结直肠癌变相关基因表达谱的研究
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摘要
研究表明胚胎发育与肿瘤发生在组织、细胞及分子水平有很多相似之处。因此,可以借助胚胎发育来研究肿瘤,为探索肿瘤发生发展机制提供新的线索。本部分研究通过检测人肠发育过程3个时间点(妊娠早期胎肠6例、妊娠中期胎肠8例、正常成人肠12例)和结直肠癌变3个阶段(正常成人肠12例、低级别腺瘤71例、高级别腺瘤33例、腺癌47例)组织的mRNA表达谱,分别分析发育和癌变过程基因表达特征,进而对两个过程的数据进行整合分析。结果显示,肠发育过程中表达上调的基因主要参与免疫应答、抗原递呈、离子转运、细胞稳态、细胞凋亡等生物学过程,表达下调的基因主要参与DNA复制、染色质组装、DNA和RNA代谢、细胞粘附、细胞组织形态发生和组织器官发育等生物学过程。结直肠癌变过程中表达上调的基因主要参与转录调控、胚胎形态发生、细胞外基质组织结构、血管生成、细胞粘附、细胞增殖等过程；表达下调的基因主要参与免疫应答、免疫细胞激活、防御反应、炎症反应、细胞离子稳态等过程。肠发育和癌变相关基因表达谱整合分析显示,结直肠肿瘤的基因表达特征与胎肠组织相似,且随着肿瘤的进展,其分子表达谱越接近胎肠发育的早期阶段,可见发育过程和癌变过程的mRNA表达具有动态联系。基于上述肠发育和结直肠癌变的分子特征,进一步筛选出两组表达趋势相反的基因簇,一簇基因在肠发育过程中表达逐渐升高而在癌变过程中表达逐渐降低,主要与免疫应答和细胞凋亡相关；另一簇基因在肠发育过程中表达逐渐降低而在癌变过程中表达逐渐升高,主要与细胞增殖相关。本部分研究结果提示,人肠发育过程与结直肠癌变过程相关mRNA表达谱具有相似性,结直肠癌变过程相关的分子事件可能是胚胎肠发育相关分子事件的再激活或再抑制。以肠发育为切入点,整合分析肠发育与结直肠癌变的内在联系,为结直肠肿瘤的研究提供了新的思路。
肿瘤细胞形态和行为等方面的异质性是由(表观)遗传学改变的持续积累所导致的,还是由肿瘤起始细胞在早期阶段就已经建立的固有特征所决定的,仍然是一个有争议的问题。多年前,有研究者提出人类肿瘤的生物先决论假说,认为肿瘤细胞的生物学潜能在癌前阶段就已经决定。本部分研究的目的就是为了探索在肿瘤起始阶段发生的异常分子事件是否可以影响结直肠癌的进展。本部分研究采用mRNA表达谱芯片和miRNA表达谱芯片分别对结直肠正常黏膜、低级别腺瘤、高级别腺瘤和腺癌组织进行了分析。结果显示,与低级别腺瘤转变为高级别腺瘤、直至腺癌的过程相比,mRNA和miRNA的表达在正常肠上皮发展为低级别腺瘤的阶段发生了更大的改变,包括2,322个差异表达mRNAs和71个差异表达miRNAs。利用这些早期的分子变化,我们构建了一个miRNA-mRNA相互作用网络,包括30个miRNA及其调控的166个靶基因。在这些基因中,由5个miRNA调控的55个基因组成的标志物,在三组独立数据集中均与结直肠癌患者的总生存时间或无病生存时间相关。因此,从正常肠上皮转化为上皮内瘤变的阶段中,转录组水平上的分子改变可能会影响结直肠癌的进展。
本部分研究通过对人结直肠正常黏膜组织及腺癌组织的基因表达谱分析,挑选出差异表达基因对,构建结直肠正常黏膜和腺癌组织中特异表达的基因簇,为揭示结直肠癌发生发展的分子机理提供新的线索。研究采用mRNA表达谱芯片,对12例正常结直肠黏膜、21例低级别腺瘤、30例高级别腺瘤和25例结直肠癌组织进行了分析。生物信息学分析筛选出结直肠癌变各阶段特异的共表达基因对,并利用正常黏膜和腺癌两个阶段的数据,构建并筛选出分别在正常结直肠组织和癌组织中特异表达的子网络/基因簇,即保守基因簇。正常黏膜相关保守基因簇的功能包括免疫细胞激活、补体激活和器官形态发生等；腺癌相关保守基因簇的功能包括细胞增殖、细胞运动和细胞粘附等。进一步预测每个保守基因簇上游潜在的调控基因,并挑选3个保守基因簇(1个正常相关和2个腺癌相关保守基因簇)中的11个基因及8个预测的调控基因在上述起始样本(12例正常和22例腺癌组织)及独立样本(13例正常和19例腺癌组织)中使用实时定量PCR的方法对其表达水平进行验证。结果显示,19个基因在起始样本和独立样本中差异表达的阳性率分别为84.21%(16/19)和42.11%(8/19)。此外,在细胞中外源过表达MAFB引起cluster16中基因(RAB31, MY05A, WIPF1和LCP1)的mRNA表达水平上调。因此,我们基于基因共表达关系构建、筛选结直肠癌变相关分子网络的方法是可行的,得到的保守基因簇可能对揭示结直肠癌变的分子机制提供线索。
It is reported that embryogenesis and tumorigenesis have many similarities at phenotypic and genetic levels, which provides new directions for investigating the mechanisms of tumorigenesis through embryogenesis. The mRNA expression profiles of three sequent subsets of human colon development tissues and three stages of colorectal tumor tissues were involved in this study, including6early embryonic colon,8middle embryonic colon,12normal adult colorectal mucosa,71low-grade adenoma,33high-grade adenoma and47adenocarcinoma tissues. The gene expression features of colorectal embryogenesis and carcinogenesis were analyzed respectively, and integrative analysis was performed using the data of two processes. The results displayed that genes up-regulated in embryogenesis referred to immune response, antigen presentation, ion transport, cellular homeostasis, apoptosis, etc.; genes down-regulated were involved in DNA replication, chromatin organization, DNA and RNA metabolic process, cell adhesion, cell morphogenesis, regulation of cell development, etc. In colorectal carcinogenesis, the up-regulated genes were associated with transcription regulation, embryonic morphogenesis, extracellular