NNK诱导肺癌发生过程中循环microRNA标志物研究
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摘要
研究背景：
众所周知,烟草类制品特别是香烟烟雾中含有大量的致肺癌化学物质,包括4-(甲基亚硝胺基)-1-(3-吡啶)-1-丁酮[4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone,NNK],多环芳烃类(如苯并芘),重金属及各类复杂有机物等。在这些致癌物中,NNK是一种烟草特异性的N-亚硝胺类物质,虽然烟草中含有大量的致肺癌物质,但NNK在吸烟导致肺癌发生过程中起着关键的作用。大量研究表明,实验动物系统给予低剂量NNK,主要诱导肺腺瘤和腺癌发生。
肺癌是目前世界上死亡率最高的肿瘤之一。大量流行病学研究表明大多数肺癌的发生与吸烟有着密切的关系。尽管我们在提高戒烟和改善肺癌病人的治疗方面做了大量的工作,但目前肺癌的5年生存率仍然较低(约15%),近30年来并没有明显改善。究其原因,缺乏有效的早期诊断肺癌的生物标志物是其中一个重要的原因。相关报道指出,有效的早期肺癌诊断标志物可能显著提高肺癌的5年生存率至50%左右。因此,探寻肺癌早期生物标志物对当前改善肺癌的治疗具有重要的意义。
MicroRNA (miRNA)是一类约19-25个核苷酸的内源性的非编码RNA。通常在转录后负性调控mRNA的表达,参与多种生物学过程,包括肿瘤发生发展、细胞生长与增殖、凋亡及发育等。众多研究数据表明,在肺癌、胰腺癌、前列腺癌、乳腺癌、肝癌及结直肠癌等多种癌症组织样本中均可检测到miRNA的表达异常,而且miRNA差异表达谱能有效分类不同类型的肿瘤。近年几个研究表明,循环miRNA对于多种癌症的检测是一种潜在的稳定的无创性的标志物。这些研究都一致性的表明循环miRNA是癌症的一种潜在的稳定的生物标志物。
近来的几个研究表明,化学致癌物可诱导miRNA表达改变。雄性F344大鼠饮水中持续喂饲NNK20周可导致大鼠肺组织miRNA表达改变。据我们所知,迄今为止化学致癌物诱导循环miRNA表达改变的相关研究还少见报道。化学致癌物诱导癌症发生一般分为四个阶段：起始阶段、促进阶段、进展阶段和恶性转化阶段。而在NNK诱导大鼠肺癌发生发展各阶段,相关循环miRNA是否发生表达改变还不清楚。.因此,我们假设某些循环miRNA在NNK诱导大鼠肺癌发生发展过程中也发生了改变而且和NNK诱导肺癌发生密切相关。以往的研究表明,雄性F344大鼠对于NNK诱导肺癌发生是一种较敏感的实验动物,而且剂量反应关系显示低剂量的NNK持续处理主要诱导肺肿瘤发生,很少出现其他器官肿瘤发生。在本研究中,雄性F344大鼠持续给予NNK以诱导大鼠肺癌发生。利用这一动物模型,分别收集NNK处理第1、5、10、20、40、60、80和95周大鼠血液并分离血清运用小分子RNA Solexa测序方法检测血清miRNA差异表达谱,接着运用定量RT-PCR方法,检测大鼠个体血清中候选差异表达miRNA的表达情况,筛选出有意义的差异表达miRNA (miR-206和miR-133b)。进一步分析miR-206和]miR-133b在NNK诱导大鼠肺癌发生发展各阶段大鼠血清中的表达情况。为进一步鉴定miR-206和miR-133b作为肺癌标志物的潜力,我们分别检测了miR-206和miR-133b在大鼠肺癌组织、肺癌细胞系及肺癌人群血清样本的表达情况。以求探寻NNK诱导肺癌发生发展过程中潜在的有效的血清miRNA标志物,为肺癌的早期检测生物标志物研究积累重要的实验数据。并通过计算机生物信息学分析预测miR-206和miR-133b的靶基因及其功能,为进一步研究循环miRNA在化学致癌物诱导癌症发生过程的功能作用提供实验依据。
研究目的：
(1)雄性F344大鼠为实验动物,皮下注射低剂量NNK(每周3次,持续20周),然后常规喂养大鼠至95周以诱导大鼠肺癌发生,建立NNK诱导大鼠肺癌发生模型；
(2)运用小分子RNA Solexa测序方法检测NNK处理组和对照组血清miRNA差异表达谱,筛选出候选差异表达miRNA;
(3)运用定量RT-PCR方法,检测大鼠个体血清中候选差异表达miRNA的表达情况,筛选出有意义的差异表达miRNA (miR-206和miR-133b);
(4)鉴定NNK诱导大鼠肺癌发生不同阶段血清中miR-206和miR-133b表达水平;
(5)从大鼠血清,大鼠肺癌组织,肺癌细胞系及肺癌患者血清等多个水平鉴定miR-206和]miR-133b作为肺癌生物标志物的潜力,为探寻致癌物诱导肺癌发生的miRNA标志物研究积累重要的实验数据。
研究方法：
(1)NNK诱导大鼠肺癌模型的建立对照组大鼠皮下注射0.3ml生理盐水,NNK处理组大鼠皮下注射NNK溶液(1.15mg/kg,0.0055mmol/kg), NNK溶液每次注射前根据大鼠体重临用前新鲜配制,溶解在0.3ml生理盐水中,每周注射3次,连续20周,之后动物常规饲养于SPF实验动物房,至第95周剖杀。
(2)大鼠血清小RNA Solexa测序在NNK处理第60周,分别从对照组大鼠和NNK处理组大鼠取等量血清混合,分别制备对照组混合血清和NNK处理组混合血清,送深圳华大基因公司进行小分子RNA Solexa测序,分析对照组和NNK处理组血清miRNA差异表达谱。
(3)大鼠个体血清候选miRNA表达分析根据miRNA差异表达谱筛选出候选miRNA,运用实时定量RT-PCR方法,在第60周大鼠个体血清中鉴定这些候选miRNA的表达水平。确定显著差异表达的niRNA (miR-206和]miR-133b)。
(4)NNK诱导肺癌发生不同阶段大鼠血清中miR-206和miR-133b表达分析运用实时定量RT-PCR方法,以miR-16为内参,分析第1、5、10、20、40、60、80和95周大鼠血清中miR-206和]miR-133b表达变化。
