【作者】 郑海音；

【导师】 林建银； 林旭；

【作者基本信息】 福建医科大学 ， 病原生物学， 2014， 博士

【摘要】 胃癌是中国人最常罹患的恶性肿瘤之一,发病率和死亡率高居不下。早期诊断和寻找到新的治疗靶标对于改善胃癌转归至关重要。MicroRNA,又称miRNA,是由19-24个核苷酸组成的短链非编码RNA,通常与mRNA3’非编码区结合导致m RNA降解或阻断mRNA转录后的翻译,从而负调控基因的表达。近来研究表明,在多种恶性肿瘤的发生发展过程中常伴随着miRNA表达水平的异常。miRNA发挥着类似于癌基因或抑癌基因的作用,参与了肿瘤的生长、分化、粘附、凋亡、侵袭及转移等生物学过程。本研究着眼于寻找胃癌组织中差异表达的mi RNA,进而阐明差异miRNA在胃癌发生发展过程中的作用及机制,这一研究不仅有助于阐明胃癌的发病机理,还可为发现胃癌早期诊断的特异性肿瘤标志物和新的治疗靶标奠定基础。本研究第一部分旨在筛选出胃癌中差异表达的miRNA并分析其临床意义。通过选取35例不同临床分期(I-IV期)胃癌患者的癌组织及对应的癌旁正常组织,采用人microRNA芯片进行miRNA表达谱分析,筛选出胃癌组织中差异表达的miRNA,芯片结果显示共有56条miRNA在胃癌组织中表达异常,其中表达上调的miRNA 32条,表达下调的miRNA 24条。进一步扩大临床样本量,通过实时定量聚合酶链反应(Real-time PCR)验证芯片结果的可靠性,结果显示mi R-214在胃癌组织中表达显著上调,miR-148a和miR-1225-5p在胃癌组织中表达显著下调,与芯片的结果相一致。将筛选并鉴定出的差异miRNA,进一步与患者的性别、年龄、肿瘤分期、淋巴结转移等临床病理参数之间进行相关性分析,分析结果表明胃癌miR-1225-5p的表达水平与胃癌的TNM分期、淋巴结转移呈负相关性。从上述研究结果显示miR-1225-5p可能在胃癌细胞的增殖、侵袭及转移中发挥一定的作用,但其功能机制未见报道。本研究第二部分旨在阐明miR-1225-5p对胃癌细胞生物学行为的影响。本部分一方面通过转染化学合成的miR-1225-5p前体分子(mimics)使胃癌细胞过表达miR-1225-5p,采用CCK-8、软琼脂克隆形成、Transwell迁移及侵袭等实验观察miR-1225-5p对胃癌细胞生长、增殖、迁移、侵袭等生物学行为的影响。体外细胞学实验结果显示胃癌细胞过表达miR-1225-5p能够抑制胃癌细胞的增殖、迁移及侵袭能力。另一方面,构建miR-1225-5p慢病毒感染MGC803胃癌细胞株进行裸鼠注射皮下成瘤实验,体内实验结果显示过表达miR-1225-5p显著抑制胃癌细胞在裸鼠体内肿瘤的生长。本研究第三部分旨在阐明miR-1225-5p调控的下游靶基因及二者如何相互作用影响胃癌细胞的生物学行为。首先,利用生物信息学软件(PicTar、MiRanda、TargetScan等)预测mi R-1225-5p可能调控的靶基因;通过双荧光素酶报告基因实验和蛋白印迹实验(Western blot)对候选靶基因进行验证,确定IRS1可能是miR-1225-5p调控的下游靶基因之一。其次,利用RNAi技术降低胃癌细胞中靶基因IRS1的蛋白表达水平,其对胃癌细胞生长、增殖、迁移、侵袭等生物学行为的作用类似于细胞内过表达miR-1225-5p;进一步通过“拯救”实验,即过表达IRS1能够部分逆转miR-1225-5p对胃癌细胞增殖、迁移和侵袭的抑制作用,证实了miR-1225-5p可能通过调控IRS1的表达影响胃癌的发生发展。更多还原

