CKS2在结直肠癌中的表达和临床意义

背景：细胞周期蛋白依赖性激酶亚基（CKS）家族在细胞周期调节中起重要作用。研究发现其家族成员CKS2在一些恶性肿瘤中呈高表达,并与肿瘤的高侵袭性行为和预后不良相关。目前关于CKS2与结直肠癌关系的文献报道较罕见。目的：研究CKS2在结直肠癌中的表达和临床意义。方法：应用realtimeliT—PCR和蛋白质印迹法检测23例临床结直肠癌手术标本癌组织、癌旁非癌组织和正常组织中的CKS2mRNA和蛋白表达。结果：CKS2mRNA和蛋白在结直肠组织中的相对表达量依次为：癌组织〉癌旁组织〉正常组织,癌组织与正常组织问差异有统计学意义（P〈0．01）。性别对CKS2mRNA在不同结直肠组织中的表达趋势无明显影响。癌组织中的CKS2蛋白表达与肿瘤大小和pTNM分期呈正相关（P〈0．01）,与肿瘤部位无关。结论：CKS2蛋白在结直肠癌中呈高表达并与肿瘤临床病理特征相关,有望成为结直肠癌新的分子标记物和治疗靶点。     

EYA4基因在结直肠癌中的甲基化状态及其意义

背景：EYA4（eyes absent4）是一种非巯基蛋白酪氨酸磷酸酶,参与器官发育、细胞凋亡调控、先天性免疫、DNA损伤修复、血管生成等过程。目的：研究EYA4基因在结直肠癌中的甲基化状态及其意义。方法：选取2006年6月～2007年1月上海交通大学医学院附属仁济医院31例结直肠癌患者,采集其癌组织和相应癌旁非癌组织。以5-氮杂-2’-脱氧胞苷（5-Aza—dC）处理人结肠癌细胞株HT29、HCT116、SW480、SW1116。应用MSP技术检测结直肠癌组织、癌旁非癌组织、HT29、HCT116、SW480、SW1116细胞中EYA4基因启动子的甲基化状态。分析结直肠癌患者临床病理特征与EYA4基因甲基化关系。采用RT—PCR和蛋白质印迹法分别检测结肠癌细胞株中EYA4基因和蛋白的表达水平。结果：结直肠癌组织EYA4基因启动子甲基化率显著高于癌旁组织（77．4％对6．5％,P〈0．01）。EYA4基因和蛋白在启动子甲基化的HT29、HCT116、SW480细胞中不表达,在启动子非甲基化的SW1116细胞中显著表达。以5-Aza—dC处理后EYA4基因和蛋白在LIT29和SW480细胞中表达上调。结论：EYA4基因是结直肠癌发生的甲基化相关基因,有望成为结直肠癌的诊断标记物。     

胰岛素样生长因子Ⅱ基因印迹丢失在结直肠腺瘤中的意义

背景：胰岛素样生长因子Ⅱ（IGF-Ⅱ）可促进多种细胞增殖,抑制细胞凋亡,其表达受基因印迹调控,IGF-Ⅱ基因印迹丢失（LOI）与多种肿瘤的发生相关。目前较少见IGF-Ⅱ与结直肠腺瘤关系的研究报道。目的：分析结直肠腺瘤中IGF-Ⅱ的表达及其基因印迹状态,初步探讨两者在结直肠腺瘤发生、发展中的作用。方法：以免疫组化方法检测14例正常结直肠黏膜、12例增生性息肉和53例结直肠腺瘤组织中IGF-Ⅱ的表达,以聚合酶链反应（PCR）-限制性片段长度多态性（RFLP）方法分析IGF-Ⅱ基因印迹状态。结果：IGF—Ⅱ的表达主要定位于细胞质,其表达在正常结直肠黏膜、增生性息肉和腺瘤组织中逐级递增（P＜0．05）。正常黏膜、增生性息肉和腺瘤组织的IGF—Ⅱ杂合子基因型比例分别为42．9％、41．7％和60．4％。杂合子标本中,腺瘤组织的IGF-ⅡLOI发生率显著高于正常黏膜和增生性息肉（68．8％对16．7％和20．0％,P＜0．05）。结论：IGF-Ⅱ LOI可能是导致IGF—Ⅱ蛋白表达增加,促进结直肠腺瘤发生以及向腺癌演变的重要机制。     