(5)大鼠肺组织中miR-206和miR-133b表达分析运用实时定量RT-PCR方法,以RNUB6为内参,检测大鼠肺组织及肺癌组织中miR-206和miR-133b表达水平。
(6)肺癌细胞系中miR-206和miR-133b表达分析运用实时定量RT-PCR方法,以RNUB6为内参,检测肺癌细胞系A549、QG56、H446、H1299、95-D及16HBE-T细胞中miR-206和miR-133b表达水平。
(7)鉴定健康人和肺癌患者血清中miR-206和miR-133b表达水平。运用实时定量RT-PCR方法,以miR-16为内参,检测健康人和肺癌患者血清中miR-206和miR-133b表达水平。
(8)生物信息学分析：为了预测miR-206和miR-133b在肺癌发生发展过程可能产生的功能作用,我们选择miRNA靶基因预测软件(http://www.microma.org/microma/)对miR-206和]miR-133b进行靶基因预测,并筛选出miR-206和miR-133b共同的靶基因,分析是否有些共同靶基因参与了肺癌或肿瘤的发生。另外通过网络计算机生物信息学资源http://acgt.cs.tau.ac.il/fame/index.html预测miR-206和miR-133b的功能。同时通过http://www.genome.jp/kegg/pathway/对miR-206和miR-133b参与调控的信号通路进行分析,以发现miR-206和miR-133b参与到的和癌症通路或非小细胞肺癌通路相关一些靶基因。
(9)统计分析采用SPSS13.0统计分析软件进行相关统计学分析。用Shapiro-Wilk正态性检验进行各组数据的正态性检验。对于参数比较,两独立样本比较采用两独立样本t检验；配对样本采用配对样本t检验；多组样本比较采用单因素方差分析。对于非参数比较,两样本比较采用Mann-Whitney检验；多组样本比较采用Kruskall-Wallis检验。肿瘤发生率的比较采用Fisher's确切概率检验；两变量的相关分析采用Spearman秩相关检验。为评价血清miRNA作为标志物的预测值,采用受试者工作特征曲线(Receiver operating characteristic, ROC)分析评价其区分正常人与肺癌病人的能力。计量资料均表示为均数±标准差,所有统计分析均为双侧检验,设定P<0.05为具有统计学意义。
研究结果：
(1)NNK诱导肺癌动物模型的建立在NNK处理后第95周,对照组7只大鼠仅发现1个肺部肿瘤,NNK处理10只大鼠发现19个肺部肿瘤,对照组大鼠组织病理切片结果显示为正常肺支气管上皮细胞形态；NNK处理组肺肿瘤显示为中低分化的肺腺癌。比较对照组与NNK处理组肿瘤发生率分别为1/7和9/10,两组肿瘤发生率之间有显著的统计学差异(P=0.004)；两组间肿瘤大小比较也具有统计学显著性意义(t=6.286,P<0.001)。
(2)血清miRNA差异表达谱387个已知的大鼠miRNA中181个miRNA在对照组和NNK处理组血清中能检测到。在对照组与NNK处理组血清之间82个miRNA表达具有明显统计学差异。与对照组血清相比,NNK处理组血清中,37个miRNA表达显著性上调；45个miRNA表达显著性的下调；99个miRNA表达在两组之间无统计学差异。在能检测到的181个miRNA中进行两组血清之间公共及特有表达的miRNA分析显示,20个miRNA特有性表达的在对照组血清中；8个miRNA特有性表达在NNK处理组血清中；其余153个miRNA在两组血清均有表达。
(3)大鼠个体血清中候选miRNA表达分析对照组血清相比,NNK处理组血清中miR206(U=21.00, P=0.012), miR-133b (U=26.00, P=0.030)和miR-382(U=24.00, P=0.021)表达水平均显著性上调(Mann-Whitney检验)。MiR-365, miR-34c, miR-20a, miR-29b, miR-30e, miR-183, miR-331和miR-195在两组大鼠个体血清中表达水平未见显著性差异(P>0.05)。同时,我们对3个有显著性差异的miRNA (miR-206, miR-133b和miR-382)进一步分析发现,在大鼠个体血清中miR-206和miR-133b的表达水平具有明显的相关性,Spearman秩相关检验显示二者的表达水平呈明显的正相关,Spearman相关系数r=0.758,95%可信区间为0.339-0.926,P=0.003。
(4)NNK诱导大鼠肺癌发生不同阶段血清miR-206和miR-133b表达分析相对于对照组血清,NNK处理组血清中miR-206和miR-133b表达水平在第1、5、10、20、40、60和80周均上调,miR-206上调倍数分别为1.43±0.11(t=-6.744, P=0.003);1.59±0.32(t=-3.215, P=0.032);2.17±0.35(t=-5.735, P=0.029);4.82±0.88(t=-7.497, P=0.017);3.23±0.99(t=-3.876, P=0.018);2.41±0.15(t=-16.250, P=0.004):1.85±0.12(t=-12.035, P=0.007):miR-133b上调倍数分别为1.08±0.10(t=-1.485, P=0.276);1.45±0.08(t=-9.208, P=0.011);2.12±0.25(t=-7.774, P=0.016);2.68±0.03(t=-97.887, P<0.001);2.33±0.17(t=-13.238, P<0.001);1.82±0.16(t=-8.953, P=0.001);1.30±0.16(t=-3.156, P=0.034)。可见在20周前大鼠血清中miR-206和miR-133b(?)目对表达水平呈上升趋势,在20周达高峰,之后其表达水平又逐渐下降。