matrix organization, angiogenesis, cell adhesion, cell proliferation, etc.; the down-regulated genes were associated with immune response, activation of immune cells, defense response, inflammatory response, cellular ion homeostasis, etc. Integrative analysis revealed that the gene expression characteristics of colorectal neoplasm were similar to the embryonic colon tissues, and along with colorectal tumor progression, their expression profiles were more approximate to those of the early stage embryonic colon tissues. These results indicate that there is a dynamic connection of mRNA expression between embryogenesis and carcinogenesis. Based on the molecular features described above, two gene clusters with opposite expressing patterns were screened, one was up-regulated during development and down-regulated during carcinogenesis, correlating with immune response and apoptosis; the other was down-regulated during development and up-regulated during carcinogenesis, associating with cell proliferation. In this part, it is concluded that the expression profiles of human colon development and colorectal tumorigenesis are highly similar, and molecular events of colorectal carcinogenesis may be the re-activation or re-inhibition of molecular events related to colon embryogenesis. Therefore, integrative and comparative analysis of colon development and colorectal tumorigenesis will be a promising strategy for colorectal tumor research.
Part II:Genes involved in the transition from normal epithelium to intraepithelial neoplasia are associated with colorectal cancer patient survival
Whether the heterogeneity in tumor cell morphology and behavior is the consequence of a progressive accumulation of (epi)genetic alterations or an intrinsic property of cancer-initiating cells established at initiation remains controversial. The hypothesis of biological predetermination in human cancer was proposed many years ago and states that the biological potency of cancer cells is predestinated in the precancerous stage. The present study aimed to investigate whether the aberrant molecular events occurring in initial cancer stages could eventually influence colorectal cancer progression. We analyzed the mRNA and miRNA expression profiles of colorectal normal mucosa, low-grade adenoma, high-grade adenoma, and adenocarcinoma tissues. Compared with the transitions from low-grade to high-grade adenoma to invasive carcinoma, the transition from normal epithelium to low-grade adenoma appeared to be associated with greater changes in the number and expression levels of mRNAs and miRNAs, with a differential expression of2,322mRNAs and71miRNAs detected. Utilizing these early molecular changes, a miRNA-hub network analysis showed that166genes were identified as targets regulated by30miRNAs. Among these genes, a55-gene signature regulated by5miRNAs was shown to be associated with overall survival or disease-free survival in three independent sample sets. Thus, the molecular changes in the transcriptome associated with the transition from normal to intraepithelial neoplasm may influence colorectal cancer progression.
