as operation,radiotherapy and chemotherapy has been used for cancer treatments,the prognosis of patients with glioma has remained dismal.As nearly half of gliomas are histologically malignant and most of gliomas are infiltrative growth,patients still cannot avoid relapse even when the fully excision of tumor under the microscope and imaging is performed.Therefore,new diagnostic and therapeutic strategies are required in order to control this devastating disease. Chemotherapy is still the main protocols for glioma other than surgery and radiation.But there are still some barriers for this therapy,mainly because of the adverse side effects of the anticancer agents and the development of chemoresistance. So it requires sequential use of a number of therapeutic agents in an attempt to increase efficacy while maintaining anticancer drugs tolerable toxicity.Studies of molecular biology for tumor are key issue to get insight into the mechanism underlying the pathogenesis of glioma and thus,lead to more effective methods.It is might be of important value to seek for target genes which could enhance chemosensitivity of glioma.
Programmed cell death 5(PDCD5),also designated as TF-1 cell apoptosis-related gene-19(TFAR19),is a new identified gene which facilitates apoptosis triggered by certain stimuli.Recent evidences indicate that the expression of PDCD5 protein is down-regulated in some human tumors such as ovarian cancer, gastric cancer,and renal clear cell carcinoma.How about the status of PDCD5 in gliomas and what an effect of PDCD5 is on the progression of glioma? Whether PDCD5 could be one of valuable target genes for glioma chemosensitivity? All these problems remain to be investigated.
In this study,we analyzed the relationship between PDCD5 expression and glioma progression according to four main points:
1.The status of PDCD5 expression in human primary gliomas and its clinical significance
2.The effect of PDCD5 expression on the growth and chemosensitivity of glioma derived cell lines
3.The mechanism for PDCD5 influencing chemosensitivity
4.The relationship between PDCD5 and other apoptosis related genes(such as PDCD4)
Method
1.The expression status and clinical significance of PDCD5 in human primary gliomas
(1) Case collection:a total of 88 glioma specimens including 30 frozen and 58 paraffin-embedded tissues obtained from patients aged between 30 and 60 years who underwent surgery at the department of Neurosurgery in Qilu Hospital of Shandong University during year 2003-2006.None of the patient had received adjuvant treatments such as radiotherapy or chemotherapy treatment prior to surgery in order to eliminate their effects on gene expression.Also 3 normal nervous tissues were excised from tumor-adjacent sites.
(2) The expression of PDCD5 mRNA and protein in 88 glioma tissues and glioma derived cell lines U251 and U87 were detected by RT-PCR and western blot.
(3) The correlation between PDCD5 expression and the pathological characteristics was analyzed byχ~2 test.Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test.P<0.05 was considered statistically significant.The calculation was performed using the SPSS statistical software.
2.The effect of exogenous PDCD5 overexpression or silenced expression on the growth and chemosensitivity of glioma cell lines
1.To determine the effect of PDCD5 on the growth and survival of glioma cells, a recombinant plasmid carrying the full-length PDCD5 cDNA transfected into U87 glioma cells which with low expression of PDCD5.Then transfected cells were selected by G418.The analysis of PDCD5 expression after transfection was by RT-PCR,Western blot and immunocytochemistry.
2.The morphology detection of the glioma cell line after PDCD5 transfection by microscope.The growth curve was detected by viable cell counts.
3.Analysis of the chemosensitivity of PDCD5 transfected cells and controlled cells at different concentration of chemotherapeutic agents(such as cisplatin) by MTT assay.
4.The morphology detection for the growth of transfected cells treated with long-term low concentration cisplatin by microscope.
5.Synthesis of siRNA targeted PDCD5(HPLC purity >95%) and non-silencing control siRNA.Transfected U251 cells and U87 cells with siRNA,then examined the expression of PDCD5 by RT-PCR and Western blot after 48 hours.
6.The morphology detection for the growth of cells transfected with siRNA,and viable cell counts for the proliferation of glioma cells.
7.Analysis of the chemosensitivity of U87 cells and U251 cells with silenced expression of PDCD5 by MTT assay.