到第95周,相对于对照组血清,NNK处理组血清中miR-206和miR-133b表达水平下调,其下调倍数分别为2.09±0.64(t=5.769, P=0.004),2.78±0.30(t=26.813, P=0.001)。(5)大鼠肺组织及肺肿瘤中miR-206和miR-133b表达分析在9对NNK处理组肺肿瘤及其对应的正常肺组织中,相对于正常肺组织,在配对的肺肿瘤组织中miR-206和miR-133b均出现低水平表达,且差异均具有统计学意义(配对t检验)。同时我们检测了7只对照组大鼠肺组织中miR-206和：miR-133b的表达水平,采用单因素方差分析检验各组间的统计学差异,三组间miR-206(F=16.376, P<0.001)和miR-133b (F=11.001, P<0.001)表达水平具有显著性差异。如图3-16C和D所示,与对照组正常肺组织相比,NNK处理组中正常肺组织miR-206(P=0.001)和miR-133b(P=0.019)显著性低表达；在NNK处理组肺肿瘤组织中miR-206(p<0.001)和miR-133b (p<0.001)显著低表达。相对于NNK处理组正常肺组织,NNK处理组肺肿瘤组织中miR-206(P=0.048)和miR-133b (P=0.031)也显著低表达。
(6)肺癌细胞系中miR-206和miR-133b表达分析相对16HBE细胞,肺癌细胞系A549, QG56, H446, H1299,95-D和16HBE-T细胞中miR-206表达水平分别下调28.341±3.435,97.638±16.922,33.999±11.882,33.055±16.138,23.424±3.899和31.688±12.306倍；相对于16HBE细胞,肺癌细胞系A549, QG56, H446, H1299,95-D和16HBE-T细胞中miR-133b(图3-12B)表达水平分别下调1.793±0.410,3.926±2.014,2.894±2.135,3.768±0.734,2.176±0.710和2.652±1.186倍。
(7)肺癌和健康人血清中miR-206和miR-133b表达分析与健康人血清相比,肺癌患者血清中miR-206(图3-13A)和miR-133b(图3-13B)显著性低表达,差异具有统计学意义(miR206-U=61.00, miR133b-U=80.00, P<0.001, Mann-Whitney检验)。miR-206和miR-133b的ROC曲线分析结果显示miR-206和miR-133b ROC曲线下面积(Areas under curve, AUC)分别为0.9024(95%CI=0.8125-0.9923)和0.8720(95%CI=0.7734-0.9706)。(8)通过靶基因预测,人类miR-206和miR133b分别预测有7201和5314个靶基因,其中2577个基因是他们共同的靶基因。通过功能预测,我们发现miR-206参与了6个KEGG信号通路,包括VEGF信号通路、P53信号通路、烟酸和烟酰胺的代谢、囊泡转运的SNARE相互作用、谷胱甘肽代谢和嗅觉传导；；miR-133b参与了2个KEGG信号通路,包括Notch信号通路和脂质代谢。其中,P53信号通路和Notch信号通路与癌症发生密切相关。
结论：
(1)NNK处理第60周大鼠血清进行小分子RNA Solexa测序分析,结果.表明NNK处理可诱导血清miRNA表达发生改变。
(2)通过大鼠个体血清中差异表达miRNA的实时定量分析,发现NNK处理组大鼠血清miR-206和miR-133b较对照组血清显著性高表达,而且血清miR-206和miR-133b的表达水平呈明显正相关；在大鼠肺癌组织,肺癌细胞系以及肺癌患者血清中,miR-206和miR-133b也呈现协同变化的关系,可能作为肿瘤抑制,niRNA参与NNK诱导的肺癌发生发展。
(3)通过对NNK诱导大鼠肺癌发生发展各阶段血清中miR-206和miR-133b的实时定量检测,发现NNK处理组血清在早期呈上升趋势,在20周表达水平达高峰,之后逐渐降低,最后在第95周NNK处理组血清中呈低表达。
(4)通过在大鼠肺癌组织,肺癌细胞系及有吸烟史的肺癌患者血清中进一步得以鉴定,发现血清miR-206和miR-133b是NNK诱导肺癌发生发展的潜在生物标志物。
Background:
As is known to all, cigarette smoke contains substantial amounts of lung carcinogens, including4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), polycyclic aromatic hydrocarbons (PAHs), metals and miscellaneous organic compounds etc. Of these lung carcinogens, NNK is a tobacco-specific N-nitrosamine and the most important lung carcinogen. Although many carcinogens in cigarette smoke contribute to lung cancer, the key compound NNK plays a major role in lung carcinogenesis. Many studies showed that NNK is a potent lung carcinogen in rats, mice, and hamsters. NNK induced lung tumorigenesis have remarkable organospecificity. The systemic administration of NNK to rats is a reproducible and robust method for the induction of lung tumors.
