p53选择性剪接异构体在肾细胞癌中的表达及其临床意义的研究
摘要
研究背景和目的
肾细胞癌是常见的发生在肾脏的恶性肿瘤。每年在欧洲大约有2万例新增肾癌病人,而在美国每年新增的肾癌患者达到3万人,近年来发病率呈上升的趋势。在中国肾癌的发病率低于欧美,但其发病率和检出率也在逐年上升。肾癌是多基因相关的肿瘤,其发病机制至今不清,在遗传学上可分为散发性肾癌和家族性肾癌。肾癌的发病机制极其复杂,无论是家族性还是散发性肾癌其发生与体内抑癌基因的失活密不可分。p53是迄今为止已发现的和人类肿瘤相关性最高的抑癌基因,也是研究最多的抑癌基因,它在调节细胞的生长周期、发育、DNA的修复、细胞的凋亡等方面起着重要作用。野生型p53基因(wild tipe wtp53)具有维持基因组稳定、抑制或阻止细胞转化从而抑制肿瘤发生的作用。正常p53功能的改变和缺失与不同种类的肿瘤有密切关系。很多研究已经证实,约有50%以上的肿瘤患者发现p53基因的突变,p53基因的突变与肿瘤的发生、化疗药物的抵抗性以及不良的预后密切相关。目前在人类很多组织的肿瘤中检测到了p53基因的突变,有报道约80%以上的小细胞肺癌存在p53基因的突变,结肠癌中超过70%发生p53基因突变,肝癌中也有超过50%以上的p53基因突变。但与其他肿瘤相比,肾癌患者中p53的突变率并不高,有报道肾癌中p53的基因突变率大概为3.33%。由此可见在肾癌的发生中可能存在其他的机制导致了抑癌基因p53的活性的改变或缺失,了解p53基因失活的途径有助于研究。肾癌的发病机理,从而为肾癌的早期诊断和治疗提供新的思路,这也是本课题选题的基础和依据。
近年来随着p53家族基因p63、p73以及它们各自选择性剪接异构体的相继发现,对p53作用以及其调节机制的研究逐渐深入。与p53密切相关的家族性基因p63、p73,它们在DNA结合区(DNA-binding domain DBD)的序列与p53有很大的相似性,并对p53的易感基因具有反式激活作用,可引起细胞周期的停滞和凋亡。现在的研究还证实虽然p53、p63和p73三者作用密切相关但他们自己还有独特的生物学作用,p63、p73基因中有些剪接异构体可以显性负调节p53的活性,起着类似癌基因的作用。
选择性剪接是指一个前体mRNA通过选择不同的剪接位点组合产生不同mRNA剪接变异体的过程,经常发生在隐蔽的位点,致使外显子遗漏和(或)内含子保留。选择性剪接是基因表达的转录后水平的调控,是控制基因表达的重要机制,它使基因表达呈现多样性,很多研究证实选择性剪接和细胞的生理、发育调控以及疾病的发生有关。通过选择性启动子和多种剪接形式,p53现今比较明确的至少可以表达包括有p53、p53β、p53γ、Δ133p53、Δ133p53p、Δ133p53γ、Δ40p53、Δ40p53β和Δ40p53γ九种剪接异构体。这些异构体在人体组织中的表达情况和作用机制尚不完全清楚。以往的研究显示,这些剪接异构体在正常人类组织中有不同程度的表达,而这种表达有组织特异性的特点,还证实p53剪接异构体和全长p53之间有着及其复杂的相互作用机制,它们不但可以调节p53的转录活性而且本身还具有不同生物学作用。有关p53剪接异构体在乳腺癌、神经细胞肿瘤、急性白血病、头颈部鳞癌等肿瘤组织中的表达情况的研究,提示p53异构体的异常表达可能与人类多种肿瘤的发生、发展有密切关系。
据我们所知,目前国内外尚没有具体关于p53剪接异构体在肾癌中的表达情况和作用机制的研究。本课题借鉴以往的研究经验,选取相对比较稳定表达的包括p53、p53β、p53γ、Δ133p53、Δ133p53β和Δ133p53γ六种异构体为研究对象,旨在分析这六种p53剪接异构体在肾癌组织以及癌旁正常肾脏组织中mRNA和蛋白的表达情况,并通过联系肾癌的临床、病理指标进行统计学分析,探讨p53及其选择性剪接异构体与肾癌的发生发展的关系,进一步研究肾癌的发病机制,争取为肾癌的早期诊断和免疫基因治疗探索出新的思路和方法。
材料与方法:
选取2007年6月至2008年3月间,在山东省立医院泌尿外科行肾癌根治术的肾细胞癌患者47例。所有标本均行病理学检查,根据WHO 1997年制定的肾实质上皮性肿瘤分类标准,其中透明细胞癌41例,嗜色细胞癌5例,嫌色细胞癌1例。所取癌旁肾组织,距肿瘤边缘至少大于2cm,而且行病理检查均未发现癌细胞。所有患者术前均未接受任何的放疗、化疗。患者中,男性32例,女性15例,年龄在15-84岁之间,平均年龄56岁。肿瘤的分期应用2002年国际抗癌协会TNM分期标准,T1期:24人;T2期:8人;T3期:11人;T4期;4人。核分级应用Fuhrman分级标准,G1级:7人;G2级:32人;G3级:5人:G4级:3人。所取每一标本分为3份,其中一份用10%福尔马林固定后,石蜡包埋做病理切片用于免疫组化分析,另外两份新鲜组织标本经液氮快速冷冻后保存到-80℃冰箱中,用于逆转录PCR(RT-PCR)以及Western Blot检测。
本研究分为两部分进行。
第一部分应用nested RT-PCR法分析p53剪接异构体在肾细胞癌和癌旁正常肾组织中mRNA的表达情况和临床意义。首先采用Trizol法提取肾癌组织和正常肾组织总RNA,应用20gl逆转录反应体系(其中含总RNA 1μg)反转录合成cDNA,应用特定的引物和巢式PCR技术在适当条件下扩增。共35个循环后,然后产物应用琼脂糖凝胶电泳,分析电泳结果。以β-actin作为内参照。对比肾癌组织和癌旁肾组织的剪接异构体mRNA的表达情况,统计学分析其表达差异,并结合肾癌的细胞类型、分级和临床分期分析各剪接异构体的表达与肾癌分级、分期的关系,进而探讨p53异构体在肾细胞癌和正常肾组织中的表达情况和意义。
第二部分应用免疫组化法以及Western Blot分析p53及其剪接异构体在。肾癌组织以及癌旁正常。肾组织中的蛋白水平的表达。首先行免疫组化实验,对47例肿瘤标本分别应用Anti-p53(识别位点在372-382位氨基酸序列,故可以识别p53和Δ133p53)、DO-1(识别位点在20-25位氨基酸序列,故可以识别p53、p53β和p53γ,三种异构体)和DO-12(识别位点DNA结合区256-270位氨基酸序列,故可以识别所有p53异构体)三种抗体作为一抗,采用SABC方法进行免疫组化染色。β-actin作为内参照。不加入一抗作阴性对照。按照染色肿瘤细胞占总细胞的百分比将评分分为1-4级:其中1级为0-10%;2级为11-25%;3级为26-50%;4级为＞50%,1-2级为低表达,3-4级为高表达。按照一抗分为三组,每一组根据p53蛋白的表达情况分为不表达、低表达、高表达。统计学分析三组抗体p53蛋白阳性率与肾癌的临床分期、病理分级的关系。Western Blot实验分别应用DO-1和DO-12两种抗体进行,过氧化物酶标的山羊抗小鼠IgG为二抗。应用高分辨率化学发光胶片进行信号检测。通过检测异构体蛋白条带来研究各p53异构体在肾癌组织以及癌旁正常肾组织的内源性蛋白水平的表达。