【Abstract】 Gastric cancer is one of the most common malignant tumors with high morbidity and mortality. Early diagnosis and new therapeutic targets finding are crucial for better prognosis. MicroRNAs(miRNAs) are a class of single-strand small non-coding RNAs with 19–24 nucleotides in length, which can negatively regulate genes by triggering either miRNAs degradation or translational repression through binding to the3’-untranslated region of their target mRNAs. Emerging evidence has showed the association of aberrantly expressed miRNA with the oncogenesis and development in many kinds of malignant tumors. MiRNA can play a role similar to oncogenes or tumor suppressors, which is involved in tumor growth, differentiation, adhesion, apoptosis,invasion and metastasis. In the study, we focused on looking for differential expression of microRNA in gastric cancer tissue, and revealed their role in carcinogenesis and progressing of the gastric cancer. This study not only would help for deep understanding the pathogenesis of gastric cancer, but also could be informative for discovery of specific and sensitive molecular biomarkers and drug targets of gastric cancer.The first part of this study is to screen the differentially expressed mi RNAs in gastric cancer, and analyze their correlations with clinicopathologic characteristics. To obtain the aberrant expressions of miRNAs between gastric cancer and adjacent normal tissues, we employed miRNA expression chips to study the differences of mi RNA expression profiles in 35 pairs of gastric cancer tissues and matched normal tissues adjacent to the tumor with different clinical stage(stage I to IV). 56 mi RNAs were found to be aberrantly expressed,of which 32 were down-regulated and 24 up-regulated in gastric cancer tissues adjacent normal tissues. We enlarge clinical samples to verify the reliability of chip results by quantitative real-time PCR,and the results show that miR-214 expression is significantly raised, while miR-148 a and miR-1225-5p expression is lower in gastric cancer tissues,which were consistent with the microarray data. We further analyze the correlations ofmiRNAs expression with clinicopathologic characteristics, such as age, gender, tumor stage and lymph node metastasis etc, and results indicate a negative correlation of the miR-1225-5p expression level with TNM stage and lymph node metastasis in gastric cancer. These results suggest that miR-1225-5p may play a role in proliferation, invasion and metastasis of gastric cancer, yet the involved mechanism have not been elucidated.The second part of the study is to investigate the effect of miR-1225-5p on the gastric cancer cells. To address this issue, MiR-1225-5p is over-expressed by transfecting gastric cancer cells with miR-1225-5p precursor molecules(mimics), and the effect of miR-1225-5p on cell proliferation, invasion and metastasis were determined by CCK-8assay, soft agar colony formation assay, transwell migration assay and matrigel invasion assay respectively. Furthermore, MGC803 gastric cell lines over-expressing miR-1225-5p were established by infection of recombinant lentivirus and used to inoculated BALB/c nude mice, and the xerograft growth was measured. The results demonstrated that over-expression of miR-1225-5p inhibits gastric cancer cell proliferation, migration and invasion.The third part of the study is to explore the target genes of miR-1225-5p and regulation of the biological behavior of gastric cancer cells by interreaction between miR-1225-5p and their target mRNAs. Firstly, the prediction of the candidate target genes for miR-1225-5p was performed by the programs of Targetscan, PicTar and MiRanda. Then the luciferase reporter assay and westem blot analysis were adapted to confirm the IRS1 as a target of miR-1225-5p. Secondly, the effect of knockdown of IRS1 on gastric cancer cell growth, proliferation, migration, and invasion is similar to those of over-expression of miR-1225-5p. Furthermore, overexpression of IRS1 can partially rescue the inhibition effect of miR-1225-5p on gastric cancer cell proliferation, migration and invasion. These findings indicated that miR-1225-5p may play important role in carcinigenesis and development of gastric cancer.更多还原