Part Ⅲ:A study on colorectal normal mucosa and adenocarcinoma specific conserved gene clusters
Utilizing the mRNA expression profiles of colorectal normal mucosa and adenocarcinoma tissues, differentially expressed gene pairs were screened and normal and carcinoma specific conserved gene clusters were constructed, providing new clues to explore the molecular mechanism of colorectal carcinogenesis. The mRNA expression profiles of12normal mucosa,21low-grade adenoma,30high-grade adenoma and25adenocarcinoma tissues were analyzed. Through bioinformatics analysis, the co-expressed gene pairs in each stage of colorectal cancer development were screened and normal mucosa and carcinoma specific co-expression networks (conserved gene clusters) were established. The functions of normal mucosa specific conserved gene clusters were activation of immune cells, complement activation, organ morphogenesis, etc.; the functions of adenocarcinoma specific conserved gene clusters were cell proliferation, cell motility, cell adhesion, etc. We also predicted the potential regulatory genes of the conserved gene clusters. Then, we selected11genes of three clusters (including one normal specific cluster and two carcinoma specific clusters) and8predicted regulatory genes for further validation in initial samples (12normal tissues and22adenocarcomoma tissues) and independent samples (13normal tissues and19adenocarcinoma tissues) by Real time PCR. The results revealed that the positive rates of the19genes differentially expressed between normal and carcinoma were84.21%(16/19) and42.11%(8/19) in initial and independent samples respectively. Additionally, we verified that over-expression of MAFB increased the mRNA expression of the genes in cluster16(RAB31, MYO5A, WIPF1, LCP1) in colorectal cancer cell lines. Therefore, the method of co-expression network construction is feasible, and the colorectal normal mucosa and adenocarcinoma specific conserved gene clusters may contribute to explore the molecular mechanisms of colorectal cancer.
肿瘤细胞形态和行为等方面的异质性是由(表观)遗传学改变的持续积累所导致的,还是由肿瘤起始细胞在早期阶段就已经建立的固有特征所决定的,仍然是一个有争议的问题。多年前,有研究者提出人类肿瘤的生物先决论假说,认为肿瘤细胞的生物学潜能在癌前阶段就已经决定。本部分研究的目的就是为了探索在肿瘤起始阶段发生的异常分子事件是否可以影响结直肠癌的进展。本部分研究采用mRNA表达谱芯片和miRNA表达谱芯片分别对结直肠正常黏膜、低级别腺瘤、高级别腺瘤和腺癌组织进行了分析。结果显示,与低级别腺瘤转变为高级别腺瘤、直至腺癌的过程相比,mRNA和miRNA的表达在正常肠上皮发展为低级别腺瘤的阶段发生了更大的改变,包括2,322个差异表达mRNAs和71个差异表达miRNAs。利用这些早期的分子变化,我们构建了一个miRNA-mRNA相互作用网络,包括30个miRNA及其调控的166个靶基因。在这些基因中,由5个miRNA调控的55个基因组成的标志物,在三组独立数据集中均与结直肠癌患者的总生存时间或无病生存时间相关。因此,从正常肠上皮转化为上皮内瘤变的阶段中,转录组水平上的分子改变可能会影响结直肠癌的进展。
本部分研究通过对人结直肠正常黏膜组织及腺癌组织的基因表达谱分析,挑选出差异表达基因对,构建结直肠正常黏膜和腺癌组织中特异表达的基因簇,为揭示结直肠癌发生发展的分子机理提供新的线索。研究采用mRNA表达谱芯片,对12例正常结直肠黏膜、21例低级别腺瘤、30例高级别腺瘤和25例结直肠癌组织进行了分析。生物信息学分析筛选出结直肠癌变各阶段特异的共表达基因对,并利用正常黏膜和腺癌两个阶段的数据,构建并筛选出分别在正常结直肠组织和癌组织中特异表达的子网络/基因簇,即保守基因簇。正常黏膜相关保守基因簇的功能包括免疫细胞激活、补体激活和器官形态发生等；腺癌相关保守基因簇的功能包括细胞增殖、细胞运动和细胞粘附等。进一步预测每个保守基因簇上游潜在的调控基因,并挑选3个保守基因簇(1个正常相关和2个腺癌相关保守基因簇)中的11个基因及8个预测的调控基因在上述起始样本(12例正常和22例腺癌组织)及独立样本(13例正常和19例腺癌组织)中使用实时定量PCR的方法对其表达水平进行验证。结果显示,19个基因在起始样本和独立样本中差异表达的阳性率分别为84.21%(16/19)和42.11%(8/19)。此外,在细胞中外源过表达MAFB引起cluster16中基因(RAB31, MY05A, WIPF1和LCP1)的mRNA表达水平上调。因此,我们基于基因共表达关系构建、筛选结直肠癌变相关分子网络的方法是可行的,得到的保守基因簇可能对揭示结直肠癌变的分子机制提供线索。
It is reported that embryogenesis and tumorigenesis have many similarities at phenotypic and genetic levels, which provides new directions for investigating the mechanisms of tumorigenesis through embryogenesis. The mRNA expression profiles of three sequent subsets of human colon development tissues and three stages of colorectal tumor tissues were involved in this study, including6early embryonic colon,8middle embryonic colon,12normal adult colorectal mucosa,71low-grade adenoma,33high-grade adenoma and47adenocarcinoma tissues. The gene expression features of colorectal embryogenesis and carcinogenesis were analyzed respectively, and integrative analysis was performed using the data of two processes. The results displayed that genes up-regulated in embryogenesis referred to immune response, antigen presentation, ion transport, cellular homeostasis, apoptosis, etc.; genes down-regulated were involved in DNA replication, chromatin organization, DNA and RNA metabolic process, cell adhesion, cell morphogenesis, regulation of cell development, etc. In colorectal carcinogenesis, the up-regulated genes were associated with transcription regulation, embryonic morphogenesis, extracellular matrix organization, angiogenesis, cell adhesion, cell proliferation, etc.; the down-regulated genes were associated with immune response, activation of immune cells, defense response, inflammatory response, cellular ion homeostasis, etc. Integrative analysis revealed that the gene expression characteristics of colorectal neoplasm were similar to the embryonic colon tissues, and along with colorectal tumor progression, their