统计学分析应用χ~2检验或Fisher精确检验。检验水准α=0.05。所有统计学分析使用统计软件SPSS 11.0完成。
结果
第一部分,通过设计特定的引物并利用巢式PCR技术,在肿瘤组织中我们检测出所有p53、p53β、p53γ、Δ133p53、A133p53β和Δ133p53γ,六种比较确定的p53剪接异构体,而在正常肾组织中检测出了除Δ133p53β以外的另外5种异构体。p53 mRNA无论是在肾癌组织中还是癌旁组织中全部表达。对比各剪接异构体在肾癌组织和癌旁正常肾组织的表达,我们发现p53β(35T/16N)在肾癌组织中表达高于癌旁组织,其差别经卡方检验有统计学意义(p＜0.001)。六种异构体中Δ133p53βmRNA表达最低,在正常肾组织中未检测到,并且在肿瘤组织中也只检测到2例弱阳性表达。Δ1331953(38T/30N)在肿瘤组织和癌旁组织中的表达也很高,表达率分别为,80.9%、63.8%,二者相比无统计学意义。其余异构体在正常组织和肿瘤组织中有不同程度的表达,p53γ(21T/25N)、Δ133p53γ,(15T/10N),但其差异均无统计学意义。将异构体mRNA的表达情况联系肾癌的病理类型、分级以及临床分期,发现p53β表达与肾癌的临床分期有关,与病理类型和分级无关。
第二部分,免疫组化结果显示在正常肾组织中,所有标本对这三种抗体均无免疫显色反应,p53蛋白均为阴性。在肾癌组织中应用这三种抗体进行免疫染色,在Anti-p53组有18例出现核深染的阳性反应,DO-1抗体组11例阳性,DO-12抗体组13例阳性,统计学分析三组差别无意义。对同一标本应用三种不同抗体为一抗行免疫组化,其染色结果差别不大。联系临床病理指标,Anti-p53和DO-12抗体组显示p53蛋白阳性率与肿瘤的分期、分级都有相关性,而DO-1抗体组显示p53蛋白阳性率只与肿瘤的分期有相关性。Western Blot结果还显示出了p53剪接异构体蛋白的表达与mRNA的表达存在不一致性。在一些肿瘤组织标本中,应用DO-12抗体检测到了在分子量分别约53KD、46KD、35KD以及25KD的特征性的条带,而应用DO-1抗体检测到了分子量分别位于53KD和46KD的特征性条带,这些条带分别对应着相应分子量的p53蛋白异构体,证实了p53剪接异构体在肾癌组织中有内源性的蛋白表达。
结论:
(1)肾癌组织和在正常肾组织中p53剪接异构体mRNA的表达有差异,Δ133p53β在正常肾组织中不表达,p53β在肾癌组织中的表达显著高于正常肾组织,差异具有统计学意义。其余异构体均有不同程度的表达。
(2)p53βmRNA的表达与肾细胞癌的分期有关,提示它可能和肾细胞癌的发生、发展有密切关系,p53β的表达情况可协助肾癌的分期。
(3)p53剪接异构体在肾细胞癌组织中有不同程度的蛋白内源性表达,蛋白的表达和mRNA的表达不一致。
(4)p53蛋白的表达的阳性率与肾细胞癌的分期有关,与细胞类型无关
(5)p53β可以进一步研究作为肾细胞癌新的诊断分子标记和基因治疗的新的靶点
Background and objects
p53 gene is one of the most important human tumor suppressor and transcription factor genes to tumorigenesis.It is a key regulator in preventing cancer formation and plays a vital role in cell cycling,growth,DNA repair,cell cycle arrest,or apoptosis.Thus WTp53 has been classed as a "guardian of the genome" due to its ability to coordinate multiple and diverse signaling pathways involved in this response.Mutation of p53 is a common occurrence in many cancers and is associated with tumor progression,resistance to chemotherapy,and poor prognosis.
Renal cell carcinoma(RCC) is the most common malignancy in adult kidney,with 30,000 new cases per year in the U.S.and 20,000 cases in the European Union.In China,the incidence of RCC is second only to bladder turnout in all malignant tumours in the urinary system. According to a new report,there are more than 23,000 new case of RCC per year in china,and the incidence is increasing rapidly due to the aging population and high smoking and obesity rates,p53 appears mutation in about 50%of many human cancers,while in RCC,there is a pretty low incidence of p53 mutation.It has been reported in 3-33%of patients with renal-cell carcinoma.There may be a postulated the existence of a novel dominant mechanism of inactivation of p53 in renal cells carcinoma.