葡萄糖醛酸转移酶1A基因的组织差异性表达及多态调节

目的从mRNA、蛋白质及细胞水平分析尿苷二磷酸葡萄糖醛酸转移酶（UGT）1A基因的组织差异性表达及多态凋节。方法采用逆转录聚合酶链反应分析结直肠癌组40例、正常人群肠道黏膜20例、正常肝组织10例的UGT 1A mRNA表达；免疫印迹检测各组UGT 1A蛋白表达；高效液相色谱法测定各组UGT 1A酶的催化活性。结果①结直肠癌组织中UGT 1A mRNA表达量明显低于其周围正常黏膜，而后者低于正常人群肠黏膜组织，正常人群结直肠黏膜中UGT 1A mRNA表达量总体低于肝组织，P〈0．01。结直肠癌组、正常人群结直肠黏膜组织呈现个体差异表达，而肝组织的表达较均质。②癌组织、癌周正常黏膜及正常人群肠道黏膜中UGT 1A各同工酶的表达例数均不相同。③UGT 1A蛋白表达在癌组织显著低于癌周正常组织，后者又低于正常人肠黏膜（P〈0．01），各组蛋白表达量亦各不相同。而肝组织呈均质表达。④癌组织的UGT 1A酶活性低于癌周正常黏膜．而后者的微粒体酶活性低于正常人群肠黏膜微粒体蛋白，P〈0．01。结论①UGT 1A基因位点呈组织差异性表达。②结直肠黏膜上皮UGT 1A基因位点在转录水平及功能水平均存在多态调节，不同个体对致癌物的易感性不同可能是这种差异表达的结果。     

p73和p51基因在结直肠癌中的表达和意义

背景：p53基因是一种肿瘤抑制基因，其家族成员p73和p51在结构上与p53具有高度同源性，影响细胞转录和凋亡的功能与p53相似。目的：研究p73和p51基因在结直肠癌中的表达及其与细胞凋亡和肿瘤临床病理特征的关系，探讨两者在结直肠癌发生、发展中的可能作用。方法：以逆转录聚合酶链反应（RT—PCR）检测60例结直肠癌组织和相应癌旁组织中p73、p51mRNA表达，以原位末端标记（TUNEL）法检测细胞凋亡。结果：结直肠癌组织p73、p51AmRNA表达阳性率显著高于相应癌旁组织（p73：71．7％对5．0％，P〈0．01：p51A：46．7％对11．7％，P〈0．01）：p51B mRNA在结直肠癌组织与相应癌旁组织中的相对表达量无明显差异（0．7318±0．3628对0．6836±0．3516，P〉0．05）。p73、p51A mRNA表达阳性者肿瘤细胞凋亡指数分别显著低于p73、p51A mRNA表达阴性者（p73：3．2％±2．5％对5．5％±2．8％．P=0．003；p51A：2．6％±2．3％对4．9％±2．7％，P=0．001）。p73mRNA表达与结直肠癌的分化程度、TNM分期和淋巴结转移相关（P〈0．05），p51A mRNA表达仅与淋巴结转移相关（P〈0．05）。结论：结直肠癌中p73、p51A基因表达上调，两者可能通过抑制肿瘤细胞凋亡而参与了结直肠癌的发生、发展。p73过表达可能与结直肠癌预后不良有关。     