Lung cancer is one of the most common cancers and a leading cause of cancer death in the world. Many studies indicate that cigarette smoking causes most lung cancers. Despite great efforts to improve smoking cessation and the treatment of patients with lung cancer, the5years survival rate for lung cancer patients has not significantly improved over the past30years. Scarcity of diagnostic biomarkers for early stage lung cancer is the most important causes. Discovery and use of efficient biomarker for early stage lung cancer will be able to improve the5years survival rate of lung cancer.
MicroRNAs (miRNAs) are a class of small endogenous noncoding RNA (about19-25nt) that usually post-transcriptional negative regulate the expression of mRNA. Previous studies showed that miRNAs involve in a variety of biological processes including cancer development and progression, cell growth and proliferation, apoptosis etc. Increasing evidences have demonstrated that expression of miRNAs is dysregulated in a variety of cancers, such as lung cancer, pancreatic cancer, prostate cancer, breast cancer and hepatocellular carcinoma etc. MiRNAs differential expression profile can classify variety of cancers. Several recent studies indicated that miRNAs are stable and detectable in serum or plasma. These data consistently indicate that circulating miRNAs are potential biomarker for cancer.
Previous several studies declared that chemical carcinogens can cause the alteration of miRNAs expression. Male F344rats continuously fed with NNK up to20weeks resulted in alteration of miRNA expression in lung tissues of rat. To our knowledge, relative study on alteration of circulating miRNAs expression in carcinogenesis induced by chemical carcinogens is not still reported. Chemical carcinogenesis usually includes four stages:initiation; progression; development and transformation. During NNK-induced lung carcinogenesis, the alteration of serum miRNAs expression is not still unclear. We hypothesized that the levels of serum miRNAs were change and associated with lung carcinogenesis induced by NNK. Previous studies showed that male F344rat is highly sensitive to NNK-induced lung carcinogenesis. The dose-response relationship indicated that administration of low dose NNK mainly induce lung carcinogenesis. Therefore, in the present study, NNK was systemically administered to rats to induce lung carcinogenesis. Using this animal model, Blood of rats were collected at1,5,10,20,40,60,80and95weeks after NNK treatment and sera of rats were isolated. Serum miRNAs differential expression profile was analyzed by small RNA solexa sequence. Using Quantitative real-time PCR (qRT-PCR), the differential expression serum miRNAs were identified in each rat individuals. Significant differential expression miRNAs (miR-206and miR-133b) were selected for further study. The expression levels of miR-206and miR-133b were identified in rat serum at different stages of NNK induced lung carcinogenesis. The potential of miR-206and miR-133b as biomarker for lung cancer was identified in rat sera, rat lung tumors, lung cancer lines and human sera, respectively. Our study will provide important data for explore the biomarker of chemical carcinogens induced lung carcinogenesis. We predicted the barget genes and functions of miR-206and miR133b by computational bioinformatic analysis. These studies will provide experimental evidences for further study on the functions of circulating miRNAs.
Objective:
(1) Male F344rats were administered with subcutaneous injections of low-dose NNK (3times weekly for20weeks), and animals were maintained at SPF animal house until the95th week to induce lung carcinogenesis.
(2) MiRNAs differential expression profile of rat serum in control and NNK treatment group was analyzed by small RNA solexa sequencing and candidate miRNAs were screened.
(3) Using Quantitative real-time PCR (qRT-PCR), the differential expression serum miRNAs were identified in each rat individuals. Significant differential expression miRNAs (miR-206and miR-133b) were selected for further study.
(4) The expression levels of miR-206and miR-133b were identified in rat serum at different stages of NNK induced lung carcinogenesis.