Recently p53-related genes,p63,p73 and p53 splicing isoforms have been discovered,the role of the p53 regulatory mechanism was gradually in depth study.The high level of sequence similarity in the DNA-binding domain between p53 protein family members allows p63 and p73 to transactivate p53-responsive genes causing cell-cycle arrest and apoptosis.However, p53,p63 and p73 proteins are not entirely functionally redundant,each of them has specific biological functions and some of their isomers can be a negative regulator of p53 function.
Alternative splicing is the RNA splicing variation mechanism in which the exons of the primary gene transcript,the pre-mRNA,are separated and reconnected so as to produce alternative ribonucleotide arrangements.It is an important mechanism for controlling gene expression which allows large proteomic complexity from a limited number of genes.Lots of research deemed that the variation of the alternative splicing have close relationship with cell physiology,development regulation,occurrence of disease,p53 gene family members can express multiple mRNA variants due to multiple splicing and alternative promoters.Hence, p53 gene family members express different forms of p53 protein containing different domain of the protein(isoforms).To date,at least nine different p53 isoforms have been identified in humans,including p53,p53β,p53γ,Δ133p53,Δ133p53β,Δ133p53γ,Δ40p53,Δ40p53βandΔ40p53γ.However,the function and expression of these isoforms are still not completely clear. In previous studies,these isoforms were differentially expressed in normal human tissue in a tissue-dependent manner.Furthermore,the expression of these isoforms in human cancers, such as breast cancer,neuroblastoma,acute myeloid leukemia,and squamous cell carcinoma of the head and neck,suggests that they may involve in tumor development or progression.
The present study has confirmed that there are complex interactions mechanism between p53 splicing isoforms and WTp53.p53 splicing isoforms can negatively regulate the activity of WTp53.The expression of p53 splicing isoforms in many human cancers have been reported however,to our knowledge,no study has addressed the expression of p53 and its isoforms in renal cell carcinoma till now.Therefore,in this paper we aimed at analyzing the expression patterns of p53 isoforms in RCC at the mRNA and protein levels,and their associations with clinical and pathologic factors to explore the mechanism of p53 isoforms in RCC.