3.The mechanism for PDCD5 enhancing CDDP chemosensitivity on glioma derived cell line
(1) The morphology apoptosis was analyzed by Hoechst and late apoptosis was detected by TUNEL assay.
(2) The apoptosis pathway was tested by Western blot of cleaved caspase-3, cleaved caspase-8,cleaved caspase-9,Bcl-2 and Bax.
4.The facilitating effect of PDCD5 on apoptosis related gene PDCD4
(1) Transfected glioma cell line(with high expression of PDCD5 or low expression of PDCD5) with PDCD4 plasmid or empty vector,and then examined PDCD4 expression by RT-PCR or Western blot.
(2) The morphology detection for the growth of glioma cells transfected with PDCD4 or empty vector by fluorescence microscope.
(3) Detection of changes of apoptosis related protein Bcl-2,Bax.
Result
1.The expression of PDCD5 in human primary gliomas and its clinical significance
(1) Expression of PDCD5 at mRNA and protein levels in glioma cell lines
We first examined the expression of PDCD5 in glioma derived cell lines U87 and U251 by semi-quantitative RT-PCR and Western blot.The result showed that U87 cells with high expression of PDCD5 while U251 cells with low expression of PDCD5,indicating that PDCD5 may involve in the development of glioma.
(2) The expression of PDCD5 in human primary gliomas
In order to explore the role of PDCD5,we then collected samples of human primary gliomas.PDCD5 expression at mRNA and protein levels in primary gliomas was also detected by semi-quantitative RT-PCR,Western blot and immunohistochemi -stry.
It showed that adjacent tissues of gliomas expressed high levels of PDCD5 both at mRNA and protein levels.Decreased expression of PDCD5 at mRNA level was detected in 16 fresh samples,the decreased rated was 53.33%(16/30);The expression of PDCD5 at protein level also decreased,about 83.33%(25/30),which consistent with the result of mRNA.
In order to validate the decreased expression of PDCD5 in gliomas,we explored the status and location of PDCD5 expression by imunohistochemistry in all 88 samples.The result also showed the consistency with mRNA and protein result: adjacent tissues expressed high levels of PDCD5,while tumor tissues expressed low levels of PDCD5.
(3) Relationship between PDCD5 expression and pathological characteristics for patients with gliomas
To examine the prognostic value of PDCD5 expression in primary gliomas,we studied a large cohort of patients with this disease.No significant correlation between PDCD5 expression and age,gender and histological type.However,the expression of PDCD5 correlated significantly with the pathological grade(p<0.001).Within the same histological glioma(astrocytoma) type,the rate of reduced expression of PDCD5 was higher in high-grade gliomas(Ⅲ-Ⅳ) than that in low-grade gliomas(Ⅰ-Ⅱ) (86.00%,43/50 vs.45.16%,14/31).Furthermore,we analyzed the association of the overall survival of the astrocytoma patients with low or high PDCD5 expression. However,as judged by the Kaplan-Meier analysis,PDCD5 expression had no statistically significant impact on the prognosis of astrocytoma patients(p>0.1). Similarly,no prognostic value of PDCD5 expression can be detected for patients with same grades of gliomas.
2.The effect of exogenous PDCD5 expression on growth and chemosensitivity of glioma derived cell line
To further determine effect of PDCD5 expression on glioma progress,we overexpressed PDCD5 in glioma cell line U87 by transfection with exogenous PDCD5 and analyzed its effect on tumor growth.
(1) The expression of PDCD5 in glioma cells stably transfected with PDCD5 recombinant plasmid
High level expression of PDCD5 was detected in U87 cells transfected with PDCD5 compared with untreated control or mock-transfected groups by RT-PCR, Western blot and immunocytochemistry.
(2) The exogenous expression of PDCD5 has no influence on morphology and growth of glioma cells
The morphology of U87 cells transfected with PDCD5 showed little variation compared with untreated control or empty vector transfected groups,also we found no significance for the growth curve among the U87 cells transfected with PDCD5, untreated control or empty vector transfected groups(p>0.05).
