基因芯片技术筛选和鉴定结直肠癌腹膜种植转移相关基因
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摘要
研究背景和目的
腹膜种植转移是结直肠癌常见的转移方式,文献报道结直肠癌腹膜转移的发生率约13%,有7%的结直肠癌患者在首次手术时就发现腹膜种植转移,另有4%-19%的患者在根治术后出现腹膜种植转移。结直肠癌腹膜转移的预后很差,中位生存期只有5-9个月,因此,鉴定结直肠癌腹膜转移的生物学标记对早期诊断及判断预后都具有重要意义。基因芯片技术由于具有高通量检测基因表达变化的特点,所以被广泛应用于肿瘤生物学标记的筛查。目前,大量研究证实结直肠癌原发肿瘤组织和肝脏、淋巴结转移组织中存在大量差异表达基因,但腹膜转移组织和原发肿瘤组织中有哪些基因存在表达差异,国内外报道甚少。理论上,根据肿瘤平行生长模型,在肿瘤发生的早期,一些不具有全部恶变基因的肿瘤细胞就可以出现播散,并在远处其他组织内继续通过基因突变来适应新的微环境的变化,最终形成转移灶,由于外部环境选择的压力和内在基因的不稳定性,使这些转移细胞的基因和原发肿瘤的基因差异很大。因此,借助于基因芯片技术比较结直肠癌原发肿瘤组织与腹膜转移组织全基因组mRNA表达水平的差异,就可以找到结直肠癌腹膜转移相关的基因。为此,我们首先从南方医科大学附属南方医院普外科自主开发的结直肠癌外科病例管理与分析系统软件中提取748例结直肠癌病人数据,对临床病理资料进行回顾性分析,应用SPSS统计软件探讨结直肠癌腹膜转移发生的危险因素,并确定基因芯片实验所需的病人及标本收集范围。采用基因芯片技术比较4例结直肠腺癌原发肿瘤组织和配对的4例腹膜转移组织全基因组mRNA表达水平的差异,通过倍数法确定差异表达的基因,并对这组基因进行功能注释聚类分析,通过对肿瘤进展相关的功能类中基因的分析,探讨结直肠癌腹膜转移的分子机制。由于基因芯片技术有一定的假阳性率,因此对部分基因芯片技术筛选出的差异表达基因进行RT-PCR验证。通过上部分实验,我们最终确定胶原三股螺旋重复蛋白1(Collagen Triple Helix Repeat Containing1, CTHRC1)与结直肠癌腹膜种植转移密切相关,为近一步验证CTHRC1蛋白表达水平的差异,采用免疫组化的方法检测了40例正常组织、66例结直肠原发癌组织、42例腹膜转移癌组织及5年随访期内无腹膜转移发生的68例结直肠原发癌组织的CTHRC1蛋白表达情况,探讨CTHRC1蛋白表达与临床病理数据之间的关系,比较不同强度CTHRC1蛋白表达组间病人术后累积总生存率及无病生存率的差异,并以病人的临床病理数据及CTHRC1表达水平为自变量,构建Cox回归模型,最终证实CTHRC1蛋白表达阳性是影响病人术后生存的危险因素。除比较原发肿瘤组织与腹膜转移组织间差异表达基因外,还通过基因芯片技术比较了原发肿瘤组织和正常组织间的差异表达基因,并对部分基因进行RT-PCR验证。通过不同组织间差异表达基因的比较,有助于了解结直肠癌腹膜转移的分子机制,寻找理想的腹膜种植转移标记物,从而为早期发现、预防及治疗结直肠癌的腹膜种植转移提供可靠的理论依据。
方法
第一章回顾分析2003年1月～2008年12月南方医院普外科收治的结直肠癌患者的临床病理资料,根据术中探查及术后病理结果,将病人分为腹膜转移组和无转移组。通过比较两组病人间年龄、性别、肿瘤部位、大小、病理类型、分化程度、T分期、N分期、术前癌胚抗原(CEA)、血清糖链抗原19-9(CA19-9)的差异,寻找结直肠癌腹膜种植转移的危险因素。
第二章2010年4月到2010年12月在中国广州南方医科大学附属南方医院手术治疗结直肠腺癌伴腹膜种植转移病人4例,术中收集原发肿瘤组织和腹膜转移组织标本,浸泡于RNA Later中常温保存。应用Trizol法提取总mRNA,经逆转录合成cDNA后经体外扩增合成aRNA后用Cy3荧光分子标记,标记后与人类全基因组表达谱芯片杂交,待芯片完全干燥后,利用扫描仪(LuxScan3.0,北京博奥)进行扫描,扫描后将图像转化为基于荧光强度的数字信号(GenePix Pro6.0),随后进行数据分析和处理。采用倍数法(≥2或≤0.5)确定配对标本间差异表达基因,通过DAVID (The Database for Annotation, Visualization and Integrated Discovery)分析工具对这组差异表达基因进行基因功能的注释和基因富集分析,筛选出与肿瘤进展相关的功能类,在这些功能类中挑选差异倍数较高且参与多个功能类的基因进行半定量荧光RT-PCR验证。
第三章收集2003年1月～2010年12月间广州南方医院普外科行手术治疗的结直肠腺癌伴腹膜转移病人的组织蜡块,获取正常组织蜡块标本40块,肿瘤原发组织蜡块标本66快,腹膜转移组织蜡块标本42块,另外从数据库中选取5年随访期内无腹膜转移发生的病人原发肿瘤蜡块标本68块,作为对照组。采用SP法检测CTHRC1蛋白在不同组织标本间表达水平的差异,一抗采用兔抗人CTHRC1多克隆抗体,购于美国AbCam公司,实验步骤按试剂盒说明书进行。CTHRC1蛋白主要分布于细胞膜及胞浆中,呈棕黄色颗粒,细胞核中不表达。在细胞膜及胞浆中有棕黄色颗粒判定为阳性,无棕黄色颗粒判定为阴性。
第四章2010年4月到2010年12月间收集的结直肠癌伴腹膜种植转移病人的手术标本中,选取3例中分化腺癌病人的原发肿瘤组织和正常组织进行下一步芯片实验,术中收集原发肿瘤组织和正常组织标本,浸泡于RNA Later中常温保存,应用Trizol法提取总mRNA,经逆转录合成cDNA后经体外扩增合成aRNA后用Cy3荧光分子标记,标记后与人类全基因组表达谱芯片杂交,待芯片完全干燥后,利用扫描仪(LuxScan3.0,北京博奥)进行扫描,扫描后将图像转化为基于荧光强度的数字信号(GenePix Pro6.0),随后进行数据分析和处理。采用经验贝叶斯法(P<0.05)确定配对标本间差异表达基因,通过GO TermFinder分析工具对差异表达的基因进行基因本体论(gene ontology, GO)分析,对部分差异表达基因进行共表达网络的构建及半定量荧光RT-PCR验证。
统计学处理计数资料采用x2检验或fisher确切概率法,计量资料采用独立样本t检验。对腹膜种植转移危险因素分析采用Binary Logistic回归分析,生存分析采用Kaplan-Meier法,用log-rank法检验,通过构建Cox回归模型,判定CTHRC1表达是否是影响术后病人生存的危险因素。所有计算采用SPSS13.0软件包进行,P值取双尾,P<0.05为差异具有统计学意义。
结果
第一章从数据库中共选取符合上述要求病例共748例,其中,腹膜转移组81例(10.8%),无转移组667例(89.2%)。单因素分析显示,肿瘤大小、肿瘤分化程度、肠壁浸润深度、淋巴结转移、术前癌胚抗原(CEA)和血清糖链抗原19-9(CA19-9)水平与结直肠癌腹膜种植转移有关。Logistic多因素回归分析显示,肠壁浸润深度、淋巴结转移以及术前癌胚抗原(CEA)和血清糖链抗原19-9(CA19-9)水平与结直肠癌腹膜种植转移有关。
第二章根据倍数法,在结直肠癌原发肿瘤组织和腹膜转移组织中共筛选出差异表达基因217条,其中,在腹膜转移组织中表达上调90条,表达下调127条。GO注释分析分析发现在这217条差异表达基因中,有27条与分子功能相关,占25.71%；38条和生物学过程相关,占36.19%；40条和细胞组份有关,占38.1%,在分子功能中,84%与结构分子活性相关,16%和催化活性相关；在生物学过程中,27.94%与生物黏附相关,27.94%与细胞过程相关,5.88%与代谢过程相关,19.12%与结构区域构建相关,19.12%与结构区域相关；在细胞组分中,65.57%与细胞外区域相关,34.43%与细胞外区域部分相关。通过功能聚类分析,发现这些基因主要集中在细胞外区域及细胞间黏附等15个GO terms中。进一步对90条表达上调基因进行功能注释富集分析,结果发现共有17个基因功能分类Terms出现在富集分数>4的3个功能注释簇中,其功能类型涉及细胞间黏附、细胞外基质连接、生物黏附等。在这17个功能类中挑选人白细胞活化黏附因子(activated leukocyte cell adhesion molecule, ALCAM)、钙黏附素11(Cadherin-11,CDH11)、基质金属蛋白酶-7(matrix metallopeptidase7, MMP7)、组织金属蛋白酶抑制剂1(tissue metallopeptidase inhibitor1, TIMP1)、胶原三股螺旋重复蛋白1(collagen triple helix repeat containing1, CTHRC1)这5条基因作为下一步研究方向。通过半定量荧光RT-PCR验证,无假阳性结果。
第三章腹膜转移组病人正常组织CTHRC1阳性表达率为20%,原发肿瘤组织中为68.2%,腹膜转移组织中为85.7%,无腹膜转移组病人CTHRC1阳性表达率为22.1%。腹膜转移组病人原发肿瘤组织(x2=23.127,P=0.000)及腹膜转移组织(x2=35.580,P=0.000)CTHRC1表达水平高于正常组织,差异有统计学意义,且腹膜转移组织中CTHRC1表达水平高于原发肿瘤组织,差异有统计学意义(x2=4.208,P=0.040)。另外腹膜转移组病人原发肿瘤组织中CTHRC1表达水平高于无腹膜转移组病人(χ2=28.814,P=0.040),差异有统计学意义。生存分析显示CTHRC1表达阳性组的累积总生存率(χ2=18.689,P=0.000)及无病生存率(x2=17.057,P=0.000)低于CTHRC1表达阴性组,Cox回归证实CTHRC1表达阳性组患者术后死亡风险是CTHRC1表达阴性组患者的2.061倍,CTHRC1表达阳性组患者术后复发和转移的风险是CTHRC1表达阴性组患者的1.946倍。