Materials and Methods:
Materials
Tumor samples were taken after informed consent from 47 patients with renal cell carcinoma (41patients were clear cell carcinoma,5 patients were chromophil cell carcinoma and 2 patients were chromophobe cell carcinoma),who underwent radical nephrectomy from June 2007 to March 2008 in Shandong Provincial Hospital.Patients were staged according to the 2002 staging system of the International Union Against Cancer(UICC) and the nuclear grade of tumors was determined by Fuhrman grading scheme.The nonneoplastic renal tissue resected from adjacent regions of each renal cell carcinoma were also analysed for comparison.Tissue samples were divided into three,1/3 were fixed in formalin and embedded in paraffin,the other 2/3 were frozen with liquid nitrogen after being taken from the patient quickly and then reserved in the refrigerator(-80℃) until used.
Methods
This research was divided into two parts.
Part 1.Dectet the mRNA expression of each pair of tumor specimens and the adjacent nonneoplastic tissue.Total RNA was extracted using Trizol reagent according to the manufacturer protocol,then reverse transcribed RNA into cDNA.At last cDNA was amplified by nested PCR using special primer.PCR cycling conditions for p53,p53βand p53γwere 94℃for 1min,60℃for 50sec,and 72℃for 1 min for 35 cycles using Taq DNA polymerase and the recommended protocol(Fermentas International Inc.,CA).PCR cycling conditions forΔ133p53,Δ133p53βandΔ133p53γwere 94℃for 1min,58℃for 50sec,and 72℃for 1 min for 35 cycles.Expression intensity analysis was performed by 1D Image Analysis Software (Kodak,USA).The expression of mRNA in RCC tissues and normal renal tissues were compared and the difference was compared by statistic analysis.
Part 2.Dectet the isoform protein expression of each pair of tumor specimens and the adjacent nonneoplastic tissue.Immunohistochemical staining was performed in formalin fixed,paraffin embedded tissue sections.We used the avidin-biotin-peroxidase method with 1:100 diluted DO-1,1:40 diluted anti-p53 antibodies,1:50 diluted DO-12 antibody.As negative controls,normal serums were used in place of the primary antibodies.All samples were scored independently by two of the authors who were blinded to patient status.The percentage of the total number of tumor cells staining positively was categorized and given a score of 1 to 4:1,0-10%with weak intensity;2,11-25%with weak or moderate intensity; 3,26-50%with weak or moderate intensity;and 4,more than 50%with strong intensity. Western Blot analysis using DO-1 and DO-12 as primary antibodies.The membranes were hybridized with a secondary antibody conjugated with peroxidase and the chemoluminescent signal was detected using the enhanced chemiluminescence system with high-performance chemiluminescence film.
Statistical analysis was performed usingχ~2 test or Fisher's exact test as appropriate. Correlations between variables were tested according to the Spearman correlation test.P values less than 0.05 were considered significant.Two-sided tests were used throughout all the analyses.All calculations were performed using SPSS 13.0 statistical software package
Results
ⅠBy using specific primers and nested RT,all six isoforms were detected in the tumor specimens,however only the p53βmRNA was significantly overexpressed compared with the adjacent nonneoplastic tissue(P＜0.001),p53 mRNA were found in all the tumor and nonneoplastic tissues.Nevertheless,the expression ofΔ133p53βmRNA was not detectable in adjacent nonneoplastic tissue,and even in the tumor tissues it was detected at very low levels (2/47).The other isoforms expressed at different level in both the tumor and nonneoplastic tissues without statistical significance.
ⅡThe immunohistochemistry for p53 with three antibodies were performed in both tumor and nonneoplastic tissue.In nonneoplastic renal tissue,all specimens lacked immunoreactivity for the three antibodies.The expression was noted in 18,11,13 specimens for anti- p53 group, DO-1 group and DO-12 group respectively,with no significant difference.Both anti- p53 group and DO-12 group showed that p53 was associated with the stage and the grade, however,only DO-1 antibody group showed that p53 was associated with both the stage. Western Blot analysis showed that DO-1 antibody could detect p53 at 53 kD,p53βand(or) p53γat 46 kD respectively.The DO-12 antibody could detect all these six isoforms.When the tumors and clinically normal tumor adjacent tissues were analysed with DO-12,we noted that some bands located at the approximate sizes of 53KD,46KD,35KD and 25KD respectively which correspondenced with the predicted sizes of these six isoforms.The results were consistent with the immunohistochemistry result.In these immunoBlots,β-actin was used as an internal control whose Corresponding bands located at the approximate sizes of 43KD clearly.
As lack of specific antibodies for each p53 isoforms,we used the mRNA level to correlate with the clinicopathological factors.The results showed that only p53βmRNA was significant associated with tumor stage(P=0.009).
Conclusion
Ⅰp53 isoforms are expressed in RCC and the normal tissue adjacent to the tumour with different levels,p53βmRNA was significantly overexpressed compared with the adjacent nonneoplastic tissue.Δ133p53βmRNA might not be expressed in normal kidneys.
ⅡThe expression of p53βmRNA was significant associated with tumor stage.
Ⅲp53βmay not only be the most easily identified isoform in RCC,but also play a vital role in tumor formation among these isoforms.
ⅣThe expression of p53 was associated with the stage of the RCC.
