【作者】 李琼；

【导师】 王阁；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2009， 硕士

【摘要】 背景和目的:MircoRNAs(miRNAs)是一类短序列、非编码、具有调控功能的单链小分子RNA,长约20~24 nt,由一段具有发夹样环状结构、长约70~80 nt的单链RNA前体(pre-miRNA)经过Dicer酶加工后生成后生成。miRNAs通过与其靶mRNA分子的3’端非编码区(3’-UTR)互补结合,在翻译水平上特异性抑制基因表达,参与调控生物生长和发育等许多复杂的生命过程。许多miRNAs的序列在起源甚远的物种之间相对保守,虽然这些miRNAs的生物学功能目前还不十分清楚,但可以肯定的是它们有不同的表达模式,同时参与调节各种生理病理过程。异常的miRNAs表达与人类某些疾病包括肿瘤的发生发展都密切相关,miRNAs被认为是一组新的癌或抑癌基因,其表达具有特异性的肿瘤组织表达谱。原发性肝细胞肝癌(HCC)是我国最常见的恶性肿瘤之一,其发病隐匿、预后差、死亡率高。已有研究发现miRNAs在肝癌组织中异常表达,它可能参与了肝细胞癌变的病理过程。在我们的研究中利用miRNA芯片技术筛选肝癌细胞HepG2和正常肝细胞LO2中miRNAs的差异表达谱,通过定量RT-PCR和Northern blot等方法验证芯片结果的可靠性。结合文献报道中的肝癌及癌旁肝组织中miRNAs表达模式,筛选出在HepG2细胞中高表达的miR-224作为研究的靶标,均表达明显上调。利用MTT、流式细胞仪(FACS)、划痕实验、Transwell细胞迁移和Metrigel细胞侵袭实验等技术和方法分析miR-224是否对肝癌细胞的细胞周期、增殖与凋亡、迁移与侵袭等生物学行为有无影响,其结果表明miR-224参与调节肝癌细胞的增殖与凋亡,特别是能明显的促进肝癌细胞的迁移和侵袭力,但对细胞周期的进程无明显影响。研究miRAN功能的关键是寻找其调控的下游靶基因。目前,有多个应用:生物信息学方法预测miRNA靶基因的软件,包括MIRANDA,PICTAR,Target Scan等算法对目的miRNAs的3′端序列搜索靶基因,寻找miRNAs可能在mRNA水平起作用的下游靶基因。应用MIRANDA和PICTAR的生物信息学方法对miR-224的靶基因进行预测,并以miR-224参与调节肝癌细胞的迁移和侵袭力为突破口,在预测的靶基因中挑选出PAK4进行分析和初步验证,目的在于寻找miR-224直接调控的靶基因,为阐明miR-224参与调节肝癌细胞的迁移和侵袭力参与调控的分子机制提供实验证据,进一步使我们更加全面地理解miRNA在肝癌发生机制中的作用,为寻找肝癌治疗的新靶点提供有效的途径。方法:1.应用miRNA基因芯片技术,通过基因杂交、T4-RNA连接酶标记的方法,检测肝癌细胞株HepG2和正常肝LO2细胞中miRNA表达的差异谱。通过定量RT-PCR和Northern blot的方法证实了miRNA基因芯片结果的真实可靠性。2.利用MTT、流式细胞仪(FACS)、划痕实验、Transwell细胞迁移和Metrigel细胞侵袭实验等技术和方法分析miR-224对细胞周期、细胞增殖与凋亡、迁移与侵袭等生物学行为的影响。3.应用不同的生物信息学方法对在肝癌组织和细胞中表达上调的miR-224的靶基因进行预测。4.应用免疫组化方法和组织芯片技术分析预测的靶基因PAK4在肝癌组织中的表达情况,并分析PAK4及其磷酸化状态与肿瘤的临床病理学因素之间的关系。5.应用Western blot法分析miR-224与PAK4和MMP-9蛋白表达的相关性。结果:1. MiRNA基因芯片结果显示与LO2细胞相比,在HepG2肝癌细胞中有143个miRNAs的表达具有显著差异,Fold Change在4倍以上(其中66个上调和77个下调),miR-224在不同的肝癌细胞株和肝癌组织中表达模式一致,均表达明显上调。2. MiR-224参与调节肝癌细胞的增殖与凋亡、迁移与侵袭等生物学过程,但对细胞周期的进程无明显影响。3. MiR-224对肝癌细胞的迁移和侵袭力有明显的促进作用。4.预测miR-224的靶基因有数百个,其多个基因涉及细胞周期、信号转导、细胞增殖、分化和细胞凋亡等众多生物学过程,说明miR-224在肝癌发生发展中有着重要的生物学功能。但预测的PAK4基因不是miR-224直接调控的靶基因。5.在肝癌组织中PAK4的表达强度明显高于正常肝组织,而同样在肝癌组织中磷酸化PAK4的表达水平又明显高于非磷酸化PAK4的表达。而且,磷酸化PAK4主要在胞核表达,其核表达的强阳性率与细胞分化程度的高低和有无淋巴结转移等临床病理因素具有明显相关性。6. MiR-224与PAK4和MMP-9等肿瘤迁移侵袭相关分子的表达具有明显的相关性。结论:1. MiR-224的高表达是肝癌发生发展过程中的一个重要分子事件。2. MiR-224可促进肝癌细胞的增殖与凋亡活性,但对细胞周期的进程无明显影响。3. MiR-224是调控肝癌细胞迁移和侵袭能力的一个新型分子。更多还原