第四章满足P≤0.05的基因共有98个,相对于正常组织,在肿瘤组织中表达上调的基因有41个,肿瘤组织表达下调的基因有57个。通过向GO TERM FINDER服务器提交的是最后的98个差异表达基因符号列表,通过选择Function Ontology得出p<0.01的GO term,发现基因主要集中在protein binding(GO：0005515)这一功能类中。从此功能类中挑选S100钙结合蛋白P (S100calcium binding protein P,S100P)、抗氧化蛋白l(peroxiredoxin1,PRDX1)、分泌型白细胞蛋白酶抑制因子(secretory leukocyte peptidase inhibitor, SLPI)。对三个基因采用GENE MANIA工具进行共表达网络的构建,发现三个基因在网络中有共表达趋势,SLPI在S100P和PRDX1中起到桥梁的作用。对这三条基因进行半定量荧光RT-PCR验证,无假阳性结果。
结论
1肿瘤浸润浆膜层、淋巴结转移、CEA≥10ug/L及CA19-9≥74U/ml是结直肠癌腹膜种植转移的危险因素。
2结直肠癌原发癌组织与腹膜转移组织间存在大量差异表达基因,提示在肿瘤发生转移的过程中,转移的肿瘤细胞为适应转移部位微环境的变化继连续发生特有基因的集聚,对不同组织间基因表达差异的比较,有助于揭示结直肠癌腹膜转移发生的机制,为寻找生物学标记及治疗靶向提供依据。
3在腹膜转移组织中筛选出的ALCAM、CDH1、MMP7、TIMP1、CTHRC1这5条基因的表达上调与结直肠癌腹膜转移密切相关。
4结直肠肿瘤组织中存在着CTHRC1的高表达,并与肿瘤分化程度、侵润深度及淋巴结转移相关。
5CTHRC1在结直肠癌腹膜转移的发生和发展过程中发挥重要作用,并可能成为早期诊断的生物学标记之一
6CTHRC1表达强度和结直肠癌的病人术后生存及复发呈明显负相关,可作为判断预后的生物学指标之一
7在出现腹膜转移的结直肠癌原发肿瘤组织中,存在SLPI与S100P、SLPI与PRDX1共同表达,对这些在同一条件下共同表达基因的研究有助于揭示在肿瘤发生发展过程中基因相互作用的机理。
Background and Objection:
Peritoneal carcinomatosis(PC) is common in patients with colorectal cancer. Approximately13%of patients with colorectal cancer develop peritoneal carcinomatosis, and PC is encountered in approximately7%of patients at primary surgery, in approximately4%to19%of patients during follow-up after curative surgery. For its unfavorable prognosis with a median survival of5-9months, it is significant to identify the biomarkers of PC in the early diagnosis and prognosis. Because it can detect gene expression changes with high-throughput, gene microarray technology is widely used in tumor biological marker screening. A lot of research confirmed that there were a great number of genes with different expression between colorectal cancer primary tumour and liver metastases or lymph node metastases now, but there are seldom researches to report the different expression between primary tumour and peritoneal carcinomatosis. According to parallel progression model, tumour cells depart the primary lesion before the acquisition of fully malignant phenotypes to undergo somatic progression and metastatic growth at a distant site. Therefore, owing to selection pressure and the inherent genetic instability of tumour cells, parallel progression predicts greater disparity between metastatic founder and primary tumour cells. So we can find related genes of peritoneal metastasis in colorectal cancer by the gene microarray technology. First, the data of748patients with colorectal cancer in the Nan fang Hospital of Southern Medical University was extracted from the colorectal cancer surgery cases management and analysis system software, which was developed by general surgery of Nanfang Hospital independently, the clinicalpathological data were retrospectively analyzed. SPSS statistical software was used to analysis the risk factors of PC in colorectal cancer, and eventually the range of patients and specimens which was used in the experiment of gene microarray were determined.The differences of expression level in the whole genome mRNA between4pairs of colorectal cancer tissues and corresponding peritoneal metastasis were compared by gene chips, the differentially expressed genes, which was screened by method of fold change, were analyzed by functional annotation bioinformatics microarray analysis. The results of functional annotation bioinformatics microarray analysis can help us realize the molecular mechanism of PC in colorectal cancer. Because there was certain false-positive rate in the experiment of gene chip, the RT-PCR test was applied to verify the differences of expression level. From above experiments, we finally confirmed Collagen Triple helical Repeat proteins1(CTHRC1) that was closely related with PC in colorectal cancer. The Immunohistoehemistry for CTHRC1protein was performed by SP method. The formalin-fixed paraffin-embedded tissues of40normal tissues,66primary tumour lesions as well as42PCs were stained by relevant antibodies, at the same time the formalin-fixed paraffin-embedded tissues of68primary tumour lesions without PC in the five years' follow-up were stained by the same method and antibodies. Though comparing the differences of CTHRC1protein between