Ⅴp53βmay be used as a new predictor and therapy target for RCC.
肾细胞癌是常见的发生在肾脏的恶性肿瘤。每年在欧洲大约有2万例新增肾癌病人,而在美国每年新增的肾癌患者达到3万人,近年来发病率呈上升的趋势。在中国肾癌的发病率低于欧美,但其发病率和检出率也在逐年上升。肾癌是多基因相关的肿瘤,其发病机制至今不清,在遗传学上可分为散发性肾癌和家族性肾癌。肾癌的发病机制极其复杂,无论是家族性还是散发性肾癌其发生与体内抑癌基因的失活密不可分。p53是迄今为止已发现的和人类肿瘤相关性最高的抑癌基因,也是研究最多的抑癌基因,它在调节细胞的生长周期、发育、DNA的修复、细胞的凋亡等方面起着重要作用。野生型p53基因(wild tipe wtp53)具有维持基因组稳定、抑制或阻止细胞转化从而抑制肿瘤发生的作用。正常p53功能的改变和缺失与不同种类的肿瘤有密切关系。很多研究已经证实,约有50%以上的肿瘤患者发现p53基因的突变,p53基因的突变与肿瘤的发生、化疗药物的抵抗性以及不良的预后密切相关。目前在人类很多组织的肿瘤中检测到了p53基因的突变,有报道约80%以上的小细胞肺癌存在p53基因的突变,结肠癌中超过70%发生p53基因突变,肝癌中也有超过50%以上的p53基因突变。但与其他肿瘤相比,肾癌患者中p53的突变率并不高,有报道肾癌中p53的基因突变率大概为3.33%。由此可见在肾癌的发生中可能存在其他的机制导致了抑癌基因p53的活性的改变或缺失,了解p53基因失活的途径有助于研究。肾癌的发病机理,从而为肾癌的早期诊断和治疗提供新的思路,这也是本课题选题的基础和依据。
近年来随着p53家族基因p63、p73以及它们各自选择性剪接异构体的相继发现,对p53作用以及其调节机制的研究逐渐深入。与p53密切相关的家族性基因p63、p73,它们在DNA结合区(DNA-binding domain DBD)的序列与p53有很大的相似性,并对p53的易感基因具有反式激活作用,可引起细胞周期的停滞和凋亡。现在的研究还证实虽然p53、p63和p73三者作用密切相关但他们自己还有独特的生物学作用,p63、p73基因中有些剪接异构体可以显性负调节p53的活性,起着类似癌基因的作用。
选择性剪接是指一个前体mRNA通过选择不同的剪接位点组合产生不同mRNA剪接变异体的过程,经常发生在隐蔽的位点,致使外显子遗漏和(或)内含子保留。选择性剪接是基因表达的转录后水平的调控,是控制基因表达的重要机制,它使基因表达呈现多样性,很多研究证实选择性剪接和细胞的生理、发育调控以及疾病的发生有关。通过选择性启动子和多种剪接形式,p53现今比较明确的至少可以表达包括有p53、p53β、p53γ、Δ133p53、Δ133p53p、Δ133p53γ、Δ40p53、Δ40p53β和Δ40p53γ九种剪接异构体。这些异构体在人体组织中的表达情况和作用机制尚不完全清楚。以往的研究显示,这些剪接异构体在正常人类组织中有不同程度的表达,而这种表达有组织特异性的特点,还证实p53剪接异构体和全长p53之间有着及其复杂的相互作用机制,它们不但可以调节p53的转录活性而且本身还具有不同生物学作用。有关p53剪接异构体在乳腺癌、神经细胞肿瘤、急性白血病、头颈部鳞癌等肿瘤组织中的表达情况的研究,提示p53异构体的异常表达可能与人类多种肿瘤的发生、发展有密切关系。
据我们所知,目前国内外尚没有具体关于p53剪接异构体在肾癌中的表达情况和作用机制的研究。本课题借鉴以往的研究经验,选取相对比较稳定表达的包括p53、p53β、p53γ、Δ133p53、Δ133p53β和Δ133p53γ六种异构体为研究对象,旨在分析这六种p53剪接异构体在肾癌组织以及癌旁正常肾脏组织中mRNA和蛋白的表达情况,并通过联系肾癌的临床、病理指标进行统计学分析,探讨p53及其选择性剪接异构体与肾癌的发生发展的关系,进一步研究肾癌的发病机制,争取为肾癌的早期诊断和免疫基因治疗探索出新的思路和方法。
材料与方法:
选取2007年6月至2008年3月间,在山东省立医院泌尿外科行肾癌根治术的肾细胞癌患者47例。所有标本均行病理学检查,根据WHO 1997年制定的肾实质上皮性肿瘤分类标准,其中透明细胞癌41例,嗜色细胞癌5例,嫌色细胞癌1例。所取癌旁肾组织,距肿瘤边缘至少大于2cm,而且行病理检查均未发现癌细胞。所有患者术前均未接受任何的放疗、化疗。患者中,男性32例,女性15例,年龄在15-84岁之间,平均年龄56岁。肿瘤的分期应用2002年国际抗癌协会TNM分期标准,T1期:24人;T2期:8人;T3期:11人;T4期;4人。核分级应用Fuhrman分级标准,G1级:7人;G2级:32人;G3级:5人:G4级:3人。所取每一标本分为3份,其中一份用10%福尔马林固定后,石蜡包埋做病理切片用于免疫组化分析,另外两份新鲜组织标本经液氮快速冷冻后保存到-80℃冰箱中,用于逆转录PCR(RT-PCR)以及Western Blot检测。
本研究分为两部分进行。
第一部分应用nested RT-PCR法分析p53剪接异构体在肾细胞癌和癌旁正常肾组织中mRNA的表达情况和临床意义。首先采用Trizol法提取肾癌组织和正常肾组织总RNA,应用20gl逆转录反应体系(其中含总RNA 1μg)反转录合成cDNA,应用特定的引物和巢式PCR技术在适当条件下扩增。共35个循环后,然后产物应用琼脂糖凝胶电泳,分析电泳结果。以β-actin作为内参照。对比肾癌组织和癌旁肾组织的剪接异构体mRNA的表达情况,统计学分析其表达差异,并结合肾癌的细胞类型、分级和临床分期分析各剪接异构体的表达与肾癌分级、分期的关系,进而探讨p53异构体在肾细胞癌和正常肾组织中的表达情况和意义。