TMX基因在结直肠癌中的表达研究

目的探讨TMX基因在结直肠癌及癌旁组织中的表达是否存在差异。方法对结直肠癌的的石蜡标本及切缘组织各33例,将肿瘤组织和对应的切缘正常组织放置于同一张载玻片上,采用免疫组化方法,利用IPP6．0软件进行半定量分析．研究TMX基因在结直肠癌组织中及手术切缘的表达情况,并比较这两组是否存在表达差异。结果TMX基因在结直肠癌组织及癌旁组织均表达．在结直肠癌组织的表达量明显低于正常切缘（0．142-．t-O．038VS0．210±0．037．P〈0．0001）。结论TMX基因在结直肠癌组织的表达量显著低于正常癌旁组织,提示TMX基因可能参与结直肠癌的发病。     

hClock基因mRNA及其蛋白在结直肠肿瘤中表达的研究

目的探讨人类生物钟基因hClockmRNA及其蛋白在不同Dukes分期结直肠肿瘤中的表达,研究它们的表达与结直肠肿瘤的侵袭及转移的关系。方法采用原位杂交检测结直肠癌与相应癌旁组织中hClock基因mRNA的表达,并采用免疫组织化学检测相应标本中hCloek基因蛋白产物（CLOCK蛋白）的表达。结果21例结直肠肿瘤中hCloekmRNA弱阳性表达率47．62％,中或强阳性表达率52．38％,且与Dukes分期相关（P〈0．05）;CLOCK蛋白均呈中或强阳性表达。相应癌旁组织中hClockmRNA及蛋白呈弱阳性表达（P〈0．01）。结论hCloek基因可能与结直肠肿瘤的发生、发展及侵袭、转移有相关性。     

Beclin1和PTEN蛋白在结直肠癌中的表达及预后价值

目的 研究Beclin1和PTEN蛋白在结直肠癌组织和癌旁组织中表达的差异及其与结直肠癌预后的相关性。方法 收集2007～2017年在解放军第九四医院就诊并手术的结直肠癌患者127例的结直肠癌癌灶组织和相对应的癌旁组织及每个患者的临床资料，行免疫组化检测癌组织和癌旁组织的Beclin1、PTEN的定位，并检测两个基因的表达阳性率，用实时PCR方法检测组织中Beclin1、PTEN mRNA表达，用Western印迹检测其中Beclin1、PTEN的蛋白表达，并比较2个基因在癌组织和癌旁组织中的表达差异。对Beclin1和PTEN的蛋白表达进行相关性分析，判断两个基因之间是否存在可能的调控关系。并对患者的临床资料进行生存分析，了解Beclin1、PTEN与结直肠癌患者预后间的关系。结果 Beclin1、PTEN均主要定位于结直肠细胞的胞质中，胞核中有少量表达；Beclin1在癌灶组织中的基因和蛋白表达显著高于癌旁组织，而PTEN在癌组织中的mRNA和蛋白表达显著低于癌旁组织(P<0.01)。双变量直线分析结果显示，在癌和癌旁组织中Beclin1和PTEN的蛋白表达呈负相关(P<...     

应用实时荧光定量PCR检测结直肠癌与癌旁组织中miRNA-221的差异表达状况

目的分析结直肠癌与毗邻癌旁组织中miRNA-221差异表达状况。方法常规抽提30例结直肠癌及对照癌旁组织中总RNA,应用实时荧光定量PCR方法检测标本中miRNA-221的表达状况。结果与癌旁组织相比,结直肠癌组织中miRNA-221表达明显上调（P〈0．01）。结论实时荧光定量PCR是一种较精确的miRNA定量检测方法;miRNA-221可能通过转录后基因沉默机制促进结直肠癌的发生发展。     