different tissues, we explored the relation between the expression of CTHRC1and the clinicalpathological data, Kaplane Meier survival and Cox regression analysis were done for CTHRC1and periostin as prognostic factors of peritoneal carcinomatosis. Besides screening the differentially expressed genes between colorectal cancer tissues and corresponding peritoneal metastasis, the microarray was applied to screen the differentially expressed genes between colorectal cancer tissues and normal tissues, and part of the differentially expressed genes were verified by RT-PCR test. By comparing the different of expression between different tissues, we can realize the molecular mechanism of PC in colorectal cancer more clearly and find the biomarkers of PC in colorectal cancer. The screening of different genes can help us find the PC in colorectal cancer more early, and It can provide us with reliable theory bases to treat the PC in colorectal cancer.
Methods and materials
The first chapter
The clinicalpathological data of patients with colorectal cancer in the Nanfang Hospital of Southern Medical University from January2003to December2008were retrospectively analyzed. The patients were divided into two classes according to the results of exploration in operation and pathology reports after operation. The associations between peritoneal metastasis and clinicpathological factors were evaluated by univariate analysis and multivariate regression analysis, these factors included age, gender, location of tumor, tumor size, the type of pathology, tumor differentiation, T-stage, N-stage, serum carcinoembryonic antigen (CEA) level and serum carbohydrate antigen19-9(CA19-9) level. The aim of this study was to search the risk factors of the PC in colorectal cancer.
The second chapter
A total of4pairs of colorectal cancer tissues and corresponding peritoneal metastasis tissues were obtained from4patients with sporadic colorectal adenocarcinoma who underwent surgical resection at the Nanfang Hospital of Southern Medical University (Guangzhou, china) between April2010and December2010. Samples were soaked in the RNAlater then stored at normal temperature until further use. The total RNA were extracted by Trizol reagent and subsequently inversely transcribed into cDNA, and in vitro RNA syntheses was applied to synthetize aRNA, then Cy3dye was applied for labeling reaction of aRNA, after which, the Cy3labeled aRNA samples were hybridized with the Whole Human Genome Oligo Microarray. Hybridized slides were scanned using the LuxScan3.0Scanner (Beijing CapitalBio Biotech, Ltd, China), and images were acquired by GenePix Pro v6.0software and transferred into digital signal then analyzed. Significantly different expression between tumor and PC was estimated using fold change, and fold change of greater or less than2were considered statistically significant. The DAVID (The Database for Annotation, Visualization and Integrated Discovery) analysis software was applied to functional annotations and functional categorization. From the cancer-related terms, the genes with highly fold change were choosed and then verified by RT-PCR, the selected genes always took part in many terms.
The third chapter
The clinicalpathological data of patients with colorectal cancer in the Nanfang Hospital of Southern Medical University from January2003to December2010were collected. The formalin-fixed paraffin-embedded tissue samples of of40normal tissues,66primary tumour lesions as well as42PCs were obtained, at the same time the formalin-fixed paraffin-embedded tissues of68primary tumour lesions without PC in the five years'follow-up were obtained. Immunohistoehemieal staining were performed by SP method to detect the different expression of CTHRC1protein between different tissues, the Primary antibodies were mouse monoelonal anti-human antigen CTHRC1and produced by AbCam Corp American, all the Procedures were followed by instruction of kit. CTHRC1was predominantly positive in cancer cell membrane and cytoplasm and absent in the cancer cell nuclear. The brown to yellow staining in cancer cell membrane and cytoplasm were defined as positive, the others were defined as negative.