(5) The potential of miR-206and miR-133b as biomarker for lung cancer was identified in rat sera, rat lung tumors, lung cancer lines and human sera, respectively. Our study will provide important data for explore the biomarker of chemical carcinogens induced lung carcinogenesis.
Methods:
(1) The construction of rat model for lung cancer induced by NNK:Rats from control group and NNK treatment group were respectively administered with subcutaneous injections of0.3ml saline and NNK (1.15mg/kg,0.0055mmol/kg) in0.3ml saline (newly prepared before each injection). Each rat was given injections3times weekly for20weeks. Afterward, animals were maintained at SPF animal house until the95th week to be sacrificed.
(2) Small RNA solexa sequencing of rat serum:Equal volume serum from each rat of control or NNK treatment group at the60th week was polled together, respectively. Pooled serum samples of control and NNK treatment group were send to Beijing Genomics Institute at Shenzhen for small RNA solexa sequencing and miRNAs differential expression profile was analyzed.
(3) Candidate serum miRNAs expression analysis for rat individuals by qRT-PCR:According to miRNAs differential expression profile to select candidate miRNAs, Using Quantitative real-time PCR, The differential expression serum miRNAs were identified in each rat individuals. Significant differential expression miRNAs (miR-206and miR-133b) were selected for further study.
(4) qRT-PCR analysis of the expression of miR-206and miR-133b at different stages of NNK treatment:Using Quantitative real-time PCR, miR-16was selected as endogenous control gene and the level of miR-206and miR-133b were detected in rat sera of1,5,10,20,40,60,80and95week.
(5) The expression of miR-206and miR-133b in lung tissues of rats:Using Quantitative real-time PCR, RNUB6was selected as endogenous control gene and the level of miR-206and miR-133b were detected in lung tissues of rats.
(6) The expression of miR-206and miR-133b in lung cancer cell lines:Using Quantitative real-time PCR, RNUB6was selected as endogenous control gene and the level of miR-206and miR-133b were detected in lung cancer cell lines.
(7) The level of miR-206and miR-133b in human sera of health control and lung cancer patients:Using Quantitative real-time PCR, miR-16was selected as endogenous control gene and the level of miR-206and miR-133b were detected in human sera of health control and lung cancer patients.
(8) Bioinformatic analysis:To predict the functions of miR-206and miR-133b, we predicted the target genes of miR-206and miR-133b from http://www.microrna.org/microrna/. The common target genes of miR-206and miR-133b were analyzed to find those target genes that are associated with carcinogenesis. The functions of miR-206and miR-133b are also predicted by http://acgt.cs.tau.ac.il/fame/index.html. Pathways of cancer or lung cancer were analyzed from http://www.genome.jp/kegg/pathway/to find those target genes involving in cancer pathway.
(9) Statistical analysis:Statistical analysis was performed using SPSS software (version13.0, IL, USA).Normality test was performed for data of each group using the Shapiro-Wilk normality test. For parametric comparisons, two independent samples t-test (for two independent samples), one-way analysis of variance (ANOVA) for multiple samples and paired t-test (for two paired samples) were employed respectively. For nonparametric comparisons, the Mann-Whitney test (for two nonparametric groups) and the Kruskall-Wallis test (for more than two nonparametric groups) were used respectively. Fisher's exact test was performed to compare the incidences of lung tumors between control and NNK treatment. Spearman correlation analysis was used to evaluate the correlation of two variables. In order to evaluate the predictive value of serum miRNAs, the receiver operating characteristic (ROC) curve were established for discriminating health control and patients with lung cancer. The area under the ROC curve (AUC) was calculated in order to better identify the potential of serum microRNAs as a diagnostic marker. All p-values are two-sided and less than0.05was considered statistically significant.
Results:
(1) The construction of rat model for lung cancer induced by NNK:At the95th week,19lung tumors were found in10rats of NNK treatment group, only1lung tumor was found in7rats of control group. No pathological changes were observed in lung tissues of rats of control. Most of lung tumors were moderately differentiated adenocarcinoma. Compared to control group, the incidence of lung tumor in NNK treatment group was statistical significance (Fisher's exact test, P=0.004).
(2) Serum miRNAs differential expression profile:We detected387known miRNAs of Rattus norvegicus (reference to miRBase Release13). Of these miRNAs,181miRNAs could be detected in control serum or NNK treatment serum and82differential expression miRNAs have significantly difference between control serum and NNK treatment serum. Compared with control serum,37miRNAs expression were up-regulated;45miRNAs expression were down-regulated;99miRNAs expression were equal. Of these181miRNAs,20miRNAs specifically expressed in the serum of control group;8miRNAs specifically expressed in the serum of NNK treatment group;153miRNAs commonly expressed in the serum of control and NNK treatment.
(3) Candidate serum miRNAs expression analysis for rat individuals by qRT-PCR:Compared with control serum, the expression level of miR-206(U=21.00, P=0.012), miR-133b (U=26.00, P=0.030) and miR-382(U=24.00, P=0.021) were up-regulated significantly in NNK treatment group (Mann-Whitney tests). MiR-365, miR-34c, miR-20a, miR-29b, miR-30e, miR-183, miR-331and miR-195have no significances between rat sera of control and NNK treatment group.The expression of miR-206and miR-133b was closely associated in serum of rat individuals. Spearman rank correlation analysis show that the expression of miR-206and miR-133b are positive correlation, Spearman r=0.758,95%CI=0.339-0.926, P=0.003.
