【作者】 康玲；

【导师】 来茂德；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 结直肠癌是严重危害人类健康的常见恶性肿瘤之一。在欧美等发达国家和地区居民恶性肿瘤发病和死因顺位中结直肠癌位居第二至第三。自Vogelstein提出经典的结直肠癌多基因、多步骤的发生发展模式以来,许多与结直肠癌发生发展相关的基因不断被揭示。近二十余年来,随着我国居民生活方式和膳食结构的改变,结直肠癌的发病率和死亡率呈上升趋势。因此,揭示隐藏在结直肠癌发生发展过程中内在的分子病理改变,将对结直肠癌发病机制的深入了解以及该病的防治具有十分重要的理论价值和现实意义。结直肠癌的发生发展是一个多基因参与、多阶段积累的复杂过程,正常结直肠上皮-腺瘤-腺癌顺序发展的分子模型过程中涉及到许多基因的改变。在这些基因中有一类属于转录因子,如TP53、DCC、SMAD4/DPC4等基因,这提示转录因子在结直肠癌发生发展过程中起了重要作用。KLF9(Krüppel-like factor 9,KLF9)锌指转录因子是我们实验室前期对结直肠癌电子表达谱进行生物信息学分析基础之上,构建整合TaqMan定量PCR和微流体技术的低密度芯片进行筛选研究时发现的在结直肠癌中低表达的基因。KLF9属于SP/KLF(specificityprotein/Krüppel-like factor)家族转录因子,它在机体重要器官的发育以及细胞增殖、分化、凋亡中发挥生物学功能。Klf9基因敲除小鼠肠隐窝细胞增殖和绒毛细胞迁移显现异常。结合我们实验室前期筛选结果,KLF9与肠隐窝细胞增殖、绒毛迁移有关以及该家族其它基因与人类常见恶性肿瘤发生发展有关的现象,使我们提出疑问,KLF9是否与结直肠癌发生发展有关呢?本课题以明确KLF9在结直肠癌中的表达为切入点,继而初步研究KLF9在结直肠癌中的生物学功能,以期探索它在结直肠癌发生发展中的作用。我们收集了2005年8月至2006年5月在浙江大学医学院附属第二医院手术并经病理学诊断为结直肠癌的病例70例,在手术过程中取得癌组织和远端切缘的正常粘膜。采用实时荧光定量PCR(Real-time PCR)、Western blot及免疫组织化学法对KLF9在结直肠癌组织及配对正常粘膜中的表达水平进行了检测。检测结果显示在mRNA水平上,正常结直肠粘膜KLF9表达明显高于相应癌组织(P＜0.0001)。Western blot结果表明KLF9蛋白在结直肠癌组织中表达水平下降或不表达(P＜0.01)。实验中我们构建了包含结直肠癌组织和正常结直肠粘膜的组织芯片。组织芯片免疫组织化学结果显示,大部分正常粘膜中可检测到KLF9阳性表达细胞(35/43),而在结直肠癌组织中,仅4例癌组织KLF9呈阳性表达(4/43),其余癌组织均无表达(P＜0.001)。KLF9表达与结直肠癌病人临床病理参数(肿瘤分化程度、浸润深度、淋巴结转移、肿瘤部位)无明显相关性(P＞0.05)。运用RT-PCR和Western blot检测了KLF9在结直肠癌细胞系中的表达。在所检测的HT29、SW480、SW620、LoVo、RKO及Caco2细胞系中,KLF9 mRNA在SW480中表达相对较强,其余细胞系中mRNA表达微弱。除Caco2细胞系外,KLF9蛋白在HT29、SW480、SW620、LoVo细胞系有微弱表达,在RKO细胞系中表达相对较强。比较所检测细胞系的KLF9 mRNA和蛋白表达水平发现,KLF9 mRNA在SW480细胞系中表达最强,但蛋白表达很微弱,RKO细胞系KLF9 mRNA表达丰度虽然较低,但蛋白表达水平在所检测细胞中却是最高的。PCR扩增结直肠癌细胞系HT29、SW480、SW620、RKOKLF9基因编码区序列后测序。测序结果进行网上比对,我们发现RKO细胞KLF9编码区存在518T＞C(F173S)突变,但此突变并不影响KLF9蛋白在RKO细胞系中的表达。采用生物信息学方法,利用NCBI、ExPASy、PBIL、SWISS-MODEL、ESyPred3D、Rosmal等在线服务网站和生物信息学软件,对RKO细胞KLF9蛋白和正常KLF9蛋白(NP 001197)分析比较结果显示,RKO细胞KLF9蛋白在一级、二级和三级结构上均与正常KLF9蛋白存在差异。HT29、SW480、SW620细胞系KLF9编码区中未检测到突变存在。基于KLF9在结直肠癌中表达下降的现象,我们对KLF9在结直肠癌中的生物学功能进行了初步探讨。在成功构建含有KLF9编码区的真核表达载体(pcDNA3.1-KLF9)基础上,借助脂质体PolyFect将KLF9转染到结直肠癌细胞系中探讨其在结直肠癌中的潜在功能。瞬时转染pcDNA3.1-KLF9到SW480、SW620、RKO及Caco2细胞系,对转染前后细胞周期和细胞凋亡进行了分析。从形态学观察转染前后的细胞形态未见明显改变。PI单染流式细胞术检测未发现KLF9对细胞周期有影响。Annexin V/PI双染流式细胞术检测也未见KLF9对细胞凋亡产生影响。Real-time PCR检测结果显示转染前后与细胞周期相关基因Cyclin D1、p21(Cip1)、CDK2、PCNA的表达水平无明显改变(P＞0.05)。稳定转染pcDNA3.1-KLF9到内源性KLF9蛋白微弱表达和不表达的SW480和Caco2细胞系中,以期筛选出稳定表达克隆进行后续研究。在三次稳定筛选过程中,当筛选进行到扩大培养阶段时,转染KLF9的细胞出现死亡,未能得到稳定表达KLF9的细胞株。通过以上关于KLF9在结直肠癌中的表达及生物学功能的初步探索,我们得出以下结论:1.KLF9 mRNA和蛋白在结直肠癌组织中的表达均下降,可能与结直肠癌的发生、发展有关;2.在结直肠癌细胞系中KLF9 mRNA和蛋白表达水平不一致,在不同细胞系中可能存在不同的转录后调控机制调控KLF9在细胞中的表达;3.无法筛选得到稳定表达KLF9基因的细胞株,推测该基因在SW480、Caco2结直肠癌细胞系中持续高表达会对细胞产生致死效应;抑或高表达KLF9使细胞对筛选药物的敏感性增加而致细胞死亡。更多还原