微 RNA-21和微 RNA-146a 在结直肠肿瘤中的表达及临床意义

目的探讨结直肠肿瘤患者组织和血清中 miRNA-21和 miRNA-146a 的表达情况,并分析其临床意义。方法收集100例结直肠癌、80例结直肠腺瘤患者和65名健康对照者的内镜活检组织,同时收集其血清标本,应用实时荧光定量 PCR 检测 miRNA-21和 miRNA-146a 的表达水平,分析其表达与临床病理特征间的关系,并评估血清差异表达的 miRNA 在结直肠肿瘤诊断中的价值。组间比较采用 Mann-WhitneyU 检验和 Kruskal-Wallis 检验;Spearman 相关性检验用于分析组织和血清中 miRNA 表达的关联性。结果在结直肠癌和结直肠腺瘤患者病变组织中 miRNA-21的表达分别为8．573±0．898和7．746±1．183,显著高于健康对照者的6．160±0．835（U ＝120．129、33．230,P 均＜0．01）;在结直肠癌和结直肠腺瘤患者血清中的表达分别为1．829±0．303和1．624±0．226,也高于健康对照者的1．391±0．221（U ＝40．353、15．512,P 均＜0．01）;miRNA-21在结直肠癌患者组织中和血清中的表达均高于结直肠腺瘤患者（U＝11．384、10．189,P 均＜0．01）。miRNA-21在结直肠癌患者中的高表达与肿瘤临床分期、淋巴结转移有关;miRNA-21在结直肠腺瘤中的表达水平与病理类型有关。Spearman 分析结果示,miRNA-21在结直肠癌患者组织和血清中的表达呈正相关（r ＝0．459,P ＜0．01）。miRNA-146a 在结直肠癌患者组织中为2．556±0．351,血清中为0．249±0．038,低于健康对照者的3．428±0．328和0．279±0．053（U ＝102．134、30．111,P 均＜0．01）;其在结直肠腺瘤患者组织中为3．255±0．332,血清中为0．290±0．036,与健康对照者比较差异无统计学意义（U＝3．936、3．180,P 均＞0．05）。miRNA-146a 在结直肠癌患者组织中和血清中的表达均低于结直肠腺瘤患者（U＝73．809、21．123,P 均＜0．01）。结直肠癌患者miRNA-146a 的下降程度与肿瘤临床分期、肿瘤分化程度有关,而在结直肠腺瘤中的表达与临床特征间无明显关系。根据 ROC 曲线分析,鉴别结直肠癌与健康者,血清 miRNA-21、miRNA-146a 及二者联合检测 ROC 的 AUC 分别为0．889、0．791和0．863;鉴别结直肠腺瘤与健康者,其 AUC 分别为0．784、0．692和0．761;鉴别结直肠癌与结直肠腺瘤患者,其 AUC 分别为0．705、0．820和0．713。结论血清 miRNA-21和 miRNA-146a 的差异表达在结直肠癌的早期诊断中具有潜在的价值。     

Expression of ST13 in colorectal cancer and adjacent normal tissues

AIM: To investigate the in situ expression of suppression of tumorigenecity 13 (ST13) mRNA in both colorectal cancer and adjacent normal tissues, METHODS: Colorectal cancer cell lines SW1116, SW620 and CoLo205 were enrolled to confirm the feasibility of the in situ hybridization procedure. Seven colorectal cancer and adjacent normal tissues were included for RNA-RNA in situ hybridization. RESULTS: The expression of ST13 in the seven normal colon tissues was positive and the positive signals appeared in mucosal cells. Only three of the seven colorectal cancer tissues had positive hybridization signals that appeared in adenocarcinoma cells. CONCLUSION: The expression of ST13 decreases in colorectal cancer tissue compared with that in adjacent normal tissue. ST13 is mostly expressed in colorectal epithelia and adenocarcinoma cells.     

Ornithine decarboxylase gene is overexpressed in colorectal carcinoma

AIM: To investigate the ornithine decarboxylase (ODC) gene expression in colorectal carcinoma, ODC mRNA was assayed by RT-PCR and ODC protein was detected by a monoclonal antibody against fusion of human colon ODC prepared by hybridoma technology. METHODS: Total RNA was extracted from human colorectal cancer tissues and their normal counterpart tissues. ODC mRNA levels were examined by RT-PCR. ODC genes amplified from RT-PCR were cloned into a prokaryotic vector pQE-30. The expressed proteins were purified by chromatography. Anti-ODC mAb was prepared with classical hybridoma techniques and used to determine the ODC expression in colon cancer tissues by immunohistochemical and Western blotting assay. RESULTS: A cell line, which could steadily secrete anti-ODC mAb, was selected through subcloning four times. Western blotting reconfirmed the mAb and ELISA showed that its subtype was IgG2a. RT-PCR showed that the ODC mRNA level increased greatly in colon cancer tissues (P<0.01). Immunohistochemical staining showed that colorectal carcinoma cells expressed a significantly higher level of ODC than normal colorectal mucosa (98.6±1.03% vs5.26±5%,P<0.01). CONCLUSION: ODC gene overexpression is significantly related to human colorectal carcinoma. ODC gene expression may be a marker for the gene diagnosis and therapy of colorectal carcinoma.     

