【作者】 王鹤；

【导师】 王朝霞；

【作者基本信息】 南京医科大学 ， 肿瘤学， 2012， 硕士

【摘要】 研究背景：非小细胞肺癌(NSCLC)占原发性肺癌80%以上,超过60%的患者为中晚期,常失去手术机会,化疗是Ⅲb-Ⅳ期NSCLC的主要治疗手段。顺铂(DDP)仍然是NSCLC一线化疗方案中的重要组成药物,但其获得性耐药制约了其疗效的发挥。microRNA (miRNA)是一类非编码小RNA分子,通常在转录后水平调控基因表达。成熟miRNA通过与mRNA完全或不完全配对,降解靶mRNA或阻遏其转录后翻译。miRNA作为肿瘤抑制基因或癌基因参与多种肿瘤发生发展。随着miRNA与肿瘤之间的关系逐渐被揭示,有证据表明,肿瘤细胞对抗肿瘤药物的敏感性受miRNA的调控。miR-224在多种人类恶性肿瘤中高表达,作为癌基因参与肿瘤的增殖、凋亡及转移等生物学过程；亦有研究提示miR-224与结肠癌耐甲氨蝶呤有关。然而目前尚未有miR-224影响NSCLC化疗敏感性的相关报道。研究目的：探讨miR-224在人NSCLC细胞中的表达及对DDP敏感性的影响,并探讨其作用机制。研究方法：(1)microRNA芯片检测人NSCLC耐顺铂细胞株A549/DDP与敏感亲代细胞株A549中microRNA表达谱,并利用荧光实时定量PCR (qRT-PCR)进行验证。(2)人工合成miR-224-inhibitor,通过LipofectamineTM2000瞬时转染入A549/DDP细胞并检测其转染效率。MTT实验检测DDP对耐顺铂细胞株A549/DDP、敏感亲代细胞株A549及转染后A549/DDP细胞的半数抑制浓度(half inhibition concentration, IC50)。流式细胞术分析转染后A549/DDP细胞联合DDP处理的细胞凋亡与细胞周期分布。(3)生物信息学分析miR-224可能作用的靶基因,发现miR-224序列上存在p21WAFl/CIP13’UTR结合位点。qRT-PCR与Western blot检测p21WAF1/CIP1在耐顺铂细胞株A549/DDP、敏感亲代细胞株A549、A549/DDP/miR-224-inhibitor、 A549/DDP/miR-NC中的mRNA与蛋白表达水平。构建含p21WAF1/CIP13’URT荧光素酶报告载体并通过双重荧光素酶报告实验分析其活性。(4)构建pcDNA3.1/p21WAF1/CIP1过表达载体转染入耐顺铂细胞株A549/DDP并通过qRT-PCR与Western blot检测其表达。MTT实验检测DDP对转染后A549/DDP细胞的IC50。流式细胞术分析转染后A549/DDP细胞联合DDP处理的细胞凋亡与细胞周期分布。(5)活体实验检测抑制miR-224表达的A549/DDP细胞对DDP敏感性；qRT-PCR与免疫组化检测miR-224与p21WAF1/CIP1在活体水平的表达相关性。(6)Western blot检测P53通路及下游靶分子Bcl-2, Bax在A549/DDP/miR-224-inhibitor+/-DDP、A549/DDP/miR-NC+/-DDP的表达情况。研究结果：(1)相较于敏感亲代细胞株A549, miR-224在耐顺铂细胞株A549/DDP显著高表达；(2)抑制miR-224表达的A549/DDP细胞对DDP的敏感性增强：DDP对A549/DDP/miR-224-inhibitor、A549/DDP/miR-NC的IC50分别为16.57±1.62μg/ml、29.47±2.15相较于对照组,转染miR-224inhibitor的A549/DDP细胞联合DDP处理后细胞凋亡增加、细胞周期阻滞于G1期增多；(3)体外水平p21WAF1/CIP1与miR-224表达存在负相关：相较于敏感亲代细胞株A549,p21WAF1/CIP1在耐顺铂细胞株A549/DDP明显低表达；抑制miR-224在A549/DDP细胞中的表达后,p21WAF1/CIP1表达明显升高。双重荧光素酶报告实验证实p21WAF1/CIP1为miR-224直接靶基因；(4)过表达p21WAF1/CIP1可模拟抑制miR-224表达生物学效应；(5)活体实验证实抑制miR-224表达的A549/DDP细胞对DDP敏感性增强；活体水平miR-224与p21WAF1/CIP1表达呈负相关；(6)在给予DDP处理的A549/DDP细胞抑制miR-224表达可激活P53,诱导下游靶分子Bax表达上调、Bcl-2表达下调。结论：体内外实验证实：抑制miR-224表达可逆转非小细胞肺癌A549/DDP细胞对DDP的耐药性,这一作用可能通过靶作用p21WAF1/CIP1以及调控P53通路来实现的。更多还原