(4) qRT-PCR analysis of the expression of miR-206and miR-133b at different stages of NNK treatment:Compared with control serum, the levels of miR-206and miR-133b expression were significantly up-regulated in rat serum of NNK treatment group at1,5,10,20,40,60and80weeks, respectively. Fold change up-regulated of miR-206were respectively1.43±0.11(t=-6.744, P=0.003);1.59±0.32(t=-3.215, P=0.032);2.17±0.35(t=-5.735, P=0.029);4.82±0.88(t=-7.497, P=0.017);3.23±0.99(t=-3.876, P=0.018);2.41±0.15(t=-16.250, P=0.004);1.85±0.12(t=-12.035, P=0.007). Fold change up-regulated of miR-133b were respectively1.08±0.10(t=-1.485, P=0.276);1.45±0.08(t=-9.208, P=0.011);2.12±0.25(t=-7.774, P=0.016);2.68±0.03(t=-97.887, P<0.001);2.33±0.17(t=-13.238, P<0.001);1.82±0.16(t=-8.953, P=0.001);1.30±0.16(t=-3.156, P=0.034). At95weeks after NNK treatment, miR-206and miR-133b were significantly down-regulated compared to rat serum of control. Fold change were2.09±0.64(t=5.769, P=0.004);2.78±0.30(t=26.813, P=0.001), respectively.
(5) The expression of miR-206and miR-133b in lung tumors tissues of rats: In9rat lung tumor tissues and corresponding normal lung tissues. Compared with normal lung tissues, miR-206and miR-133b was significantly low-expression in9paired lung tumor tissues (paired t-test). Meanwhile, we examined the levels of miR-206and miR-133b expression in7normal rat lung tissues of control group. Statistically significant differences were analyzed using ANOVA. Among three groups, the levels of miR-206(F=16.376, P<0.001) and miR-133b (F=11.001, P<0.001) have significantly difference. Compared with normal lung tissues of control, miR-206(P=0.001) and miR-133b(P=0.019) were low-expression significantly in normal lung tissues of NNK treatment group and lung tumor tissues of NNK treatment group (p<0.001). Compared to normal lung tissues of NNK treatment group, miR-206(P=0.048) and miR-133b (P=0.031) were low-expression significantly in paired lung tumor tissues of NNK treatment group.
(6) The expression of miR-206and miR-133b in lung cancer cell lines:The level of miR-206was significantly down-regulated28.341±3.435,97.638±16.922,33.999±11.882,33.055±16.138,23.424±3.899and31.688±12.306fold in A549, QG56, H446, H1299,95-D and16HBE-T cell relative to16HBE cell, respectively. The level of miR-133b was down-regulated1.793±0.410,3.926±2.014,2.894±2.135,3.768±0.734,2.176±0.710and2.652±1.186fold in A549, QG56, H446, H1299,95-D and16HBE-T cell relative to16HBE cell, respectively.
(7) The level of miR-206and miR-133b in human sera of health control and lung cancer patients:Compared with health controls, the expression of serum miR-206and miR-133b were significantly down-regulated in lung cancer patients (miR206-U=61.00, miR133b-U=80.00, P<0.001, Mann-Whitney test). Receiver operating characteristic (ROC) curve analyses show that the areas under curve (AUC) of serum miR-206and miR-133b were0.9024(95%CI=0.8125-0.9923) and0.8720(95%CI=0.7734-0.9706), respectively.
(8) Bioinformatic analysis:has-miR-206and has-miR-133b were predicted7201and5314target genes, respectively. And of these target genes,2577genes are common target genes of miR-206and miR-133b. By functions prediction, we found that miR-206involve in6KEGG pathways, including VEGF signaling pathway, p53signaling pathway, Nicotinate and nicotinamide metabolism, SNARE interactions in vesicular transport, Glutathione metabolism, Olfactory transduction. MiR-133b involve in2KEGG pathways, including Notch signaling pathway, Sphingolipid metabolism. Of these pathways, P53signaling pathway and notch signaling pathway are closely related to carcinogenesis.
Conclusions:
(1) Small RNA solexa sequencing of rat serum at the60th week indicate that NNK can induce the alteration of the expression of serum miRNAs.
(2) qRT-PCR analysis of differential expression miRNAs in sera of rats individuals showed that miR-206and miR-133b were significantly up-regulated in rat sera of NNK treatment group. Moreover, the expression of miR-206and miR-133b was positive correlation. The expression of miR-206and miR-133b in lung tumors and lung cancer cell lines has significantly covariant. They may involve in NNK induced lung carcinogenesis as tumor suppressor.
(3) The level of serum miR-206and miR-133b at different stages of NNK induced lung carcinogenesis indicated that alteration of serum miRNAs expression followed a two-step evolution. At early stage, the level of miR-206and miR-133b was progressively increased and the peak of expression occurs at the20th week. Afterward, the level of miRNAs was progressively decreased and low expression was observed at the95th week.