The fourth chapter
A total of3pairs of colorectal cancer tissues and normal tissues were obtained from3patients with sporadic colorectal adenocarcinoma who underwent surgical resection at the Nanfang Hospital of Southern Medical University (Guangzhou, china) between April2010and December2010. Samples were soaked in the RNAlater then stored at-80℃until further use. The total RNA were extracted by Trizol reagent and subsequently inversely transcribed into cDNA, and in vitro RNA syntheses was applied to synthetize aRNA, then Cy3dye was applied for labeling reaction of aRNA, after which, the Cy3labeled aRNA samples were hybridized with the Whole Human Genome Oligo Microarray. Hybridized slides were scanned using the LuxScan3.0Scanner (Beijing CapitalBio Biotech, Ltd, China), and images were acquired by GenePix Pro v6.0software and transferred into digital signal then analyzed. The Empirical Bayes method was used to screen differentially expressed genes(p≤0.05), and the GO Term Finder analysis software was applied to functional annotations. GENEMANIA software was applied to constuct co-expression network, and evently the selected genes were verified by RT-PCR.
Statistical analysis
Chi-squared test and Fisher exact test were used in enumeration data, and Independent-Samples T test was used in quantitative data. Binary Logistic regression was used to analyze the risk factors of PC in colorectal cancer, Kaplane-Meier method was used in survival analysis, and the cumulative survival rate between different expressive levels was compared by log-rank test. Cox regression analysis were done for CTHRC1as prognostic factors. The SPSS software13.0analyzed all the data. Probability values<0.05were considered statistically significant.
Results:
The first chapter
The748patients were extracted from the colorectal cancer surgery cases management and analysis system software, there were81(10.8%) patients in the peritoneal metastasis group and667(89.2%) patients in non peritoneal metastasis group. Univariate analysis indicated tumor size, tumor differentiation, depth of bowel wall invasion, lymph node metastasis, serum carcinoembryonic antigen (CEA) level and serum carbohydrate antigen19-9(CA19-9) level were related with peritoneal metastasis. After logistic multivariate regression analysis, depth of bowel wall invasion, lymph node metastasis, serum carcinoembryonic antigen (CEA) level and serum carbohydrate antigen19-9(CA19-9) level remained significant risk factors.
The second chapter
The217differentially expressed genes between colorectal cancer tissues and corresponding peritoneal metastasis were screened by fold change method,90genes were up-expression and127genes were down-expression. Though GO functional annotations,27(25.71%) genes were related to molecular function,38(36.19%) genes were related to biological process,40(38.1%) genes were related to cellular component. To molecular function,84percent genes were related to structural molecule activity, and16percent genes were related to catalytic activity. To biological process,27.94%percent genes were related to biological adhesion,27.94%percent genes were related to cellular process,5.88%percent genes were related to metabolic process,19.12%percent genes were related to establishment of localization,19.12%percent genes were related to localization. To cellular component,65.57%percent genes were related to extracellular region,34.43%percent genes were related to extracellular region part. GO analysis found the differential expression genes were associated with15GO Terms, which included extracellular region, cell adhesion and other functional terms. We futher did functional categorization by methods of Enrichment to90up-expressed genes, there were17terms in the annotation clusterings with the Enrichment Score were more than4, these terms included cell adhesion, extracellular matrix organization and cell adhesion so on. Activated leukocyte cell adhesion molecule(ALCAM), Cadherin-11,(CDH11), matrix metallopeptidase7(MMP7), tissue metallopeptidase inhibitor1(TIMP1) and collagen triple helix repeat containing1(CTHRC1) were selected by us to further study. These five genes were verified by the semi-quantitative reverse transcription and polymerase chain reaction (SqRT-PCR), and there were no false positive.
The third chapter
To the patients of PC group,20percent patients were positive expression in normal tissue,68.2percent patients were positive expression in primary tumor, and85.7percent patients were positive expression in peritoneal carcinomatosis. Expressions of CTHRC1were significantly higher in primary tumor (χ2=23.127, P=0.000) and peritoneal carcinomatosis (χ2=35.580, P=0.000) than normal tissue, and Expressions of CTHRC1were significantly higher in peritoneal carcinomatosis than primary tumor (χ2=4.208, P=0.040). Expressions of CTHRC1were significantly higher in peritoneal carcinomatosis group than non peritoneal carcinomatosis group(χ2=28.814,P=0.040). In survival analysis, cumulative overall survival (χ2=18.689, P=0.000) and disease-free survival (χ2=17.057, P=0.000) were significant lower cumulative in group of positive expression of CTHRC1than negative expression of CTHRC1. In univariate Cox regression analysis, to patients with positive expression of CTHRC1, the risk of death was2.061times than patients with negative expression of CTHRC1and risk of the recurrence and metastasis was1.946times than patients with negative expression of CTHRC1.
The fourth chapter
A threshold of P≤0.05was determined,98genes were differentially expressed in primary cancer lesion including41up-regulated and57down-regulated. We input the list of official gene name to GO TERM FINDER software and we selected Function Ontology software to look for the GO term with p≤0.01. Finally,we found that a great deal of genes were in the term of protein binding (GO:0005515) S100P、RDX1、SLPI were selected from the GO term by us to further study, these genes were verified by the semi-quantitative reverse transcription and polymerase chain reaction (SqRT-PCR), and there were no false positive.
Conclusion
1Depth of bowel wall invasion, lymph node metastasis, serum carcinoembryonic antigen (CEA)≥10ug/L and serum carbohydrate antigen19-9(CA19-9)≥74U/ml are main risk factors for peritoneal metastasis in colorectal cancer.
2There were a great number of different expressed genes between the primary tumor of colorectal cancer and peritoneal metastasis. This finding confirmed that dissemination of tumor cells can adapted to specific microenvironments by gather of special gene continuity. By comparing the different expression between different tissues, we can realize the molecular mechanism of PC in colorectal cancer more clearly and find the biomarkers of PC in colorectal cancer. Finally, it can provide theory bases with treatment of PC in colorectal cancer.
3The up-expressed genes, which included ALCAM, CDH11, MMP7, TIMP1, CTHRC1, in peritoneal metastasis tissues were related to the course of PC in colorectal cancer.
4Expressions of CTHRC1were significantly higher in colorectal cancer, and it was associated with differentiation degree, depth of bowel wall invasion and lymph node metastasis.
5CTHRC1played important roles in peritoneal metastasis of colorectal cancer, and it can be as an important biomarker of early diagnosis.
6The expression of CTHRC1was negatively related to survival and recurrence in patients of colorectal cancer, it can be as an important biomarker affecting prognosis.
7The three genes, including SLPI, S100P and SLPI, were co-expression in the primary tissue of colorectal cancer with peritoneal metastasis, through the study of co-expression of genes, we can realize the mechanism of interaction in different genes under the same background.