第二部分应用免疫组化法以及Western Blot分析p53及其剪接异构体在。肾癌组织以及癌旁正常。肾组织中的蛋白水平的表达。首先行免疫组化实验,对47例肿瘤标本分别应用Anti-p53(识别位点在372-382位氨基酸序列,故可以识别p53和Δ133p53)、DO-1(识别位点在20-25位氨基酸序列,故可以识别p53、p53β和p53γ,三种异构体)和DO-12(识别位点DNA结合区256-270位氨基酸序列,故可以识别所有p53异构体)三种抗体作为一抗,采用SABC方法进行免疫组化染色。β-actin作为内参照。不加入一抗作阴性对照。按照染色肿瘤细胞占总细胞的百分比将评分分为1-4级:其中1级为0-10%;2级为11-25%;3级为26-50%;4级为＞50%,1-2级为低表达,3-4级为高表达。按照一抗分为三组,每一组根据p53蛋白的表达情况分为不表达、低表达、高表达。统计学分析三组抗体p53蛋白阳性率与肾癌的临床分期、病理分级的关系。Western Blot实验分别应用DO-1和DO-12两种抗体进行,过氧化物酶标的山羊抗小鼠IgG为二抗。应用高分辨率化学发光胶片进行信号检测。通过检测异构体蛋白条带来研究各p53异构体在肾癌组织以及癌旁正常肾组织的内源性蛋白水平的表达。
统计学分析应用χ~2检验或Fisher精确检验。检验水准α=0.05。所有统计学分析使用统计软件SPSS 11.0完成。
结果
第一部分,通过设计特定的引物并利用巢式PCR技术,在肿瘤组织中我们检测出所有p53、p53β、p53γ、Δ133p53、A133p53β和Δ133p53γ,六种比较确定的p53剪接异构体,而在正常肾组织中检测出了除Δ133p53β以外的另外5种异构体。p53 mRNA无论是在肾癌组织中还是癌旁组织中全部表达。对比各剪接异构体在肾癌组织和癌旁正常肾组织的表达,我们发现p53β(35T/16N)在肾癌组织中表达高于癌旁组织,其差别经卡方检验有统计学意义(p＜0.001)。六种异构体中Δ133p53βmRNA表达最低,在正常肾组织中未检测到,并且在肿瘤组织中也只检测到2例弱阳性表达。Δ1331953(38T/30N)在肿瘤组织和癌旁组织中的表达也很高,表达率分别为,80.9%、63.8%,二者相比无统计学意义。其余异构体在正常组织和肿瘤组织中有不同程度的表达,p53γ(21T/25N)、Δ133p53γ,(15T/10N),但其差异均无统计学意义。将异构体mRNA的表达情况联系肾癌的病理类型、分级以及临床分期,发现p53β表达与肾癌的临床分期有关,与病理类型和分级无关。
第二部分,免疫组化结果显示在正常肾组织中,所有标本对这三种抗体均无免疫显色反应,p53蛋白均为阴性。在肾癌组织中应用这三种抗体进行免疫染色,在Anti-p53组有18例出现核深染的阳性反应,DO-1抗体组11例阳性,DO-12抗体组13例阳性,统计学分析三组差别无意义。对同一标本应用三种不同抗体为一抗行免疫组化,其染色结果差别不大。联系临床病理指标,Anti-p53和DO-12抗体组显示p53蛋白阳性率与肿瘤的分期、分级都有相关性,而DO-1抗体组显示p53蛋白阳性率只与肿瘤的分期有相关性。Western Blot结果还显示出了p53剪接异构体蛋白的表达与mRNA的表达存在不一致性。在一些肿瘤组织标本中,应用DO-12抗体检测到了在分子量分别约53KD、46KD、35KD以及25KD的特征性的条带,而应用DO-1抗体检测到了分子量分别位于53KD和46KD的特征性条带,这些条带分别对应着相应分子量的p53蛋白异构体,证实了p53剪接异构体在肾癌组织中有内源性的蛋白表达。
结论:
(1)肾癌组织和在正常肾组织中p53剪接异构体mRNA的表达有差异,Δ133p53β在正常肾组织中不表达,p53β在肾癌组织中的表达显著高于正常肾组织,差异具有统计学意义。其余异构体均有不同程度的表达。
(2)p53βmRNA的表达与肾细胞癌的分期有关,提示它可能和肾细胞癌的发生、发展有密切关系,p53β的表达情况可协助肾癌的分期。
(3)p53剪接异构体在肾细胞癌组织中有不同程度的蛋白内源性表达,蛋白的表达和mRNA的表达不一致。
(4)p53蛋白的表达的阳性率与肾细胞癌的分期有关,与细胞类型无关
(5)p53β可以进一步研究作为肾细胞癌新的诊断分子标记和基因治疗的新的靶点
Background and objects
p53 gene is one of the most important human tumor suppressor and transcription factor genes to tumorigenesis.It is a key regulator in preventing cancer formation and plays a vital role in cell cycling,growth,DNA repair,cell cycle arrest,or apoptosis.Thus WTp53 has been classed as a "guardian of the genome" due to its ability to coordinate multiple and diverse signaling pathways involved in this response.Mutation of p53 is a common occurrence in many cancers and is associated with tumor progression,resistance to chemotherapy,and poor prognosis.
Renal cell carcinoma(RCC) is the most common malignancy in adult kidney,with 30,000 new cases per year in the U.S.and 20,000 cases in the European Union.In China,the incidence of RCC is second only to bladder turnout in all malignant tumours in the urinary system. According to a new report,there are more than 23,000 new case of RCC per year in china,and the incidence is increasing rapidly due to the aging population and high smoking and obesity rates,p53 appears mutation in about 50%of many human cancers,while in RCC,there is a pretty low incidence of p53 mutation.It has been reported in 3-33%of patients with renal-cell carcinoma.There may be a postulated the existence of a novel dominant mechanism of inactivation of p53 in renal cells carcinoma.