【Abstract】 Background:Non-small cell lung cancer (NSCLC) accounts for more than80%of primary lung cancer, more than60%of patients with advanced, often unresectable. Chemotherapy is the main treatment for stage Ⅲb-Ⅳ NSCLC. Cisplatin (DDP), an important component of chemotherapy in NSCLC, is still first-line drug. However, its acquired resistance restricts the play of its efficacy.MicroRNAs (miRNAs) are small, endogenous noncoding RNAs that have been identified as post-transcriptional regulators of gene expression. MiRNAs exert their functions through imperfect base-pairing with the3’-untranslated region (3’-UTR) of target mRNAs. In human cancer, miRNAs can act as oncogenes or tumour suppressor genes during tumourigenesis. As the relationship between miRNA and cancer being revealed, there is evidence that tumor cell sensitivity to anticancer drugs by the miRNA. MiR-224is up-regulated in a vatiety of human malignancies, as an oncogene involved in the biological porcess of proliferation, apoptosis and metastasis. Recent researches have shown the expression of miR-224is relative to methotrexate resistance to colon cancer. To date, however, the relationship between the expression of miR-224and chemoresistance in NSCLC remains unexplored.Materials and Methods:(1) A microarray technique was used to determine the miRNA profile in resistant A549/DDP and parental A549cells. Real-time quantitative PCR (qRT-PCR) was used to confirm the array results.(2) miR-224inhibitors was synthesized and introduced to cells using LipofectamineTM2000. The expression of miR-224in transfected cells was detected by qRT-PCR. The half maximal inhibitory concentration (IC50) values of DDP in resistant A549/DDP, parental A549and transfection A549/DDP cells were determined by MTT assay. Flow cytometric analysis was used to analyze the apoptosis and cell cycle distribution of transfection A549/DDP cells treated with DDP.(3) Bioinformatis analysis showed that there were p21WAF1/CIP13’UTR binding sites in miR-224sequence. qRT-PCR and western blot were used to detecte the expression of p21WAF1/CIP1in resistant A549/DDP, parental A549, A549/DDP/miR-224-inhibitor, A549/DDP/miR-NC cells at the level of mRNA and protein. P21WAF1/CIP13’UTR luciferase reporter was constructed and a dual luciferase assay was used to detect the activity.(4) PcDNA3.1/p21WAF1/CIP1vector was constructed and transfected into A549/DDP cells. The expression of p21WAF1/CIP1was detected by qRT-PCR and wesitern blot in transfection A549/DDP cells. The half IC50value of DDP in transfection A549/DDP cells was determined by MTT assay. Flow cytometric analysis was used to analyze the apoptosis and cell cycle distribution of transfection A549/DDP cells treated with DDP.(5) In vivo experiments were used to observe the effect of decreased miR-224on A549/DDP cell sensitivity to DDP. The in vivo levels of miR-224and p21WAF1/CIP1were detected by qRT-PCR and Immunohistochemistry.(6) Western blotting was used to detecte P53signaling pathway and the downstream targets Bcl-2, Bax in A549/DDP/miR-224-inhibitor, A549/DDP/miR-NC cells treated with or without DDP.Results:(1) The level of miR-224was significantly higher in resistant A549/DDP cells than that in sensitive A549cells.(2) Inhibition of miR-224in A549/DDP cells sensitized the cells to DDP. The IC50values of DDP in A549/DDP/miR-224-inhibitor, A549/DDP/miR-NC cells were16.57±1.62μg/ml and29.47± 2.15μg/ml, respectively. Compared with the control group, inhibition of miR-224in A549/DDP cells introduced increased apoptosis and proportion of G1cell cycle.(3) The inverse correlation between miR-224and p21WAF1/CIP1expression was found in vitro.The level of p21WAF1/CIP1was significantly lower in resistant A549/DDP cells than that in sensitive A549cells. The expression of p21WAF1/CIP1was significantly up-regulated in A549/DDP cells transfected with miR-224-inhibitor. MiR-224directly targets p21WAF1/CIP1in A549/DDP cells.(4) Ectopic expression of p21WAF1/CIP1could mimic the effect of decreased miR-224.(5) In vivo experiments confimed that inhibiton of miR-224resensitized A549/DDP cells to DDP and the expression of miR-224reversed to that of P21WAF1/CIP1.(6) Inhibition of miR-224regulates P53signaling pathway by activiation P53, introduction downstream targets Bcl-2, Bax down-regulated and up-regulated respectively in A549/DDP cells treated with DDP.Conclusions:In the present study we provide the first evidence that miR-224reverses resistance to DDP in A549/DDP cells, at least in part, by targeting p21WAF1/CIP1and mediating P53signaling pathway in vitro and vivo.更多还原