腹膜种植转移是结直肠癌常见的转移方式,文献报道结直肠癌腹膜转移的发生率约13%,有7%的结直肠癌患者在首次手术时就发现腹膜种植转移,另有4%-19%的患者在根治术后出现腹膜种植转移。结直肠癌腹膜转移的预后很差,中位生存期只有5-9个月,因此,鉴定结直肠癌腹膜转移的生物学标记对早期诊断及判断预后都具有重要意义。基因芯片技术由于具有高通量检测基因表达变化的特点,所以被广泛应用于肿瘤生物学标记的筛查。目前,大量研究证实结直肠癌原发肿瘤组织和肝脏、淋巴结转移组织中存在大量差异表达基因,但腹膜转移组织和原发肿瘤组织中有哪些基因存在表达差异,国内外报道甚少。理论上,根据肿瘤平行生长模型,在肿瘤发生的早期,一些不具有全部恶变基因的肿瘤细胞就可以出现播散,并在远处其他组织内继续通过基因突变来适应新的微环境的变化,最终形成转移灶,由于外部环境选择的压力和内在基因的不稳定性,使这些转移细胞的基因和原发肿瘤的基因差异很大。因此,借助于基因芯片技术比较结直肠癌原发肿瘤组织与腹膜转移组织全基因组mRNA表达水平的差异,就可以找到结直肠癌腹膜转移相关的基因。为此,我们首先从南方医科大学附属南方医院普外科自主开发的结直肠癌外科病例管理与分析系统软件中提取748例结直肠癌病人数据,对临床病理资料进行回顾性分析,应用SPSS统计软件探讨结直肠癌腹膜转移发生的危险因素,并确定基因芯片实验所需的病人及标本收集范围。采用基因芯片技术比较4例结直肠腺癌原发肿瘤组织和配对的4例腹膜转移组织全基因组mRNA表达水平的差异,通过倍数法确定差异表达的基因,并对这组基因进行功能注释聚类分析,通过对肿瘤进展相关的功能类中基因的分析,探讨结直肠癌腹膜转移的分子机制。由于基因芯片技术有一定的假阳性率,因此对部分基因芯片技术筛选出的差异表达基因进行RT-PCR验证。通过上部分实验,我们最终确定胶原三股螺旋重复蛋白1(Collagen Triple Helix Repeat Containing1, CTHRC1)与结直肠癌腹膜种植转移密切相关,为近一步验证CTHRC1蛋白表达水平的差异,采用免疫组化的方法检测了40例正常组织、66例结直肠原发癌组织、42例腹膜转移癌组织及5年随访期内无腹膜转移发生的68例结直肠原发癌组织的CTHRC1蛋白表达情况,探讨CTHRC1蛋白表达与临床病理数据之间的关系,比较不同强度CTHRC1蛋白表达组间病人术后累积总生存率及无病生存率的差异,并以病人的临床病理数据及CTHRC1表达水平为自变量,构建Cox回归模型,最终证实CTHRC1蛋白表达阳性是影响病人术后生存的危险因素。除比较原发肿瘤组织与腹膜转移组织间差异表达基因外,还通过基因芯片技术比较了原发肿瘤组织和正常组织间的差异表达基因,并对部分基因进行RT-PCR验证。通过不同组织间差异表达基因的比较,有助于了解结直肠癌腹膜转移的分子机制,寻找理想的腹膜种植转移标记物,从而为早期发现、预防及治疗结直肠癌的腹膜种植转移提供可靠的理论依据。
方法
第一章回顾分析2003年1月～2008年12月南方医院普外科收治的结直肠癌患者的临床病理资料,根据术中探查及术后病理结果,将病人分为腹膜转移组和无转移组。通过比较两组病人间年龄、性别、肿瘤部位、大小、病理类型、分化程度、T分期、N分期、术前癌胚抗原(CEA)、血清糖链抗原19-9(CA19-9)的差异,寻找结直肠癌腹膜种植转移的危险因素。
第二章2010年4月到2010年12月在中国广州南方医科大学附属南方医院手术治疗结直肠腺癌伴腹膜种植转移病人4例,术中收集原发肿瘤组织和腹膜转移组织标本,浸泡于RNA Later中常温保存。应用Trizol法提取总mRNA,经逆转录合成cDNA后经体外扩增合成aRNA后用Cy3荧光分子标记,标记后与人类全基因组表达谱芯片杂交,待芯片完全干燥后,利用扫描仪(LuxScan3.0,北京博奥)进行扫描,扫描后将图像转化为基于荧光强度的数字信号(GenePix Pro6.0),随后进行数据分析和处理。采用倍数法(≥2或≤0.5)确定配对标本间差异表达基因,通过DAVID (The Database for Annotation, Visualization and Integrated Discovery)分析工具对这组差异表达基因进行基因功能的注释和基因富集分析,筛选出与肿瘤进展相关的功能类,在这些功能类中挑选差异倍数较高且参与多个功能类的基因进行半定量荧光RT-PCR验证。
第三章收集2003年1月～2010年12月间广州南方医院普外科行手术治疗的结直肠腺癌伴腹膜转移病人的组织蜡块,获取正常组织蜡块标本40块,肿瘤原发组织蜡块标本66快,腹膜转移组织蜡块标本42块,另外从数据库中选取5年随访期内无腹膜转移发生的病人原发肿瘤蜡块标本68块,作为对照组。采用SP法检测CTHRC1蛋白在不同组织标本间表达水平的差异,一抗采用兔抗人CTHRC1多克隆抗体,购于美国AbCam公司,实验步骤按试剂盒说明书进行。CTHRC1蛋白主要分布于细胞膜及胞浆中,呈棕黄色颗粒,细胞核中不表达。在细胞膜及胞浆中有棕黄色颗粒判定为阳性,无棕黄色颗粒判定为阴性。
第四章2010年4月到2010年12月间收集的结直肠癌伴腹膜种植转移病人的手术标本中,选取3例中分化腺癌病人的原发肿瘤组织和正常组织进行下一步芯片实验,术中收集原发肿瘤组织和正常组织标本,浸泡于RNA Later中常温保存,应用Trizol法提取总mRNA,经逆转录合成cDNA后经体外扩增合成aRNA后用Cy3荧光分子标记,标记后与人类全基因组表达谱芯片杂交,待芯片完全干燥后,利用扫描仪(LuxScan3.0,北京博奥)进行扫描,扫描后将图像转化为基于荧光强度的数字信号(GenePix Pro6.0),随后进行数据分析和处理。采用经验贝叶斯法(P<0.05)确定配对标本间差异表达基因,通过GO TermFinder分析工具对差异表达的基因进行基因本体论(gene ontology, GO)分析,对部分差异表达基因进行共表达网络的构建及半定量荧光RT-PCR验证。
统计学处理计数资料采用x2检验或fisher确切概率法,计量资料采用独立样本t检验。对腹膜种植转移危险因素分析采用Binary Logistic回归分析,生存分析采用Kaplan-Meier法,用log-rank法检验,通过构建Cox回归模型,判定CTHRC1表达是否是影响术后病人生存的危险因素。所有计算采用SPSS13.0软件包进行,P值取双尾,P<0.05为差异具有统计学意义。
结果
第一章从数据库中共选取符合上述要求病例共748例,其中,腹膜转移组81例(10.8%),无转移组667例(89.2%)。单因素分析显示,肿瘤大小、肿瘤分化程度、肠壁浸润深度、淋巴结转移、术前癌胚抗原(CEA)和血清糖链抗原19-9(CA19-9)水平与结直肠癌腹膜种植转移有关。Logistic多因素回归分析显示,肠壁浸润深度、淋巴结转移以及术前癌胚抗原(CEA)和血清糖链抗原19-9(CA19-9)水平与结直肠癌腹膜种植转移有关。
第二章根据倍数法,在结直肠癌原发肿瘤组织和腹膜转移组织中共筛选出差异表达基因217条,其中,在腹膜转移组织中表达上调90条,表达下调127条。GO注释分析分析发现在这217条差异表达基因中,有27条与分子功能相关,占25.71%；38条和生物学过程相关,占36.19%；40条和细胞组份有关,占38.1%,在分子功能中,84%与结构分子活性相关,16%和催化活性相关；在生物学过程中,27.94%与生物黏附相关,27.94%与细胞过程相关,5.88%与代谢过程相关,19.12%与结构区域构建相关,19.12%与结构区域相关；在细胞组分中,65.57%与细胞外区域相关,34.43%与细胞外区域部分相关。通过功能聚类分析,发现这些基因主要集中在细胞外区域及细胞间黏附等15个GO terms中。进一步对90条表达上调基因进行功能注释富集分析,结果发现共有17个基因功能分类Terms出现在富集分数>4的3个功能注释簇中,其功能类型涉及细胞间黏附、细胞外基质连接、生物黏附等。在这17个功能类中挑选人白细胞活化黏附因子(activated leukocyte cell adhesion molecule, ALCAM)、钙黏附素11(Cadherin-11,CDH11)、基质金属蛋白酶-7(matrix metallopeptidase7, MMP7)、组织金属蛋白酶抑制剂1(tissue metallopeptidase inhibitor1, TIMP1)、胶原三股螺旋重复蛋白1(collagen triple helix repeat containing1, CTHRC1)这5条基因作为下一步研究方向。通过半定量荧光RT-PCR验证,无假阳性结果。
第三章腹膜转移组病人正常组织CTHRC1阳性表达率为20%,原发肿瘤组织中为68.2%,腹膜转移组织中为85.7%,无腹膜转移组病人CTHRC1阳性表达率为22.1%。腹膜转移组病人原发肿瘤组织(x2=23.127,P=0.000)及腹膜转移组织(x2=35.580,P=0.000)CTHRC1表达水平高于正常组织,差异有统计学意义,且腹膜转移组织中CTHRC1表达水平高于原发肿瘤组织,差异有统计学意义(x2=4.208,P=0.040)。另外腹膜转移组病人原发肿瘤组织中CTHRC1表达水平高于无腹膜转移组病人(χ2=28.814,P=0.040),差异有统计学意义。生存分析显示CTHRC1表达阳性组的累积总生存率(χ2=18.689,P=0.000)及无病生存率(x2=17.057,P=0.000)低于CTHRC1表达阴性组,Cox回归证实CTHRC1表达阳性组患者术后死亡风险是CTHRC1表达阴性组患者的2.061倍,CTHRC1表达阳性组患者术后复发和转移的风险是CTHRC1表达阴性组患者的1.946倍。