Recently p53-related genes,p63,p73 and p53 splicing isoforms have been discovered,the role of the p53 regulatory mechanism was gradually in depth study.The high level of sequence similarity in the DNA-binding domain between p53 protein family members allows p63 and p73 to transactivate p53-responsive genes causing cell-cycle arrest and apoptosis.However, p53,p63 and p73 proteins are not entirely functionally redundant,each of them has specific biological functions and some of their isomers can be a negative regulator of p53 function.
Alternative splicing is the RNA splicing variation mechanism in which the exons of the primary gene transcript,the pre-mRNA,are separated and reconnected so as to produce alternative ribonucleotide arrangements.It is an important mechanism for controlling gene expression which allows large proteomic complexity from a limited number of genes.Lots of research deemed that the variation of the alternative splicing have close relationship with cell physiology,development regulation,occurrence of disease,p53 gene family members can express multiple mRNA variants due to multiple splicing and alternative promoters.Hence, p53 gene family members express different forms of p53 protein containing different domain of the protein(isoforms).To date,at least nine different p53 isoforms have been identified in humans,including p53,p53β,p53γ,Δ133p53,Δ133p53β,Δ133p53γ,Δ40p53,Δ40p53βandΔ40p53γ.However,the function and expression of these isoforms are still not completely clear. In previous studies,these isoforms were differentially expressed in normal human tissue in a tissue-dependent manner.Furthermore,the expression of these isoforms in human cancers, such as breast cancer,neuroblastoma,acute myeloid leukemia,and squamous cell carcinoma of the head and neck,suggests that they may involve in tumor development or progression.
The present study has confirmed that there are complex interactions mechanism between p53 splicing isoforms and WTp53.p53 splicing isoforms can negatively regulate the activity of WTp53.The expression of p53 splicing isoforms in many human cancers have been reported however,to our knowledge,no study has addressed the expression of p53 and its isoforms in renal cell carcinoma till now.Therefore,in this paper we aimed at analyzing the expression patterns of p53 isoforms in RCC at the mRNA and protein levels,and their associations with clinical and pathologic factors to explore the mechanism of p53 isoforms in RCC.
