【作者】 吴畏；

【导师】 何剪太；

【作者基本信息】 中南大学 ， 外科学， 2011， 博士

【摘要】 结直肠癌(Colorectal cancer, CRC)是我国最常见的癌症之一,结直肠癌筛查与早期诊断是降低疾病死亡率的关键,因此,寻找结直肠癌癌变相关的生物标志物(Biomarker)具有重要的临床意义。越来越多的证据表明微小RNA(microRNA, miRNA)异常表达与癌变密切相关,miRNA可能是一类重要的肿瘤生物标志物；miRNA是在真核生物中发现的一类小分子非编码RNA,约由22-25个核苷酸组成。它在细胞分化、细胞周期与细胞凋亡的调控、癌变等多个方面起重要的作用。miRNA通过5’端第2～8核苷酸与mRNA3’端非翻译区部分序列的结合,促使靶mRNA的降解和翻译抑制。miRNA起着类似转录因子的功能,调控靶基因的表达。miRNA异常表达是癌变过程中的关键因素之鉴定肿瘤异常表达的miRNA将增进我们对癌变分子机理的理解,同时,也可为癌症筛查、早期诊断与预后提供非常有用的分子标志物。已有一些研究表明：在结直肠癌中miRNA也呈现异常下调或上调表达。采用实时RT-PCR方法鉴定了13个在结直肠癌组织中异常表达的miRNA;采用包含287个miRNA的芯片分析结直肠癌与直肠癌细胞系的miRNA表达谱,确定了37个与正常组织表达有明显差异的miRNA;采用包含455个miRNA的基因芯片发现了21个miRNA在结直肠癌组织中异常表达。据估计,人存在大约1000个miRNA,在结直肠癌中,已有的研究报告分别筛选了96、145、287及455个miRNA的异常表达,因此,完全有必要系统地鉴定在结直肠癌中异常表达的miRNA。近年来的研究表明：结直肠癌应被视为一种“复杂的”、异质性疾病。结直肠癌发生在近端和远端表现出不同的生物学与临床特征。检测发现10多个肿瘤相关标志物在近端和远端发生的结直肠癌间呈现明显的差异。同样,根据肿瘤微卫星不稳定来区分右侧结肠癌与直肠癌；E-cadherin表达差异来区分左侧结肠癌和直肠癌。连同在胚胎发育过程中整个结直肠不同区域起源和基因表达谱差异方面的证据,显示结直肠癌是包含不同疾病的“复杂病”。已有的研究混合了结肠癌与直肠癌样本,同时,没有区分不同的病理分期,因此,采用基因芯片分析就造成许多本来异常表达的miRNA被忽略。为解决以上这些问题,我们使用包含了904个miRNA的新一代基因芯片(已接近人类所有的miRNA)来确定直肠癌和非瘤组织中差异表达的miRNA。6例配对直肠癌病理TNM分期为Ⅲ期、Ⅳ期或Dukes分期为“C”、“D”期,增加了鉴定在直肠癌中异常表达miRNA的灵敏性。直肠癌组织miRNA的基因表达谱我们采用miRCURYTM Array LNA microRNA chip (v.14.0)基因芯片比较直肠癌组织和邻近非肿瘤组织之间miRNA表达差异。结果发现两者之间miRNA表达谱存在着显著不同,当设定平均上升倍数大于2倍和P值小于0.05为标准,发现了直肠癌组织中有88个miRNAs呈显著上调表达和40个miRNAs呈显著下调表达。将我们的结果与文献报道在结直肠癌(CRC)中异常表达的miRNAs比较,结果发现,在88个上调表达的miRNAs中,47个已证实在结直肠癌组织中异常表达。在40个下调表达的miRNAs中,15个miRNAs已报道在结直肠癌组织中异常表达。我们在直肠癌组织中新发现了66个miRNAs异常表达。RT-qPCR分析异常表达的miRNAs选取上调或下调差异相对高的12个miRNAs,采用RT-qPCR在27例配对直肠癌与其对应正常直肠组织中分析这些miRNAs表达水平的改变,结果显示,所有6个在芯片筛选出的上调表达的miRNAs在27个直肠癌肿瘤组织中也异常高表达,与芯片结果相比较,表达水平相差从-11.88%到39.09%；同样所有6个在芯片筛选出的下调表达的miRNAs在肿瘤组织中也呈低表达,与芯片结果相比较,表达水平相差从1.35%到29.35%。对芯片与RT-qPCR分析结果进行Pearson相关性分析,发现对比上调表达miRNAs其R值为0.707(P<0.05),下调表达miRNA其R值为0.923(P<0.01),12个miRNAs相关性的R值为0.958 (P<0.01)。miRNAs可特异性地调节靶基因的表达,这些miRNAs在肿瘤组织中的异常表达可能与直肠癌的癌变与恶性演进相关。结肠癌和直肠癌组织中miRNA表达模式与表达水平存在差异结直肠癌应该被看作是包含多种疾病的一个“复合”概念。为了分析miRNA表达模式与表达水平在结肠癌和直肠癌组织中是否存在差异,我们选择了4个miRNAs,检测25例配对结肠癌及正常组织与27例配对直肠癌及正常组织miRNAs的表达水平。结果显示：与配对的正常组织比较中,miR-452在结肠癌组织中的表达水平增加3.03倍,在直肠癌组织中也增加2.99倍,他们之间增加的表达水平非常相似。miR-126虽然在结肠癌和直肠癌组织中都呈现高表达(p<0.01),但它在结肠癌组织中增加6.34倍,而在直肠癌组织中只上升了2.01倍,两个亚型癌组织中miRNAs的表达水平存在显著性差异(p<0.01)。miR-143在结肠癌组织中下降了4.82倍(p<0.01)；虽然它在直肠癌组织中也下调,但与正常直肠组织比较无显著性差异。miR-31表达水平在结肠癌组织中增加4.06倍,但它在直肠癌组织中上升高达13.4倍,在两个亚型癌组织中miRNAs的表达水平存在显著性差异(p<0.01)。这些数据表明,miRNAs在两个亚型癌组织中呈现不同的表达模式与表达水平,这可能与两者的不同病理进程相关。miRNA的表达水平与直肠癌的恶性演进相关研究发现一些miRNAs的靶基因与肿瘤的侵袭和转移相关,并提出了"metastamir"概念,miRNAs的异常表达可能与直肠癌的恶性演进相关。我们选取了可能与肿瘤转移相关的miR-31和miR-145,采用RT-qPCR分析其在27例不同病理阶段的直肠癌组织中表达水平的变化,结果发现：与正常直肠组织比较,miR-31在A期病理标本中表达水平明显上升,但其表达水平比C期与D期病理标本明显降低,p值分别是0.012和0.015。虽然B期和C期病理标本之间miR-31表达水平没有明显差异(p值为0.115),但D期与B期病理标本间存在显著性差异(p<0.01)。相反,miR-145表达水平的降低与直肠癌的恶性进展相关。miR-145在D期病理标本中表达最低,与A、B、C期病理标本比较,p值分别为0.002、0.014和0.112。结果提示：miR-31表达水平的上调与miR-145下调程度与直肠癌的病理分期相关,它们可能参与了直肠癌恶性演进的过程。结论：在直肠癌组织中,我们鉴定了一批新的异常表达的miRNAs,并分析了一些miRNAs在结肠癌与直肠癌组织中表达模式的差异,确定了miR-31和miR-145异常表达水平与直肠癌恶性演进的关系,这些研究将为阐明直肠癌癌变的分子机制提供新的线索,并为早期诊断、判定预后提供可能的分子标志物及干预靶分子,具有潜在的临床应用价值。更多还原

【Abstract】 Colorectal cancer (CRC) is the important cause of cancer-related death in China. Screening for CRC allows early stage diagnosis of the malignancy and potentially reduces mortality of the disease. A growing number of direct and indirect evidence suggests a relationship between altered miRNA expression and cancer. miRNAs are important factors in tumorigenesis and have been the subject of research in many types of cancers. miRNAs are small 22 to 25 nucleotide sequences of RNA that participate in the regulation of cell differentiation, cell cycle progression, and apoptosis. miRNAs are believed to function primarily as negative regulators of gene expression following binding to conserved sequences within the 3’untranslated region of target mRNAs. While