第四章满足P≤0.05的基因共有98个,相对于正常组织,在肿瘤组织中表达上调的基因有41个,肿瘤组织表达下调的基因有57个。通过向GO TERM FINDER服务器提交的是最后的98个差异表达基因符号列表,通过选择Function Ontology得出p<0.01的GO term,发现基因主要集中在protein binding(GO：0005515)这一功能类中。从此功能类中挑选S100钙结合蛋白P (S100calcium binding protein P,S100P)、抗氧化蛋白l(peroxiredoxin1,PRDX1)、分泌型白细胞蛋白酶抑制因子(secretory leukocyte peptidase inhibitor, SLPI)。对三个基因采用GENE MANIA工具进行共表达网络的构建,发现三个基因在网络中有共表达趋势,SLPI在S100P和PRDX1中起到桥梁的作用。对这三条基因进行半定量荧光RT-PCR验证,无假阳性结果。
结论
1肿瘤浸润浆膜层、淋巴结转移、CEA≥10ug/L及CA19-9≥74U/ml是结直肠癌腹膜种植转移的危险因素。
2结直肠癌原发癌组织与腹膜转移组织间存在大量差异表达基因,提示在肿瘤发生转移的过程中,转移的肿瘤细胞为适应转移部位微环境的变化继连续发生特有基因的集聚,对不同组织间基因表达差异的比较,有助于揭示结直肠癌腹膜转移发生的机制,为寻找生物学标记及治疗靶向提供依据。
3在腹膜转移组织中筛选出的ALCAM、CDH1、MMP7、TIMP1、CTHRC1这5条基因的表达上调与结直肠癌腹膜转移密切相关。
4结直肠肿瘤组织中存在着CTHRC1的高表达,并与肿瘤分化程度、侵润深度及淋巴结转移相关。
5CTHRC1在结直肠癌腹膜转移的发生和发展过程中发挥重要作用,并可能成为早期诊断的生物学标记之一
6CTHRC1表达强度和结直肠癌的病人术后生存及复发呈明显负相关,可作为判断预后的生物学指标之一
7在出现腹膜转移的结直肠癌原发肿瘤组织中,存在SLPI与S100P、SLPI与PRDX1共同表达,对这些在同一条件下共同表达基因的研究有助于揭示在肿瘤发生发展过程中基因相互作用的机理。
Background and Objection:
Peritoneal carcinomatosis(PC) is common in patients with colorectal cancer. Approximately13%of patients with colorectal cancer develop peritoneal carcinomatosis, and PC is encountered in approximately7%of patients at primary surgery, in approximately4%to19%of patients during follow-up after curative surgery. For its unfavorable prognosis with a median survival of5-9months, it is significant to identify the biomarkers of PC in the early diagnosis and prognosis. Because it can detect gene expression changes with high-throughput, gene microarray technology is widely used in tumor biological marker screening. A lot of research confirmed that there were a great number of genes with different expression between colorectal cancer primary tumour and liver metastases or lymph node metastases now, but there are seldom researches to report the different expression between primary tumour and peritoneal carcinomatosis. According to parallel progression model, tumour cells depart the primary lesion before the acquisition of fully malignant phenotypes to undergo somatic progression and metastatic growth at a distant site. Therefore, owing to selection pressure and the inherent genetic instability of tumour cells, parallel progression predicts greater disparity between metastatic founder and primary tumour cells. So we can find related genes of peritoneal metastasis in colorectal cancer by the gene microarray technology. First, the data of748patients with colorectal cancer in the Nan fang Hospital of Southern Medical University was extracted from the colorectal cancer surgery cases management and analysis system software, which was developed by general surgery of Nanfang Hospital independently, the clinicalpathological data were retrospectively analyzed. SPSS statistical software was used to analysis the risk factors of PC in colorectal cancer, and eventually the range of patients and specimens which was used in the experiment of gene microarray were determined.The differences of expression level in the whole genome mRNA between4pairs of colorectal cancer tissues and corresponding peritoneal metastasis were compared by gene chips, the differentially expressed genes, which was screened by method of fold change, were analyzed by functional annotation bioinformatics microarray analysis. The results of functional annotation bioinformatics microarray analysis can help us realize the molecular mechanism of PC in colorectal cancer. Because there was certain false-positive rate in the experiment of gene chip, the RT-PCR test was applied to verify the differences of expression level. From above experiments, we finally confirmed Collagen Triple helical Repeat proteins1(CTHRC1) that was closely related with PC in colorectal cancer. The Immunohistoehemistry for CTHRC1protein was performed by SP method. The formalin-fixed paraffin-embedded tissues of40normal tissues,66primary tumour lesions as well as42PCs were stained by relevant antibodies, at the same time the formalin-fixed paraffin-embedded tissues of68primary tumour lesions without PC in the five years' follow-up were stained by the same method and antibodies. Though comparing the differences of CTHRC1protein between different tissues, we explored