【Abstract】 Background and objectiveMicroRNAs (miRNAs) are a new class of non-protein-coding, endogenous, small RNAs and of about 20-24 nucleotides in length, which are cleaved from the 70~80 nt partially duplexed precursor by the RNase III Dice. Mature miRNAs are known to negatively regulate gene expression or destroy the stability of genes via incomplete or complete matching with the 3’UTR of their target genes at the post-transcriptional level. They are involved in processes such as the regulation of organismal development, nerve differentiation, cell proliferation, apoptosis and fat metabolism. By now, it is predicted that miRNAs may regulate the two-third whole human genes. Many miRNAs are has relatively conservative sequences among species.Though the foundation of these miRNAs are not clear, it is sure that they have different expressed patterns, which can regulate many physiology and pathology processes, and abnormal expression of miRNAs may result in some human diseases including tumors. miRNAs are recognized as an oncogene or anti-oncogene, they have the specific express profiles in different tumor tissues.Human hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. It has the strongest invasion, the fastest metastasis and the highest recurrence. Abnormal expression profiles of miRNAs in HCC tissues have been detected by many studies. In our study, we detected the different expression profiles of miRNAs in HepG2 and LO2 cell lines by miRNA microarray. To determine the validity of miRNA microarray, we verified miR-224 expression in HepG2 cells by qRT-PCR and Northern blot. Along with reports of the different expression patterns of miRNAs in HCC and non-tumor tissues, this allowed us to isolate miR-224 as a study target, since it is overexpressed in both HCC tissues and cells, and at the same time it has more higher expression was found in the placenta tissues than other normal tissues. Then the different experiments including MTT, FACS, Scratch wound, Transwell migration and Matrigel invasion assays were performed to decided whether miR-224 can influence the biological behaviors of HepG2 cells or not. The results showed miR-224 plays a very important role in maintenance of malignant phenotype of HepG2 cell, which is a new regulator in the cell migration and invasion of hepatocellular carcinoma cells.The key to study the function of miRNAs is to find out its target genes. Predicting the targets of miRNAs and investigating the interaction between miRNAs and the corresponding targets are becoming the study focus. By now, there are several bioinformatics ways to predicte the target genes of miRNAs, including MIRANDA, PICTAR, Target Scan and so on. We predicted the miR-224 target genes by different bioinformatics, and analyse the expression of PAK4 by Immunohistochemistry and Tissue chip in HCC tissues, which is one of its predicted genes. MiR-224 was significantly upregulated in HepG2 cells. Cell proliferation, migration and invasion, but not cell cycles, were altered after changing the expression of miR-224. Taking invasion and migration as a breakthrough, it shows miR-224 can increased expression of PAK4/ MMP-9 in HepG2 and LO2 cell by Western blot. So, miR-224 may indirectly regulate PAK4 expression in liver cancer. The results will not only help us to understand the role of miRNA during the hepatocarcinogenesis, but also provide a valid ways to look for a new target for the treatment of HCC.Methods1. MiRNA microarray was performed to detect the different expression profiles of miRNAs in HepG2 and LO2 cell lines. qRT-PCR and Northern blot were performed to determine the validity of miRNA microarray.2. MTT, FACS, Scratch wound, Transwell migration and Matrigel invasion assays were performed to investigate the miR-224 influences on the cell cycles, cell proliferation and apoposis, cell migration and invasion.3. The different bioinformatics ways to predicted the target genes of miR-224 which is overexpressed both in the HCC tissues and cells.4. Immunohistochemistry and Tissue chip showed the expression level of PAK4 in HCC and the relationship among the phosphorylation expression level of PAK4(p-PAK4) with the clinical pathology factor of HCC.5. Western blot was performed to decide the relationship among miR-224 with PAK4 and MMP-9. Results1. The result of miRNA microarray shows the pattern of miRNA expression in HepG2 cells was markedly different from LO2 cells. After normalization, the expression profiles of 143 miRNAs were determined between HepG2 and LO2 cells, revealing 66 upregulations and 77 downregulations miRNAs. MiR-224 was overexpressed both in HCC cells and tissues.2. Cell proliferation, apoptosis, migration and invasion but no cell cycles were altered after changing the expression of miR-224.3. MiR-224 positively regulating cell migration and invasion.4. There are hundreds of target gens were predicted by two bioinformatics ways, most of genes are involved in cell cycles, cell proliferation, differentiation and apoptosis, signal transduction, and so on. MiR-224 may indirectly regulate PAK4 expression in HCC.5. There is a functional linkage among the expression of miR-224 and PAK4 /MMP-9 which were two mediators of c tumor cell migration and invasion.6. The protein of PAK4 are higher expressed level in HCC tissues compare normal liver tissues, and p-PAK4 are higher expressed level than PAK4 in HCC tissues. p-PAK4 are located mainly in cell nucleus and the expressed positive rate of p-PAK4 has a close relation with the degree of cell differentiation、lymph node metastasis or TNM stage.Conclusions1. The overexpression of miR-224 is a very important event during the hepatocarcino- genesis.2. MiR-224 is involved in the regulation of cell proliferation and apoptosis but not cell cycles in HepG2 cell.3. MiR-224 is a new regulator in the cell migration and invasion of HepG2 cell.更多还原