the biological roles of miRNA are under intense investigation, they are believed to define and maintain cellular fate in a manner similar to transcription factors, alterations in miRNA expression may be an important contributor to the development of cancers. Screening aberrant miRNAs may provide a basis for understanding the functional role of miRNAs in cancer.miRNAs are also abnormally down-regulated or up-regulated in colon-cancer tissue. Recently dysregulated miRNAs screening in CRC by gene chip analysis or real-time RT-PCR has been reported.13 miRNA is significantly altered in colorectal cancer.287 miRNAs gene chip is used to analyse miRNA expression profile in CRC and CRC cell lines and identified 37 miRNAs that were differentially expressed between CRC and normal tissue. miRNA microarray containing 455 human miRNA probes is used to identify aberrant expression miRNAs between surgically resected colorectal cancers and noncancerous tissues. They found that 21 miRNAs overexpressed in colorectal cancer tissues. It estimated that it exists about 1000 miRNAs in human, but previous studies only screened different miRNAs in CRC patients from a panel of 96,145,287,455 miRNAs respectively. It is completely necessary to use systemic method to identify new aberrant expression miRNAs by gene chip.Accumulating evidence suggests that colorectal cancer (CRC) should be viewed as a heterogeneous disease, with proximal and distal CRCs showing multiple biological and clinical differences. There is a significant difference in regional expression of 10 tumor-associated markers. Tumor diameter, pT stage and MSI status are used to distinguish right-sided colon cancers from rectal cancers, and pT stage and E-cadherin are used to discriminate left-sided colon cancers and rectal cancers. Distal sporadic MSI-high CRCs formed a distinct subgroup with distinguished clinicopathological and molecular features from proximal MSI-high CRCs. These data along with existing evidence for the presence of distinct regional embryological origin and gene expression profile are highly supportive of the concept that proximal and distal CRCs are distinct clinicopathologic entities. Previously studies work with the total concept of "CRC" and did not screen dysregulated miRNAs of specific subtype of CRC. At the same time, aberrant miRNAs are identified by comparing CRC tumor and normal tissues, but the paired samples came from different stages of CRCs. Some CRC progression-related miRNAs may be neglected in the chip analysis.To resolve the issues, we used 904 miRNAs gene chips which is near all miRNAs of human and identify new aberrant expression miRNAs between rectal cancers and non tumour tissues. All 6 paired rectal cancers were pathological classified in TNM category of III stage and IVstage or "Dukes C and D". It increases the sensitivity to identify new aberrantly expressed miRNAs in rectal cancers.miRNA expression profiles in rectal cancer tissuesWe used miRCURYTM Array LNA microRNA chip (v.14.0) to evaluate miRNA expression profiles between rectal cancer tissues and adjacent non-tumours tissues. The miRNA expression pattern was found to be significantly different. When setting average change more than 2 fold and p value less than 0.05 as a cutoff level,88 miRNAs are up-regulated and 40 miRNAs are down-regulated. Comparing with previous literature of miRNAs and CRC, we found that 47 genes have been reported in the CRC among 88 up-regulated genes. Among 40 down-regulated genes,15 genes have been reported.Validation of miRNA expression profiles by real time quantitative PCRTo validate the relative miRNA expression levels by chip analysis,12 miRNAs were selected and they are among the top 20 differentially expressed miRNAs by chip analysis. We