the relation between the expression of CTHRC1and the clinicalpathological data, Kaplane Meier survival and Cox regression analysis were done for CTHRC1and periostin as prognostic factors of peritoneal carcinomatosis. Besides screening the differentially expressed genes between colorectal cancer tissues and corresponding peritoneal metastasis, the microarray was applied to screen the differentially expressed genes between colorectal cancer tissues and normal tissues, and part of the differentially expressed genes were verified by RT-PCR test. By comparing the different of expression between different tissues, we can realize the molecular mechanism of PC in colorectal cancer more clearly and find the biomarkers of PC in colorectal cancer. The screening of different genes can help us find the PC in colorectal cancer more early, and It can provide us with reliable theory bases to treat the PC in colorectal cancer.
Methods and materials
The first chapter
The clinicalpathological data of patients with colorectal cancer in the Nanfang Hospital of Southern Medical University from January2003to December2008were retrospectively analyzed. The patients were divided into two classes according to the results of exploration in operation and pathology reports after operation. The associations between peritoneal metastasis and clinicpathological factors were evaluated by univariate analysis and multivariate regression analysis, these factors included age, gender, location of tumor, tumor size, the type of pathology, tumor differentiation, T-stage, N-stage, serum carcinoembryonic antigen (CEA) level and serum carbohydrate antigen19-9(CA19-9) level. The aim of this study was to search the risk factors of the PC in colorectal cancer.
The second chapter
A total of4pairs of colorectal cancer tissues and corresponding peritoneal metastasis tissues were obtained from4patients with sporadic colorectal adenocarcinoma who underwent surgical resection at the Nanfang Hospital of Southern Medical University (Guangzhou, china) between April2010and December2010. Samples were soaked in the RNAlater then stored at normal temperature until further use. The total RNA were extracted by Trizol reagent and subsequently inversely transcribed into cDNA, and in vitro RNA syntheses was applied to synthetize aRNA, then Cy3dye was applied for labeling reaction of aRNA, after which, the Cy3labeled aRNA samples were hybridized with the Whole Human Genome Oligo Microarray. Hybridized slides were scanned using the LuxScan3.0Scanner (Beijing CapitalBio Biotech, Ltd, China), and images were acquired by GenePix Pro v6.0software and transferred into digital signal then analyzed. Significantly different expression between tumor and PC was estimated using fold change, and fold change of greater or less than2were considered statistically significant. The DAVID (The Database for Annotation, Visualization and Integrated Discovery) analysis software was applied to functional annotations and functional categorization. From the cancer-related terms, the genes with highly fold change were choosed and then verified by RT-PCR, the selected genes always took part in many terms.
The third chapter
The clinicalpathological data of patients with colorectal cancer in the Nanfang Hospital of Southern Medical University from January2003to December2010were collected. The formalin-fixed paraffin-embedded tissue samples of of40normal tissues,66primary tumour lesions as well as42PCs were obtained, at the same time the formalin-fixed paraffin-embedded tissues of68primary tumour lesions without PC in the five years'follow-up were obtained. Immunohistoehemieal staining were performed by SP method to detect the different expression of CTHRC1protein between different tissues, the Primary antibodies were mouse monoelonal anti-human antigen CTHRC1and produced by AbCam Corp American, all the Procedures were followed by instruction of kit. CTHRC1was predominantly positive in cancer cell membrane and cytoplasm and absent in the cancer cell nuclear. The brown to yellow staining in cancer cell membrane and cytoplasm were defined as positive, the others were defined as negative.