Hydrophobic protein in colorectal cancer in relation to tumor stages and grades

AIM: To identify differentially expressed hydrophobic proteins in colorectal cancer. METHODS: Eighteen pairs of colorectal cancerous tissues in addition to tissues from normal mucosa were analysed. Hydrophobic proteins were extracted from the tissues, separated using 2-D gel electrophoresis and analysed using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS). Statistical analysis of the proteins was carried out in order to determine the significance of each protein to colorectal cancer (CRC) and als...     

Comparative study of carcinoembryonic antigen and epithelial membrane antigen expression in normal colon, adenomas and adenocarcinomas of the colon and rectum.

The heterogeneous nature of tumour antigen expression may require selection of monoclonal antibodies on an individual patient or tumour basis to allow adequate tumour localisation. Carcinoembryonic antigen (CEA) and epithelial membrane antigen (EMA) expression has not previously been compared in colorectal cancer patients. Sections of cancer (n = 52), adjacent normal colon (n = 45), synchronous adenomas (n = 11) and nodal metastases (n = 49) were examined by indirect immunoperoxidase staining in 51 consecutive patients with colorectal cancer using monoclonal antibodies to CEA and EMA. The percentage of cells with positive staining in the primary tumours was graded 1: less than 25%, 2: 25-49%, 3: 50-75%, 4 greater than 75%. All primary colorectal cancers expressed CEA and 43 of 52 expressed EMA (83%). Grading showed CEA greater than EMA in 39, equal in 11 and less in two. Well differentiated cancers were more frequently graded three or four for CEA staining (23 of 27) than moderately differentiated cancers (11 of 22) (p less than 0.01). Equivalent figures for EMA were four of 27 and three of 22 (not significant) (NS) although the majority (86%) were graded 1 and 2. Grade 1 CEA expression was found in six of 15 proximal and only two of 37 distal lesions (p less than 0.01, chi 2 test) while for EMA equivalent figures were three of 15 and six of 37 (NS). Nodal deposits all expressed CEA and 45 of 49 expressed EMA (92%); 29 of 45 normal colon sections showed CEA expression (64%) as did all adenomas. EMA was not expressed by normal colon or adenomas. These results suggest that EMA expression is more specific but less sensitive than CEA for colonic cancer and is independent of tumour differentiation and site. Thus selecting monoclonal antibodies to CEA or EMA based on tumour biopsies may allow improved tumour localisation for imaging or therapy in patents with colorectal cancer.     

ETS2在结直肠癌组织中的表达及临床意义

目的探讨转录因子ETS2（E-twenty six 2）在结直肠癌组织中的表达情况,分析其与结直肠癌患者临床病理参数之间的关系,并评估其临床预后价值。 方法收集新鲜的结直肠癌、癌旁和相应正常组织,以及结直肠癌和癌旁组织的石蜡标本,采用实时荧光定量PCR法检测组织中ETS2 mRNA的表达,免疫组化检测石蜡切片中ETS2蛋白的表达。 结果结直肠癌组织中ETS2 mRNA的表达明显高于癌旁组织和正常组织（P<0.001）,而癌旁组织和正常组织中的ETS2 mRNA表达量差异无统计学意义（P>0.05）。免疫组化结果显示ETS2在52.07%（63/121）的结直肠癌中表达,而仅在13.04%（6/46）的癌旁组织中表达,两者比较差异有统计学意义（P<0.05）。ETS2表达与肿瘤分化程度、浸润深度、淋巴结转移以及MMR状态有关（P<0.05）。ETS2表达与结直肠癌患者术后无进展生存有关（P<0.001）。采用COX多因素回归分析发现,ETS2表达（HR：0.461,95%CI：0.271~0.683,P=0.003）和浸润深度（HR：0.352,95%CI：0.113~0.769,P=0.015）是结直肠癌患者术后无进展生存的独立预后因子。 结论与癌旁组织相比,ETS2在结直肠癌组织中的转录和翻译水平都过表达,ETS2是预测结直肠癌患者术后无进展生存的潜在标记物。     