【Abstract】 Colorectal carcinoma（CRC）is one of the most common malignancies which threaten the health of human.It is the third common cancer and the second leading cause of cancer-related death in each year in western developed countries.Numerous genes,associated with the occurrence and development of CRC,have been found since the proposal of Vogelstein model of colorectal carcinogenesis.The incidence and mortality of CRC increase gradually with the changes of lifestyle and diet structure in China in recent two decades.It is of great value to explore the molecular changes related to CRC and investigate their roles in CRC for the prevention, diagnosis and treatment of this disease.Accumulation of genetic changes in the adenoma-carcinoma sequence is the basis for development of CRC.Alterations of three different classes of genes, tumor-suppressor genes（eg,APC,DCC,SMAD4/DPC4,TP53）,oncogenes（eg, K-ras,c-myc）and mismatch repair genes（MMRs）,are involved in colorectal carcinogenesis.Some of these genes,such as DCC,SMAD4/DPC4 and TP53,belong to transcription factors.The relationship between transcription factors and CRC imply that they play important roles in the occurrence and development of CRC. KLF9（Krüppel-like factor 9,KLF9）zinc finger transcription factor,screened from electronic gene expression libraries developed by our laboratory,is a differentially expressed gene in CRC.KLF9,which is a number of SP/KLF（specificity protein/Krüppel-like factor,SP/KLF）family transcription factors,is implicated in the control of cell proliferation,differentiation and apoptosis.Dysregulation of intestinal crypt cell proliferation and villus cell migration has been observed in mice lacking Klf9.There is growing evidence that some SP/KLF proteins play a critical role in the growth and metastasis of many tumor types,such as breast cancer,colorectal cancer, prostate cancer,esophageal cancer and hepatocellular carcinoma,by regulating growth-related signal transduction pathways,differentiation,apoptosis and angiogenesis.The relationship between KLF9 and CRC has not been explored.This, accompanied by the role of KLF9 in crypt cell proliferation and villus cell migration and well-characterized functions for some KLF proteins in occurrence and development of cancer,prompted us to characterize the expression and in vitro function of KLF9 in CRC.Fresh colorectal cancer tissues and the histologically normal tissues in the distant resection margin to the tumor were collected from 70 patients who underwent the curative colorectal cancer resection at the Second Affiliated Hospital,School of Medicine,Zhejiang University from August 2005 to June 2006.The levels of KLF9 mRNA were detected by quantitative real-time reverse transcriptase PCR（Q-PCR）, and the expression of KLF9 protein detected by Western blot.Tissue microarray （TMA）,containing normal,adenoma,and adenocarcinoma colorectal tissues,was constructed,and KLF9 protein expression was detected by immunohistochemistry. Of the 50 cancerous tissues examined,86%（43/50）expressed lower levels of KLF9 