(4) By identifying the expression of miR-206and miR-133b in rat sera, rat lung tumors, lung cancer cell lines and sera of lung cancer patients with smoking history, serum miR-206and miR-133b may be potential biomarker for lung carcinogenesis induced by NNK.
众所周知,烟草类制品特别是香烟烟雾中含有大量的致肺癌化学物质,包括4-(甲基亚硝胺基)-1-(3-吡啶)-1-丁酮[4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone,NNK],多环芳烃类(如苯并芘),重金属及各类复杂有机物等。在这些致癌物中,NNK是一种烟草特异性的N-亚硝胺类物质,虽然烟草中含有大量的致肺癌物质,但NNK在吸烟导致肺癌发生过程中起着关键的作用。大量研究表明,实验动物系统给予低剂量NNK,主要诱导肺腺瘤和腺癌发生。
肺癌是目前世界上死亡率最高的肿瘤之一。大量流行病学研究表明大多数肺癌的发生与吸烟有着密切的关系。尽管我们在提高戒烟和改善肺癌病人的治疗方面做了大量的工作,但目前肺癌的5年生存率仍然较低(约15%),近30年来并没有明显改善。究其原因,缺乏有效的早期诊断肺癌的生物标志物是其中一个重要的原因。相关报道指出,有效的早期肺癌诊断标志物可能显著提高肺癌的5年生存率至50%左右。因此,探寻肺癌早期生物标志物对当前改善肺癌的治疗具有重要的意义。
MicroRNA (miRNA)是一类约19-25个核苷酸的内源性的非编码RNA。通常在转录后负性调控mRNA的表达,参与多种生物学过程,包括肿瘤发生发展、细胞生长与增殖、凋亡及发育等。众多研究数据表明,在肺癌、胰腺癌、前列腺癌、乳腺癌、肝癌及结直肠癌等多种癌症组织样本中均可检测到miRNA的表达异常,而且miRNA差异表达谱能有效分类不同类型的肿瘤。近年几个研究表明,循环miRNA对于多种癌症的检测是一种潜在的稳定的无创性的标志物。这些研究都一致性的表明循环miRNA是癌症的一种潜在的稳定的生物标志物。
近来的几个研究表明,化学致癌物可诱导miRNA表达改变。雄性F344大鼠饮水中持续喂饲NNK20周可导致大鼠肺组织miRNA表达改变。据我们所知,迄今为止化学致癌物诱导循环miRNA表达改变的相关研究还少见报道。化学致癌物诱导癌症发生一般分为四个阶段：起始阶段、促进阶段、进展阶段和恶性转化阶段。而在NNK诱导大鼠肺癌发生发展各阶段,相关循环miRNA是否发生表达改变还不清楚。.因此,我们假设某些循环miRNA在NNK诱导大鼠肺癌发生发展过程中也发生了改变而且和NNK诱导肺癌发生密切相关。以往的研究表明,雄性F344大鼠对于NNK诱导肺癌发生是一种较敏感的实验动物,而且剂量反应关系显示低剂量的NNK持续处理主要诱导肺肿瘤发生,很少出现其他器官肿瘤发生。在本研究中,雄性F344大鼠持续给予NNK以诱导大鼠肺癌发生。利用这一动物模型,分别收集NNK处理第1、5、10、20、40、60、80和95周大鼠血液并分离血清运用小分子RNA Solexa测序方法检测血清miRNA差异表达谱,接着运用定量RT-PCR方法,检测大鼠个体血清中候选差异表达miRNA的表达情况,筛选出有意义的差异表达miRNA (miR-206和miR-133b)。进一步分析miR-206和]miR-133b在NNK诱导大鼠肺癌发生发展各阶段大鼠血清中的表达情况。为进一步鉴定miR-206和miR-133b作为肺癌标志物的潜力,我们分别检测了miR-206和miR-133b在大鼠肺癌组织、肺癌细胞系及肺癌人群血清样本的表达情况。以求探寻NNK诱导肺癌发生发展过程中潜在的有效的血清miRNA标志物,为肺癌的早期检测生物标志物研究积累重要的实验数据。并通过计算机生物信息学分析预测miR-206和miR-133b的靶基因及其功能,为进一步研究循环miRNA在化学致癌物诱导癌症发生过程的功能作用提供实验依据。
研究目的：
(1)雄性F344大鼠为实验动物,皮下注射低剂量NNK(每周3次,持续20周),然后常规喂养大鼠至95周以诱导大鼠肺癌发生,建立NNK诱导大鼠肺癌发生模型；
(2)运用小分子RNA Solexa测序方法检测NNK处理组和对照组血清miRNA差异表达谱,筛选出候选差异表达miRNA;
(3)运用定量RT-PCR方法,检测大鼠个体血清中候选差异表达miRNA的表达情况,筛选出有意义的差异表达miRNA (miR-206和miR-133b);
(4)鉴定NNK诱导大鼠肺癌发生不同阶段血清中miR-206和miR-133b表达水平;
(5)从大鼠血清,大鼠肺癌组织,肺癌细胞系及肺癌患者血清等多个水平鉴定miR-206和]miR-133b作为肺癌生物标志物的潜力,为探寻致癌物诱导肺癌发生的miRNA标志物研究积累重要的实验数据。