analysis expression level of these miRNAs in 27-paired samples of rectal cancer, and results showed that miRNA expression in the paired benign and tumor tissue was consistently increased or decreased in all cases. We found all 6 up-regulated miRNAs in chip analysis increase expression level in rectal cancer tissues, and 6 down-regulated miRNAs also decrease expression in normal tissues. Expression levels of 5 up-regulated miRNAs are less than that in chip analysis, range from -11.88% to 39.09% respectively. On the contrary, Expression levels of 6 miRNAs are more than that in chip analysis, range from 1.35% to 29.35%. The correlation of relative miRNAs expression levels was analyzed by cDNA array versus RT-qPCR. Pearson correlation of down-regulated miRNAs was 0.923 (P< 0.01) and Pearson correlation of up-regulated miRNAs was 0.707 (P< 0.05). Pearson correlation of both down-and up-regulated miRNAs was 0.958 (P<0.01). This suggests that the differential expression is maintained between rectal cancer and normal tissue. miRNAs function in tumor and normal at different expression levels and is related with pathogenesis of rectal cancer.Different expression pattern of miRNAs between colon and rectal cancersColorectal cancer should be viewed as a heterogeneous disease. To test if it exist different expression pattern of miRNAs between colon cancer and rectal cancer, we selected 4 aberrant miRNAs of rectal cancers and detected the expression level from 25-paired colon cancers and 27-paried rectal cancers. Comparing with paired normal tissues, miR-452 b expression level in colon cancers increase 3.03 fold and that in rectal cancer also increase 2.99 fold and they are very resemble. Although miR-126 increase expression both in colon cancers and rectal cancers and p value less than 0.01 by t-test analysis, but it increase 6.34 fold in colon cancers and only 2.01 fold in rectal cancers, and it exist significant difference between two subtype cancers (p< 0.01). miR-143 down-regulates in multiple cancers and it decrease 4.82 fold in colon cancer (p< 0.01), but it does not change in rectal cancer (p>0.05). miR-31 expression increase 4.06 fold in colon cancers and it increase 13.4 in rectal cancers, and it exist significant difference between both subtype cancers (p< 0.01). The data demonstrates that miRNAs express different pattern between two subtype cancers and it will results in different pathological progress.miRNA expression levels are related with progress of rectal cancerSome miRNAs may target invasion and metastasis-related genes and are involved in progress of cancers. Up-regulated expression miR-31 and down-regulated expression miR-145 are detected in different pathological stages of rectal cancers; Comparing with normal tissues, miR-31 expression level of A stage is significantly increase, it is lower than of C stage of rectal cancers and that of D stage, p values are 0.012 and 0.015 respectively. Although we did not find it exist significant difference between B stage and C stage (p value is 0.115), it exists statically difference between B stage and D stage. On the contrary, expression level of miR-145 decreases with progress of rectal cancers. It significantly decreases in D stage. Comparing with A, B and C stages, p values are 0.002, 0.014 and 0.112 respectively. Unlike miR-31 can not tell the difference between A stage and B stage and between C stage and D stage, miR-145 can tell every stage with others (p<0.05). Taken together, our data showed that miRNAs expression level may be related with different pathological stage of rectal cancers.ConclusionOur data may provide diagnostic biomarkers for rectal cancer and offer new clues to study the molecular mechanism of carcinogenesis of rectal cancer.更多还原