Materials and Methods:
Materials
Tumor samples were taken after informed consent from 47 patients with renal cell carcinoma (41patients were clear cell carcinoma,5 patients were chromophil cell carcinoma and 2 patients were chromophobe cell carcinoma),who underwent radical nephrectomy from June 2007 to March 2008 in Shandong Provincial Hospital.Patients were staged according to the 2002 staging system of the International Union Against Cancer(UICC) and the nuclear grade of tumors was determined by Fuhrman grading scheme.The nonneoplastic renal tissue resected from adjacent regions of each renal cell carcinoma were also analysed for comparison.Tissue samples were divided into three,1/3 were fixed in formalin and embedded in paraffin,the other 2/3 were frozen with liquid nitrogen after being taken from the patient quickly and then reserved in the refrigerator(-80℃) until used.
Methods
This research was divided into two parts.
Part 1.Dectet the mRNA expression of each pair of tumor specimens and the adjacent nonneoplastic tissue.Total RNA was extracted using Trizol reagent according to the manufacturer protocol,then reverse transcribed RNA into cDNA.At last cDNA was amplified by nested PCR using special primer.PCR cycling conditions for p53,p53βand p53γwere 94℃for 1min,60℃for 50sec,and 72℃for 1 min for 35 cycles using Taq DNA polymerase and the recommended protocol(Fermentas International Inc.,CA).PCR cycling conditions forΔ133p53,Δ133p53βandΔ133p53γwere 94℃for 1min,58℃for 50sec,and 72℃for 1 min for 35 cycles.Expression intensity analysis was performed by 1D Image Analysis Software (Kodak,USA).The expression of mRNA in RCC tissues and normal renal tissues were compared and the difference was compared by statistic analysis.
Part 2.Dectet the isoform protein expression of each pair of tumor specimens and the adjacent nonneoplastic tissue.Immunohistochemical staining was performed in formalin fixed,paraffin embedded tissue sections.We used the avidin-biotin-peroxidase method with 1:100 diluted DO-1,1:40 diluted anti-p53 antibodies,1:50 diluted DO-12 antibody.As negative controls,normal serums were used in place of the primary antibodies.All samples were scored independently by two of the authors who were blinded to patient status.The percentage of the total number of tumor cells staining positively was categorized and given a score of 1 to 4:1,0-10%with weak intensity;2,11-25%with weak or moderate intensity; 3,26-50%with weak or moderate intensity;and 4,more than 50%with strong intensity. Western Blot analysis using DO-1 and DO-12 as primary antibodies.The membranes were hybridized with a secondary antibody conjugated with peroxidase and the chemoluminescent signal was detected using the enhanced chemiluminescence system with high-performance chemiluminescence film.
Statistical analysis was performed usingχ~2 test or Fisher's exact test as appropriate. Correlations between variables were tested according to the Spearman correlation test.P values less than 0.05 were considered significant.Two-sided tests were used throughout all the analyses.All calculations were performed using SPSS 13.0 statistical software package
Results
ⅠBy using specific primers and nested RT,all six isoforms were detected in the tumor specimens,however only the p53βmRNA was significantly overexpressed compared with the adjacent nonneoplastic tissue(P＜0.001),p53 mRNA were found in all the tumor and nonneoplastic tissues.Nevertheless,the expression ofΔ133p53βmRNA was not detectable in adjacent nonneoplastic tissue,and even in the tumor tissues it was detected at very low levels (2/47).The other isoforms expressed at different level in both the tumor and nonneoplastic tissues without statistical significance.
ⅡThe immunohistochemistry for p53 with three antibodies were performed in both tumor and nonneoplastic tissue.In nonneoplastic renal tissue,all specimens lacked immunoreactivity for the three antibodies.The expression was noted in 18,11,13 specimens for anti- p53 group, DO-1 group and DO-12 group respectively,with no significant difference.Both anti- p53 group and DO-12 group showed that p53 was associated with the stage and the grade, however,only DO-1 antibody group showed that p53 was associated with both the stage. Western Blot analysis showed that DO-1 antibody could detect p53 at 53 kD,p53βand(or) p53γat 46 kD respectively.The DO-12 antibody could detect all these six isoforms.When the tumors and clinically normal tumor adjacent tissues were analysed with DO-12,we noted that some bands located at the approximate sizes of 53KD,46KD,35KD and 25KD respectively which correspondenced with the predicted sizes of these six isoforms.The results were consistent with the immunohistochemistry result.In these immunoBlots,β-actin was used as an internal control whose Corresponding bands located at the approximate sizes of 43KD clearly.
As lack of specific antibodies for each p53 isoforms,we used the mRNA level to correlate with the clinicopathological factors.The results showed that only p53βmRNA was significant associated with tumor stage(P=0.009).
Conclusion
Ⅰp53 isoforms are expressed in RCC and the normal tissue adjacent to the tumour with different levels,p53βmRNA was significantly overexpressed compared with the adjacent nonneoplastic tissue.Δ133p53βmRNA might not be expressed in normal kidneys.
ⅡThe expression of p53βmRNA was significant associated with tumor stage.
Ⅲp53βmay not only be the most easily identified isoform in RCC,but also play a vital role in tumor formation among these isoforms.
ⅣThe expression of p53 was associated with the stage of the RCC.
Ⅴp53βmay be used as a new predictor and therapy target for RCC.
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