【作者】 陈悦；

【导师】 王振宁；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2010， 博士

【摘要】 目的微小RNA (microRNA,miRNA)是近年来发现的一类21-25个核苷酸的内源性非编码小RNA, miRNA基因先转录产生miRNA的初级产物-pri-miRNA,它被RNaesⅢ内切酶切割成70个核苷酸序列的miRNA的前体-pre-miRNA, pre-miRNA再被Dicer酶切割成约22nt的miRNA的成熟体。成熟的miRNA形成RNA诱导沉默复合体,成熟miRNA的5’-非翻译区的2-7个核苷酸的“种子序列”可以与靶mRNA的3’-非翻译区互补结合,在转录后水平上抑制靶基因的表达。1993年,首个miRNA-lin-4被发现,从此揭开了对miRNA研究的序幕。随后,许多实验室从线虫、果蝇、家鼠、拟南芥及人类等多种真核生物中发现了至少500种这类小分子RNA,其中在人类染色体上就有300余种,占人类基因的1-4%。MiRNA的编码基因是目前最大的一类调节基因。它在细胞的生长、增殖、发育和凋亡过程中发挥着重要作用,并与许多肿瘤的发生、发展相关。MiRNA在肿瘤中发挥着相当于癌基因或抑癌基因的作用,调节着肿瘤细胞的多种重要生物学行为。大肠癌是世界上常见的恶性肿瘤之一,它的发病是多因素、多步骤的复杂过程。目前发现很多miRNAs在大肠癌中异常表达。MiR-143和miR-145作为较早在大肠癌中发现的miRNAs,已证实在大肠癌中存在异常表达。let-7也是较早发现在大肠癌中异常表达的miRNA,通过进一步的研究证实,它不仅在大肠癌的发生而且在大肠癌的发展过程中也发挥了重要的作用。越来越多的研究表明,miRNA与大肠癌有着非常密切的关系。随着研究的不断深入,miRNA在大肠癌的基因诊断和治疗中将发挥重要的作用。在本研究中,我们参考国内外相关文献及前期关于大肠癌的miRNA表达谱的研究,初步确定miR-148b为我们的研究重点。利用定量real-time PCR技术检测了大肠癌组织相对于癌远端非瘤组织中miR-148b的表达及与其有相同的“种子序列”的miRNAs (miR-148a和miR-152)的表达情况。通过使大肠癌细胞系中过表达miR-148b,初步探索其在大肠癌的发生、发展中的作用。方法一、MiR-148b在大肠癌组织中的表达收集101例大肠癌患者肿瘤及周围非瘤组织手术标本,经提取RNA,加尾,抽提纯化,反转录后得到cDNA模板,运用定量real-time PCR检测miR-148b的相对于癌旁非瘤组织的表达量。如果相对表达量小于1,则被认为是低表达。反之,被认为是高表达。然后运用统计学方法分析miR-148b与临床病理资料之间的相关性。二、MiR-148b在大肠癌细胞系中的表达及其对细胞增殖的影响由于没有正常的大肠粘膜细胞系,我们在大肠癌组织中随机选择了三个癌旁非瘤组织作为大肠癌细胞系的阴性对照。运用定量real-time PCR检测出miR-148b在大肠癌细胞系(HT-29和HCT-116)中的表达情况。向大肠癌细胞系中(HT-29和HCT-116)转染miR-148b mimics,使miR-148b在大肠癌细胞系过表达,通过MTS实验检测转染miR-148b mimics后对细胞增殖的影响。三、MiR-148a及miR-152在大肠癌组织及细胞系中的表达运用定量real-time PCR检测101例大肠癌组织标本中miR-148a及miR-152相对于周围非瘤组织的表达量,然后运用统计学方法分析二者与临床病理资料之间的相关性。然后,提取大肠癌细胞系HCT-116和SW-620的RNA,经加尾,抽提纯化,反转录后,运用定量real-time PCR检测这两个细胞系(HCT-116和SW-620)中miR-148a和miR-152相对于非瘤组织的表达量。运用统计学方法分析miR-148a、miR-148b和miR-152三者在大肠癌组织中表达的相关性。结果一、MiR-148b在大肠癌组织中的表达1、在人大肠癌组织中发现miR-148b较其相应周围非瘤组织低表达。在101例大肠癌组织中,其中84(83%)例miR-148b (p<0.001)低表达,miR-148b在大肠癌组织中相对于周围非瘤组织表达的中位数是：0.16。MiR-148b的低表达与肿瘤的大小(p<0.001)和浸润深度(p=0.012)相关。肿瘤越大、浸润越深,miR-148b的表达量越低。2、PCR产物经TA克隆,DNA测序后与miRBase上公布的miR-148b序列比对正确、完整。二、MiR-148b在大肠癌细胞系中的表达及其对细胞增殖的影响定量real-time PCR结果显示,miR-148b在人大肠癌细胞系HT-29(p<0.001)和HCT-116 (p<0.001)中均有不同程度的低表达。HT-29及HCT-116在转染了miR-148b mimics后,经定量real-time PCR检测验证,转染了miR-148b mimics的实验组细胞系中,miR-148b的表达水平相对于对照组均有不同水平的提高。应用MTS法绘制的生长曲线表明,对于HT-29细胞系,转染了miR-148b mimics实验组的细胞在48小时开始其增殖速率明显低于阴性对照及空白对照组(p<0.05)。对于HCT-116细胞系,转染了miR-148b mimics实验组的细胞在72小时开始其增殖速率明显低于阴性对照及空白对照组(p<0.05)。三、MiR-148a及miR-152在大肠癌组织及细胞系中的表达1、在人大肠癌组织中发现miR-148a及miR-152较其相应周围非瘤组织低表达。在101例大肠癌组织中,其中69(68%)例miR-148a(p<0.001)低表达,80(79%)例miR-152 (p<0.001)低表达,miR-148a及miR-152在大肠癌组织中相对于周围非瘤组织表达的中位数分别是：0.36和0.11。MiR-148a和miR-152的低表达与肿瘤的大小(p=0.018和p=0.004)和浸润深度(p=0.023和p=0.002)相关。肿瘤越大、浸润越深,这两种miRNAs的表达量越低。2、定量real-time PCR结果显示,miR-148a及miR-152在人大肠癌细胞系HCT-116 (allp<0.001)及SW-620 (all p<0.001)中的表达均较癌旁非瘤组织有不同程度的下降。3、MiR-148a、miR-148b及miR-152三种miRNAs的表达在大肠癌组织中存在显著的相关性(p<0.001)。4、MiR-148a及miR-152的PCR产物经TA克隆,DNA测序后与miRBase上公布的序列比对正确、完整。结论1、大肠癌组织中miR-148b的表达量显著低于其周围非瘤组织。MiR-148b的低表达与肿瘤大小及浸润深度相关,肿瘤越大、浸润越深,miR-148b的表达量越低。2、大肠癌细胞系HT-29和HCT-116中,miR-148b的表达量显著低于非瘤对照组。3、MiR-148b的过表达抑制了大肠癌细胞的增殖。4、大肠癌组织中miR-148a及miR-152的表达量显著低于其周围非瘤组织。MiR-148a和miR-152的低表达与肿瘤大小及浸润深度相关,肿瘤越大、浸润越深,这两种miRNAs的表达量越低。5、大肠癌细胞系HCT-116及SW-620中,miR-148a及miR-152的表达量显著低于非瘤对照组。6、MiR-148a、miR-148b及miR-152三种miRNAs在大肠癌组织中的表达存在显著的相关性。更多还原

