microRNA表达谱与非小细胞肺癌临床特点和预后的研究

【作者】 谭晓刚；

【导师】 赫捷；

【作者基本信息】 中国协和医科大学 ， 肿瘤学， 2009， 博士

【摘要】 近30年来随着城市化和工业化的进步,肺癌已经成为全球范围内因癌症导致死亡的首要原因,约占总的恶性肿瘤死亡病例的30%。临床医师经常面临这样的困惑,即同一分期、同一组织学类型、采用同一治疗方案的肺癌患者,其治疗效果、远期生存情况有着明显不同。当前国际广泛采用的基于普通病理学的肺癌TNM分期系统存在许多缺陷,尤其是对于开展肺癌患者个体化的治疗及预后评估等发挥的作用极为有限。miRNA是真核生物中一类长度约为22个核苷酸的参与基因转录后水平调控的小分子非编码RNA。近几年越来越多的研究发现miRNA参与肿瘤发生发展、外侵转移和转归预后等多个环节,研究肺癌中1niRNA表达谱及其与肺癌不同病理类型和预后转归的相关性,为临床更加科学的进行肺癌肿瘤分型、分期及预后判断提供了新的途径,发掘与肺癌相关的特异miRNA表达谱用于肺癌的分型分期和预后判断具有重要的临床意义。本研究应用来自中国医学科学院肿瘤医院胸外科2000年到2002年间外科手术切除的原发性非小细胞肺癌标本116例,包括43例腺癌和60例鳞癌,以及13例小细胞肺癌共116例的癌与癌旁组织与包含677条人类已知序列的miRNA和小鼠的共924条miRNA成熟的序列杂交。结果第一次通过数学模型利用5个miRNA (hsa-miR-210, hsa-miR-30a, hsa-miR-140-3p, hsa-miR-182 hsa-miR-486-5p)将肺癌／癌旁分开,准确率98.2%,肺鳞癌／癌旁分开,准确率93.3%。第一次找到一个miRNA (hsa-miR-31)与肺鳞癌预后相关。hsa-miR-31预后K-M分析,其表达越高,预后越差(p=0.007),Cox回归分析,其是独立预后因素(p=0.015)并用realtime-PCR证实。多个miRNA与临床病理(年龄、性别、吸烟)相关。如hsa-miR-205与非小细胞肺癌患者吸烟指数(=400支／年)成正相关。hsa-miR-205, hsa-miR-31与鳞癌的分化相关。hsa-miR-205, hsa-miR-181a与鳞癌患者的性别相关。另外发现hsa-miR-126和hsa-miR-20a与小细胞肺癌预后相关。当今由于缺乏有效的指导临床诊断和治疗的肿瘤分子标记物,所以非小细胞肺癌患者的疗效不佳。肽基脯氨酰顺反异构酶Pinl在许多肿瘤中过表达,也包括非小细胞肺癌,其可能成为靶向治疗靶点。为探讨Pinl表达对肺癌发生,发展以及转移的影响。我们利用慢病毒包装干涉RNA载体感染肺腺癌细胞H1299,实验表明,稳定抑制肺腺癌细胞H1299 Pinl表达,可以抑制细胞的增殖,抑制软琼脂克隆形成和裸鼠成瘤。同时,可以在体外减弱细胞的迁移和侵袭能力。Pin1在肺癌发生,发展以及转移的过程中起重要作用,有望成为肺癌靶向治疗的有意义的靶点。更多还原

【Abstract】 Lung cancer has the highest world-wide cancer mortality. Patients with apparently similar lung cancers may experience very different clinical outcomes, and it is often difficult to predict a patient’s prognosis. Tumor-node-metastasis (TNM) classification allows diagnosis of the tumor, it provides little therapeutic biological information, such as the metastatic potential or the sensitivity or resistance of the tumor to radiotherapy and chemotherapy. MicroRNAs (miRNAs) are a species of small non-coding single-stranded RNA of about 21 nucleotides that, through partial sequence homology, may interact with the 3’-untranslated region of target mRNA molecules. Recent evidence has shown that miRNA may function as tumor supressors or oncogenes, and alterations in miRNA expression may play a critical role in the initiation and progression of a number of cancers. Therefore, miRNA expression profiles may also be useful for diagnosis of lung cancer, for sub-type classification, or for predicting patient prognosis and guiding personalized disease management. While microRNA expression profiling has recently been used to characterize the prognosis for patients with adenocarcinoma of lung cancer, description of the microRNA patterns in lung cancer, especially the prognosis for squamous cell carcinoma of lung cancer is lacking.To define the microRNA expression patterns associated with lung cancer and the microRNA associated with prognosis for squamous cell carcinoma of lung cancer. MicroRNA microarray expression profiling of tumors and paired adjacent normal tissues was performed on a cohort of 116 patients,60 squamous cell carcinomas,43 adenocarcinomas and 13 small cell lung cancers, recruited between 2000 and 2002. We evaluated microRNA associations with cancer versus adjacent normal tissue, clinicopathological status. Survival association was validated in an independent cohort of 20 squamous cell carcinomas, using quantitative reverse transcription polymerase chain reaction assays. Five microRNAs classifier could distinguish malignant lung cancer lesions from adjacent normal tissues. Some miRNAs showed correlations with several different clinicopathological parameters. High hsa-miR-31 expression was associated with poor survival in squamous cell carcinoma of lung cancer. High hsa-mir-20a and low hsa-mir-126 expression correlated with poor survival of SCLC. Lung cancer may have a distinct miRNA expression pattern that may differentiate it from normal tissue and clinicopathological status. High hsa-miR-31 expression is associated with poor survival outcome.Current therapies for NSCLC patients are inefficient due to the lack of diagnostic and therapeutic markers. The phospho-Ser/Thr-Pro specific prolyl-isomerase Pin1 is overexpressed in many different cancers, including NSCLC, and may possibly be used as a target for cancer therapy. To validate the oncogenic potential of Pinl in lung cells, we down-regulated Pinl by RNA interference in H1299 cells. Retrovirus-mediated siRNA targeting of Pinl resulted in the stable suppression of both cell growth, anchorage-independent growth in soft agar and tumorigenic including cell migration, invasion in H1299 cells. Pinl expression may be an unfavorable prognostic factor in patients of NSCLC patients, and these results indicate that Pinl may have a role in tumor development and metastasis and thus could serve as a novel target for treatment of NSCLC.更多还原