Hypermethylation and expression regulation of secreted frizzled-related protein genes in colorectal tumor

INTRODUCTION Secreted frizzled-related proteins (sFRPs) comprise a family of five secreted glycoproteins that antagonize Wnt canonical and noncanonical signaling by different mechanisms directly or indirectly[1]. Wnt signaling regulates cell growth, motil…     

Undetectable expression of hMLH1 protein in sporadic colorectal cancer with replication error phenotype

PURPOSE: Four DNA mismatch repair genes have been identified as being susceptible genes for hereditary nonpolyposis colorectal cancer. Deficiency of one of the mismatch repair genes causes the replication error phenotype in more than 80 percent of patients with hereditary non-polyposis colorectal cancer and in 10 to 30 percent of patients with sporadic colorectal cancer. To determine which mismatch repair gene is lacking the function in patients with replication error-positive colorectal cancer, several approaches have been used at the nucleic acid and protein levels. We studied replication error in 40 samples of randomly selected colorectal cancers and expression of hMSH2 and hMLH1 proteins analyzed by immunoblot in the tumor and normal tissues of the replication error-positive and replication error-negative samples. MATERIALS AND METHODS: Frozen tumor and normal tissues were obtained from 40 Japanese patients who had colorectal cancer. According to the Amsterdam criteria, those patients were classified as having 39 sporadic and 1 unknown colorectal cancers. Genomic DNA was extracted from tumor and normal tissues for determining replication error with eight microsatellite markers. Expression of hMSH2 and hMLH1 proteins in cell lysates of tumor and normal tissues of 16 patients was analyzed by immunoblot. RESULTS: The replication error phenotype was found in 6 (15 percent) of the 39 sporadic cases. hMLH1 protein was not detected in two of the six replication error-positive tumor tissues and not in the normal tissues, indicating that the tumor cells of the two patients had severe mutations in both alleles of thehMLH1gene. Another four replication error-positive and ten replication error-negative tumors and normal tissues expressed hMLH1 protein. hMSH2 protein was detected in all samples. CONCLUSION: hMLH1 protein was undetectable in the two tumor tissues of the six replication error-positive samples of sporadic colorectal cancer. The detection procedure used here may have potential use for determining a dysfunctional mismatch repair gene product.     

D-14 Monoclonal antibody to carcinoembryonic antigen: immunohistochemical analysis of formalin-fixed,paraffin-embedded human colorectal carcinoma,tumors of non-colorectal origin and normal tissues

Summary The reactivity of D-14 monoclonal antibody (mAb) to a specific epitope of carcinoembryonic antigen (CEA) was evaluated on formalin-fixed, paraffin-embedded tissues. A total of 52 normal tissues, 90 colorectal carcinomas and 127 non-colorectal neoplasms were tested using the peroxidase/antiperoxidase technique. D-14 mAb did not react with normal tissues apart from producing a weak staining of normal colonic glands immediately adjacent to the neoplastic structures. All 61 primary and 29 metastatic colorectal carcinomas expressed the carcinoembryonic antigen. However, there was considerable heterogeneity in cellular antigen expression in both primary and metastatic colorectal carcinomas with 10%–99% of tumor cells staining. Of 22 stomach adenocarcinomas, 14 were also immunoreactive, as were 2 of 5 pancreatic carcinomas. Only 6 of 100 neoplasms of non-gastrointestinal origin expressed weak to moderate immunoreactivity. In 7 cases, colorectal micrometastases not recognized in conventional hematoxylin and eosin slides could be identified with D-14 mAb. The specificity of this antibody could be used in differentiating colorectal carcinomas from other types of tumors, including adenocarcinoma from other sites.This paper was presented at the 1989 Annual Meeting of the American Association for Cancer Research Inc., San Francisco, California