【Abstract】 ObjectiveMicroRNA(miRNA) is a class of endogenous, non-coding 21-25 nucleotides RNA, which was discovered in recent years. MiRNAs are transcribed as primary transcripts (pri-miRNA). An 70 nt precursor called the pre-miRNA, which is cleaved by Dicer to generate an 22nt mature miRNA, is excised out from the pri-miRNA by RNase-Ⅲ.The mature miRNA then gets assembled into the effector complexes. The "seed region", which is the core sequence that encompasses the first 2-7 nucleotides at the 5’portion of miRNA, is essential for the specific suppression of targe mRNA. In 1993,the first miRNA-lin-4 was discovered. Since then, increasing numbers of studies showed at least 500 miRNAs aberrantly expressed in different types of cancers, and showed that miRNAs were involved in the regulation of the proliferation, differentiation and apoptosis. Therefore, miRNAs were deemed to play a crucial role in the initiation and progression of human cancers, which could regulate many oncogenes and tumor suppressor genes.Colorectal cancer is one of the most common malignancies in the world. Altered expression of miRNAs have been reported in colorectal cancer, and may play a critical part in carcinogenesis. MiR-143 and miR-145, which were discovered in the early study, played important roles in the initiation and progression of colorectal cancer. Let-7,which was also found aberrant expression in colorectal cancer, was associated with not only carcinogenesis but also development of cancer. With more and more miRNAs were discovered in colorectal cancer, miRNAs might be involved in the diagnosis and therapy of colorectal cancer.In the present study, we detected the expression of miR-148a, miR-148b and miR-152 relative to their non-tumorous control in colorectal cancer tissues and cell lines using quantity real-time PCR. We studied the function of miR-148b in the colorectal cancer cell lines and detected preliminarily the role of miR-148b in the initiation and progression of cancer. 1. Expression of miR-148b in the colorectal cancer tissuesWe detected the relative expression of miR-148b in colorectal cancer tissues of 101 patients compared to their matched non-tumor adjacent tissues(NATs). Therefore, the value of the relative expression ratio< 1.0 was considered as low-expression in cancer relative to the non-tumorous control. We analyzed the association between miR-148b expression and clinicopathological characteristics.2. The expression of miR-148b in colorectal cancer and the effects on colorectal cancer cell proliferationWe detected the expression of miR-148b in colorectal cancer cell lines, HCT-116 and HT-29, by quantity real-time PCR. MiR-148b was over-expressed in colorectal cancer cell lines by transfecting miR-148b mimics. MTS assay was used to investigate the effect of miR-148b on the proliferation of colorectal cancer cell lines.3. Expression of miR-148a and miR-152 in the colorectal cancer tissues and cell linesWe detected the expression of miR-148a and miR-152 in the colorectal cancer tissues and cell lines(HCT-116 and SW-620) by quantity real-time