The fourth chapter
A total of3pairs of colorectal cancer tissues and normal tissues were obtained from3patients with sporadic colorectal adenocarcinoma who underwent surgical resection at the Nanfang Hospital of Southern Medical University (Guangzhou, china) between April2010and December2010. Samples were soaked in the RNAlater then stored at-80℃until further use. The total RNA were extracted by Trizol reagent and subsequently inversely transcribed into cDNA, and in vitro RNA syntheses was applied to synthetize aRNA, then Cy3dye was applied for labeling reaction of aRNA, after which, the Cy3labeled aRNA samples were hybridized with the Whole Human Genome Oligo Microarray. Hybridized slides were scanned using the LuxScan3.0Scanner (Beijing CapitalBio Biotech, Ltd, China), and images were acquired by GenePix Pro v6.0software and transferred into digital signal then analyzed. The Empirical Bayes method was used to screen differentially expressed genes(p≤0.05), and the GO Term Finder analysis software was applied to functional annotations. GENEMANIA software was applied to constuct co-expression network, and evently the selected genes were verified by RT-PCR.
Statistical analysis
Chi-squared test and Fisher exact test were used in enumeration data, and Independent-Samples T test was used in quantitative data. Binary Logistic regression was used to analyze the risk factors of PC in colorectal cancer, Kaplane-Meier method was used in survival analysis, and the cumulative survival rate between different expressive levels was compared by log-rank test. Cox regression analysis were done for CTHRC1as prognostic factors. The SPSS software13.0analyzed all the data. Probability values<0.05were considered statistically significant.
Results:
The first chapter
The748patients were extracted from the colorectal cancer surgery cases management and analysis system software, there were81(10.8%) patients in the peritoneal metastasis group and667(89.2%) patients in non peritoneal metastasis group. Univariate analysis indicated tumor size, tumor differentiation, depth of bowel wall invasion, lymph node metastasis, serum carcinoembryonic antigen (CEA) level and serum carbohydrate antigen19-9(CA19-9) level were related with peritoneal metastasis. After logistic multivariate regression analysis, depth of bowel wall invasion, lymph node metastasis, serum carcinoembryonic antigen (CEA) level and serum carbohydrate antigen19-9(CA19-9) level remained significant risk factors.
The second chapter
The217differentially expressed genes between colorectal cancer tissues and corresponding peritoneal metastasis were screened by fold change method,90genes were up-expression and127genes were down-expression. Though GO functional annotations,27(25.71%) genes were related to molecular function,38(36.19%) genes were related to biological process,40(38.1%) genes were related to cellular component. To molecular function,84percent genes were related to structural molecule activity, and16percent genes were related to catalytic activity. To biological process,27.94%percent genes were related to biological adhesion,27.94%percent genes were related to cellular process,5.88%percent genes were related to metabolic process,19.12%percent genes were related to establishment of localization,19.12%percent genes were related to localization. To cellular component,65.57%percent genes were related to extracellular region,34.43%percent genes were related to extracellular region part. GO analysis found the differential expression genes were associated with15GO Terms, which included extracellular region, cell adhesion and other functional terms. We futher did functional categorization by methods of Enrichment to90up-expressed genes, there were17terms in the annotation clusterings with the Enrichment Score were more than4, these terms included cell adhesion, extracellular matrix organization and cell adhesion so on. Activated leukocyte cell adhesion molecule(ALCAM), Cadherin-11,(CDH11), matrix metallopeptidase7(MMP7), tissue metallopeptidase inhibitor1(TIMP1) and collagen triple helix repeat containing1(CTHRC1) were selected by us to further study. These five genes were verified by the semi-quantitative reverse transcription and polymerase chain reaction (SqRT-PCR), and there were no false positive.
The third chapter
To the patients of PC group,20percent patients were positive expression in normal tissue,68.2percent patients were positive expression in primary tumor, and85.7percent patients were positive expression in peritoneal carcinomatosis. Expressions of CTHRC1were significantly higher in primary tumor (χ2=23.127, P=0.000) and peritoneal carcinomatosis (χ2=35.580, P=0.000) than normal tissue, and Expressions of CTHRC1were significantly higher in peritoneal carcinomatosis than primary tumor (χ2=4.208, P=0.040). Expressions of CTHRC1were significantly higher in peritoneal carcinomatosis group than non peritoneal carcinomatosis group(χ2=28.814,P=0.040). In survival analysis, cumulative overall survival (χ2=18.689, P=0.000) and disease-free survival (χ2=17.057, P=0.000) were significant lower cumulative in group of positive expression of CTHRC1than negative expression of CTHRC1. In univariate Cox regression analysis, to patients with positive expression of CTHRC1, the risk of death was2.061times than patients with negative expression of CTHRC1and risk of the recurrence and metastasis was1.946times than patients with negative expression of CTHRC1.
The fourth chapter
A threshold of P≤0.05was determined,98genes were differentially expressed in primary cancer lesion including41up-regulated and57down-regulated. We input the list of official gene name to GO TERM FINDER software and we selected Function Ontology software to look for the GO term with p≤0.01. Finally,we found that a great deal of genes were in the term of protein binding (GO:0005515) S100P、RDX1、SLPI were selected from the GO term by us to further study, these genes were verified by the semi-quantitative reverse transcription and polymerase chain reaction (SqRT-PCR), and there were no false positive.
Conclusion
1Depth of bowel wall invasion, lymph node metastasis, serum carcinoembryonic antigen (CEA)≥10ug/L and serum carbohydrate antigen19-9(CA19-9)≥74U/ml are main risk factors for peritoneal metastasis in colorectal cancer.
2There were a great number of different expressed genes between the primary tumor of colorectal cancer and peritoneal metastasis. This finding confirmed that dissemination of tumor cells can adapted to specific microenvironments by gather of special gene continuity. By comparing the different expression between different tissues, we can realize the molecular mechanism of PC in colorectal cancer more clearly and find the biomarkers of PC in colorectal cancer. Finally, it can provide theory bases with treatment of PC in colorectal cancer.
3The up-expressed genes, which included ALCAM, CDH11, MMP7, TIMP1, CTHRC1, in peritoneal metastasis tissues were related to the course of PC in colorectal cancer.
4Expressions of CTHRC1were significantly higher in colorectal cancer, and it was associated with differentiation degree, depth of bowel wall invasion and lymph node metastasis.
5CTHRC1played important roles in peritoneal metastasis of colorectal cancer, and it can be as an important biomarker of early diagnosis.
6The expression of CTHRC1was negatively related to survival and recurrence in patients of colorectal cancer, it can be as an important biomarker affecting prognosis.
7The three genes, including SLPI, S100P and SLPI, were co-expression in the primary tissue of colorectal cancer with peritoneal metastasis, through the study of co-expression of genes, we can realize the mechanism of interaction in different genes under the same background.
引文
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