mRNA than individually-matched normal mucosa（P＜0.0001）.Expression of KLF9 protein was down-regulated in CRC by Western blot（P＜0.01）and immunohistochemistry（P＜0.001）.Statistically,no correlation was found between KLF9 protein expression and tumor differentiation grade,infiltrating depth,location, and lymph node metastasis（P＞0.05）.Expression of KLF9 in HT29,SW480, SW620,LoVo,RKO,and Caco2 colorectal cancer cell lines was also measured by RT-PCR and Western blot.mRNA of KLF9 could be detected among the six CRC cell lines.SW480 expressed the highest levels of KLF9 mRNA,and the rest showed weak expression.Except Caco2,the other five cell lines expressed KLF9 protein. The highest KLF9 protein was observed in RKO and the other four exhibited low levels of expression.KLF9 full-length coding sequence（CDS）in HT29,SW480, SW620 and RKO cell lines was sequenced.CDS of RKO had a mutation of 518T＞C （F173S）and no mutations were detected in others.We analyzed and compared the structure of RKO and normal KLF9 protein（NP₀01197）with the help of some online services and softwares,such as NCBI,ExPASy,PBIL,SWISS-MODEL, ESyPred3D,and Rosmal.A few differences of the primary,secondary and tertiary structures exist.These differences may cause functional alteration of RKO KLF9 protein.Based on down-regulation of KLF9 in CRC,we attempt to explore the function of KLF9 in CRC cell lines,pcDNA3.1-KLF9 eukaryotic expression vector was constructed and applied in transfection by using PolyFect transfection reagent.The expression of KLF9 mRNA and protein in the transfectants was detected by RT-PCR and western blot.No morphological alterations could be observed in KLF9 transfectants using light microscopy when cells were transfected transiently. Apoptosis and cell cycle assay using flow cytometry showed that KLF9 transfection did not induce apoptosis and alteration of cell cycle.The expression of cell cycle-related genes,Cyclin D1,p21（Cip1）,CDK2 and PCNA,was detected in KLF9 transfectants using real-time PCR.The results showed that there are no differences in the levels of mRNA expression of these genes.In the stable transfection,cells were selected with G418 for getting the stable cell lines.Unfortunately,cells transfected with pcDNA3.1-KLF9 began to die during the later period of stable transfection,so we could not obtain the transfectants which keep expressing KLF9.From this study,we concluded that:1.Expression of KLF9 is down-regulated,and reduced expression might be involved in the occurrence and development of colorectal carcinoma.2.Post-transcriptional modifications may be related to KLF9 expression in CRC cell lines.3.Continuous expression of KLF9 protein may be lethal for some CRC cell lines,or high levels of KLF9 may lead to increased sensitivity to drug used in stable transfection and cause cell death.更多还原