expression profiles were more approximate to those of the early stage embryonic colon tissues. These results indicate that there is a dynamic connection of mRNA expression between embryogenesis and carcinogenesis. Based on the molecular features described above, two gene clusters with opposite expressing patterns were screened, one was up-regulated during development and down-regulated during carcinogenesis, correlating with immune response and apoptosis; the other was down-regulated during development and up-regulated during carcinogenesis, associating with cell proliferation. In this part, it is concluded that the expression profiles of human colon development and colorectal tumorigenesis are highly similar, and molecular events of colorectal carcinogenesis may be the re-activation or re-inhibition of molecular events related to colon embryogenesis. Therefore, integrative and comparative analysis of colon development and colorectal tumorigenesis will be a promising strategy for colorectal tumor research.
Part II:Genes involved in the transition from normal epithelium to intraepithelial neoplasia are associated with colorectal cancer patient survival
Whether the heterogeneity in tumor cell morphology and behavior is the consequence of a progressive accumulation of (epi)genetic alterations or an intrinsic property of cancer-initiating cells established at initiation remains controversial. The hypothesis of biological predetermination in human cancer was proposed many years ago and states that the biological potency of cancer cells is predestinated in the precancerous stage. The present study aimed to investigate whether the aberrant molecular events occurring in initial cancer stages could eventually influence colorectal cancer progression. We analyzed the mRNA and miRNA expression profiles of colorectal normal mucosa, low-grade adenoma, high-grade adenoma, and adenocarcinoma tissues. Compared with the transitions from low-grade to high-grade adenoma to invasive carcinoma, the transition from normal epithelium to low-grade adenoma appeared to be associated with greater changes in the number and expression levels of mRNAs and miRNAs, with a differential expression of2,322mRNAs and71miRNAs detected. Utilizing these early molecular changes, a miRNA-hub network analysis showed that166genes were identified as targets regulated by30miRNAs. Among these genes, a55-gene signature regulated by5miRNAs was shown to be associated with overall survival or disease-free survival in three independent sample sets. Thus, the molecular changes in the transcriptome associated with the transition from normal to intraepithelial neoplasm may influence colorectal cancer progression.
Part Ⅲ:A study on colorectal normal mucosa and adenocarcinoma specific conserved gene clusters
Utilizing the mRNA expression profiles of colorectal normal mucosa and adenocarcinoma tissues, differentially expressed gene pairs were screened and normal and carcinoma specific conserved gene clusters were constructed, providing new clues to explore the molecular mechanism of colorectal carcinogenesis. The mRNA expression profiles of12normal mucosa,21low-grade adenoma,30high-grade adenoma and25adenocarcinoma tissues were analyzed. Through bioinformatics analysis, the co-expressed gene pairs in each stage of colorectal cancer development were screened and normal mucosa and carcinoma specific co-expression networks (conserved gene clusters) were established. The functions of normal mucosa specific conserved gene clusters were activation of immune cells, complement activation, organ morphogenesis, etc.; the functions of adenocarcinoma specific conserved gene clusters were cell proliferation, cell motility, cell adhesion, etc. We also predicted the potential regulatory genes of the conserved gene clusters. Then, we selected11genes of three clusters (including one normal specific cluster and two carcinoma specific clusters) and8predicted regulatory genes for further validation in initial samples (12normal tissues and22adenocarcomoma tissues) and independent samples (13normal tissues and19adenocarcinoma tissues) by Real time PCR. The results revealed that the positive rates of the19genes differentially expressed between normal and carcinoma were84.21%(16/19) and42.11%(8/19) in initial and independent samples respectively. Additionally, we verified that over-expression of MAFB increased the mRNA expression of the genes in cluster16(RAB31, MYO5A, WIPF1, LCP1) in colorectal cancer cell lines. Therefore, the method of co-expression network construction is feasible, and the colorectal normal mucosa and adenocarcinoma specific conserved gene clusters may contribute to explore the molecular mechanisms of colorectal cancer.
引文
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