(3) The exogenous overexpression of PDCD5 enhances the chemosensitivity of glioma derived cell line
We tested the cell viability of U87 cells transfected with PDCD5 or MOCK control treated with different concentration of CDDP by MTT assay.The cell viability of PDCD5 overexpressed U87 cells was lower than that of mock control.The deference had statistics significance when the concentration of CDDP was 12.5μg/mlor 25μg/ml.
We also examined effect of PDCD5 on chemosensitivity of other chemotherapeutic drugs.Consistent with CDDP,the chemosensitivity of U87 cells transfected with PDCD5 for CBDCA and VCR was higher than mock control,having statistics significance,while no significant changes was detected in U87 cells transfected with PDCD5 when treated by VP-16.
We then treated the U87 cells with low concentration of CDDP,lower viable cell counts was detected in PDCD5 overexpressed compared with mock control.Also the morphology had the differentiation tendency toward normal glial cells. Remarkably,PDCD5 transfection significantly diminished the capacity of these cells to form colonies treated with low concentration CDDP.
3.The effect of PDCD5 silenced expression on growth and chemosensitivity of glioma derived cell line
(1) Silenced expression of PDCD5 in glioma cell lines both at mRNA and protein levels
Non-silencing siRNA or the two candidate siRNAs targeted PDCD5 (siPDCD5-1,-2) were transfected into PDCD5 transfected U87 cells or U251 cells.48 hours after transfection,both PDCD5 mRNA and protein levels were significantly decreased in those cells that were transfected with siPDCD5-1 or siPDCD5-2,as assessed by RT-PCR and Western blot.Effect of siPDCD5-2 was more obvious,so we selected it for future experiment.
(2) Silenced expression of PDCD5 has no influence on the growth of glioma derived cell line
The morphology of glioma cells transfected with siPDCD5 showed little variation compared with non-silencing or untreated groups,also we found no significance for the proliferation among the glioma cells transfected with siPDCD5 (p>0.05).
(3) Silenced expression of PDCD5 decreases the chemosensitivity of glioma cell lines to CDDP
Transfected U87 cells with exogenous PDCD5 expression with siPDCD5 or non-silencing siRNA and then subjected to CDDP and analyzed chemosensitivity by MTT assay.The viable cell rates of U87 cells transfected with PDCD5 were greatly restored at the concentration of CDDP at 10μg/ml or 12.5μg/ml.The viable cell rates of U251 cells also increased when transfected with siRNA targeted PDCD5,which means the chemosensitivity to CDDP decreased.
4.The mechanism for PDCD5 enhancing chemosensitivity of glioma cells on CDDP
(1) The effect of exogenous PDCD5 overexpression on apoptosis
To furthermore determine the mechanism of PDCD5 enhancing chemosensitivity of CDDP,we tested the apoptosis of U87 cells transfected with PDCD5 or mock control.Hoechst assay revealed that PDCD5-transfected glioma cells appeared conspicuous nuclear condense and underwent more apoptosis than the mock control.
Furthermore,TUNEL analysis also clearly showed that PDCD5-transfected glioma cells appeared underwent more apoptosis(44.22%) than the mock control (0.16%) at 25μg/ml of CDDP.Similar results were achieved when at 50μg/ml of CDDP.
(2) The influence of exogenous PDCD5 overexpression on the activity of apoptosis related protein
In order to further expatiate the mechanism for enhanced apoptosis by combination with PDCD5 and CDDP,we further detected the cleavage of caspase-3 caspase-8 and caspase-9,also protein of Bcl-2 and Bax was determined by Western blot.Compared to mock control,Western blot analysis showed that the cleavage of caspase-3,caspase-9 increased greatly in PDCD5 transfected cells compared with controlled groups,whereas caspase-8 showed little difference among the three groups. Expression of Bcl-2 protein also showed obvious decreased in PDCD5 transfected cells,while Bax had no remarkable difference,the ratio of Bcl-2/Bax decreased obviously in PDCD5 stably transfected group.