PCR. We analyzed the association between these two miRNAs expression and clinicopathologic characteristics.Results1. Expression of miR-148b in the colorectal cancer tissues(1) We found low-expression of miR-148b in colorectal cancer tissues relative to their NATs. Among 101 patients with colorectal cancer,84 (83%) cases showed low-expression of miR-148b (p<0.001). The median fold change was 0.16. Low expression of miR-148b was associated with increased tumor size (p<0.001) and advanced pT stage (p=0.012).(2) The products of PCR were confirmed by TA cloning and sequencing assay.2. The expression of miR-148b in colorectal cancer cell lines and the effects on colorectal cancer cell proliferation As there is no normal colorectal epithelial cell, we randomly selected 3 NATs as control. The results of quantity real-time PCR showed that low-expression of miR-148b in the colorectal cancer cell lines (HT-29[p<0.001] and HCT-116[p<0.001]) at different levels between them. After transfection with miR-148b mimics and negative control(NC), we found up-regulation of miR-148b in the experience group comparing to NC group. As the results of MTS assay showed, up-regulation of miR-148b suppressed the proliferation of HT-29 from 48 hours(p<0.05) and HCT-116 from 72 hours(p<0.05).3. Expression of miR-148a and miR-152 in the colorectal cancer tissues and cell lines(1) We found low-expression of miR-148a and miR-152 in colorectal cancer tissues relative to their NATs.69 (68%) cases showed low-expression of miR-148a (p<0.001) and 80 (79%) cases showed low-expression of miR-152 (p<.001) in cancer tissues.The median fold change was 0.36 and 0.11, respectively. Low expression of miR-148a and miR-152 was associated with increased tumor size (p=0.018 and p=0.004, respectively) and advanced pT stage (p=0.023 and p=0.002, respectively).(2) As there is no normal colorectal epithelial cell, we randomly selected 3 NATs as control. The results of quantity real-time PCR showed that low-expression of miR-148a and miR-152 in the colorectal cancer cell lines (HCT-116[all p<0.001] and SW-620[all p<0.001]) at different levels among them.(3) A strong correlation among the expression of miR-148a, miR-148b and miR-152 in colorectal cancer tissues(p<0.001).(4) The products of PCR were confirmed by TA cloning and sequencing assay.Conclusions1. MiR-148b was down-regulated in colorectal cancer tissues relative to their NATs. Low expression miR-148b was associated with increased tumor size and advanced pT stage.2. MiR-148b was down-regulated in colorectal cancer cell lines (HCT-116 and HT-29) relative to non-tumorous control.3. Up-regulation of miR-148b suppressed the proliferation of colorectal cancer cells in MTS assay.4. MiR-148a and miR-152 were down-regulated in colorectal cancer tissues relative to their NATs. Low expression miR-148a and miR-152 was associated with increased tumor size and advanced pT stage.5. MiR-148a and miR-152 were down-regulated in colorectal cancer cell lines (HCT-116 and SW-620) relative to non-tumorous control.6. A strong correlation among the expression of miR-148a, miR-148b and miR-152 in colorectal cancer tissues.更多还原