5.The facilitating effect of PDCD5 on apoptosis related gene PDCD4
(1) The effect of PDCD5 and PDCD4 expression on the prognosis of glioma patients
We further studied the role of PDCD5,PDCD4 in the development of glioma in order to explore their relationship.We found that in PDCD4~(positive) group,glioma patients with high PDCD5 expression had better prognosis than that with low expression of PDCD5(p<0.05),while in the PDCD4~(negative) groups,glioma patients with high PDCD5 had no better prognosis than that with low expression of PDCD5. Further in the PDCD5~(high) groups,glioma patients with PDCD4 expression had better prognosis than that with no expression of PDCD4(p<0.05),meanwhile in the PDCD5~(low) Wgroups,glioma patients with PDCD4 expression had no better prognosis than that with no expression of PDCD4,these result suggested that PDCD5 may facilitate the role of PDCD4.
(2) The effect of PDCD5 and PDCD4 expression on the growth of glioma cells in vitro
PDCD5 is a pro-apoptosis factor while PDCD4 could induce the apoptosis,but whether they had potential interaction remained unknown.Preliminary study was done in vitro.PDCD4 was transfected into U87 cells with high or low expression of PDCD5,and then the morphology was observed by fluorescence microscope.In PDCD5~(low) group,the cells with co-expression of PDCD4 had poor status than the cells with no expression of PDCD4;In PDCD5~(high)group,the cells with co-expression of PDCD4 had poor status than the cells with no expression of PDCD4,with cells turning round,shrinkage.Viable cell counts showed that the group with co-expression of PDCD4 and high PDCD5 had slower growth than another three groups,which suggested that PDCD5 and PDCD4 coeffect could enhance the inhibition of glioma cells.
(3) The impact of expression of PDCD5 and PDCD4 on the apoptosis related protein
The balance of proapoptotic and antiapoptotic Bcl-2 family members regulates the susceptibility of cells to apoptosis.The change of the ratio of Bcl-2/Bax may mean the mitochondrial dysfunction with alterations in the permeability transition pore,the release of cytochrome c and apoptosis taken place.So we further detected the expression of Bcl-2 and Bax.By semi-quantitative analysis,the value of Bcl-2/Bax decreased more obviously in PDCD5~(high)PDCD4 p~(sitive)group of cells compared with another three groups.
Conclusion
1.Expression of PDCD5 was decreased in primary human glioma tissues and the decreased expression of PDCD5 correlated significantly with the high grade (Ⅲ-Ⅳ) of astrocytoma.
2.We demonstrated that the expression of PDCD5 could enhance the chemosensitivity of CDDP on glioma cell lines from two aspects.
3.Exogenous PDCD5 expression enhanced the chemosensitivity of glioma cell line on CDDP mainly by promoting apoptosis of mitochondrial pathway.
4.PDCD5 could enhance the inhibition of PDCD4 to glioma cells in vitro.
Originality
1.We demonstrate,for the first time,that decreased expression of PDCD5 is a frequent event in human glioma and that the expression of PDCD5 significantly correlated with the grade of astrocytomas,which suggested that PDCD5 may involve in the pathological process of glioma.
2.We studied the expression of PDCD5 and chemosensitivity in gliomas for the first time.(1) PDCD5 overexpression enhanced the chemosensitivity of glioma cell line to CDDP,while silenced expression of PDCD5 decreased the chemosensitivity,which indicated the potential value of PDCD5 as a sensitizer in glioma treatment. (2)Exogenous PDCD5 overexpression mainly by promoting apoptosis of mitochondrial pathway to exert its role on chemosensitivity,which provide first hand data for the gene therapy targeted PDCD5 for glioma.
3.For the first time we found that PDCD5 and PDCD4 co-expression was valuable to the prognosis of glioma patients and better inhibited the glioma cells growth in vitro,which predict the potential role of PDCD5 and PDCD4 for prognosis and outcome of glioma patients.
Limitations of this study
1.The promoting effect of PDCD5 on CDDP chemosensitivity in vivo and potential clinical application value needs further to be demonstrated.
2.The mechanism of decreased PDCD5 expression in gliomas needs further study.
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