研究方法：
(1)NNK诱导大鼠肺癌模型的建立对照组大鼠皮下注射0.3ml生理盐水,NNK处理组大鼠皮下注射NNK溶液(1.15mg/kg,0.0055mmol/kg), NNK溶液每次注射前根据大鼠体重临用前新鲜配制,溶解在0.3ml生理盐水中,每周注射3次,连续20周,之后动物常规饲养于SPF实验动物房,至第95周剖杀。
(2)大鼠血清小RNA Solexa测序在NNK处理第60周,分别从对照组大鼠和NNK处理组大鼠取等量血清混合,分别制备对照组混合血清和NNK处理组混合血清,送深圳华大基因公司进行小分子RNA Solexa测序,分析对照组和NNK处理组血清miRNA差异表达谱。
(3)大鼠个体血清候选miRNA表达分析根据miRNA差异表达谱筛选出候选miRNA,运用实时定量RT-PCR方法,在第60周大鼠个体血清中鉴定这些候选miRNA的表达水平。确定显著差异表达的niRNA (miR-206和]miR-133b)。
(4)NNK诱导肺癌发生不同阶段大鼠血清中miR-206和miR-133b表达分析运用实时定量RT-PCR方法,以miR-16为内参,分析第1、5、10、20、40、60、80和95周大鼠血清中miR-206和]miR-133b表达变化。
(5)大鼠肺组织中miR-206和miR-133b表达分析运用实时定量RT-PCR方法,以RNUB6为内参,检测大鼠肺组织及肺癌组织中miR-206和miR-133b表达水平。
(6)肺癌细胞系中miR-206和miR-133b表达分析运用实时定量RT-PCR方法,以RNUB6为内参,检测肺癌细胞系A549、QG56、H446、H1299、95-D及16HBE-T细胞中miR-206和miR-133b表达水平。
(7)鉴定健康人和肺癌患者血清中miR-206和miR-133b表达水平。运用实时定量RT-PCR方法,以miR-16为内参,检测健康人和肺癌患者血清中miR-206和miR-133b表达水平。
(8)生物信息学分析：为了预测miR-206和miR-133b在肺癌发生发展过程可能产生的功能作用,我们选择miRNA靶基因预测软件(http://www.microma.org/microma/)对miR-206和]miR-133b进行靶基因预测,并筛选出miR-206和miR-133b共同的靶基因,分析是否有些共同靶基因参与了肺癌或肿瘤的发生。另外通过网络计算机生物信息学资源http://acgt.cs.tau.ac.il/fame/index.html预测miR-206和miR-133b的功能。同时通过http://www.genome.jp/kegg/pathway/对miR-206和miR-133b参与调控的信号通路进行分析,以发现miR-206和miR-133b参与到的和癌症通路或非小细胞肺癌通路相关一些靶基因。
(9)统计分析采用SPSS13.0统计分析软件进行相关统计学分析。用Shapiro-Wilk正态性检验进行各组数据的正态性检验。对于参数比较,两独立样本比较采用两独立样本t检验；配对样本采用配对样本t检验；多组样本比较采用单因素方差分析。对于非参数比较,两样本比较采用Mann-Whitney检验；多组样本比较采用Kruskall-Wallis检验。肿瘤发生率的比较采用Fisher's确切概率检验；两变量的相关分析采用Spearman秩相关检验。为评价血清miRNA作为标志物的预测值,采用受试者工作特征曲线(Receiver operating characteristic, ROC)分析评价其区分正常人与肺癌病人的能力。计量资料均表示为均数±标准差,所有统计分析均为双侧检验,设定P<0.05为具有统计学意义。
研究结果：
(1)NNK诱导肺癌动物模型的建立在NNK处理后第95周,对照组7只大鼠仅发现1个肺部肿瘤,NNK处理10只大鼠发现19个肺部肿瘤,对照组大鼠组织病理切片结果显示为正常肺支气管上皮细胞形态；NNK处理组肺肿瘤显示为中低分化的肺腺癌。比较对照组与NNK处理组肿瘤发生率分别为1/7和9/10,两组肿瘤发生率之间有显著的统计学差异(P=0.004)；两组间肿瘤大小比较也具有统计学显著性意义(t=6.286,P<0.001)。
(2)血清miRNA差异表达谱387个已知的大鼠miRNA中181个miRNA在对照组和NNK处理组血清中能检测到。在对照组与NNK处理组血清之间82个miRNA表达具有明显统计学差异。与对照组血清相比,NNK处理组血清中,37个miRNA表达显著性上调；45个miRNA表达显著性的下调；99个miRNA表达在两组之间无统计学差异。在能检测到的181个miRNA中进行两组血清之间公共及特有表达的miRNA分析显示,20个miRNA特有性表达的在对照组血清中；8个miRNA特有性表达在NNK处理组血清中；其余153个miRNA在两组血清均有表达。
(3)大鼠个体血清中候选miRNA表达分析对照组血清相比,NNK处理组血清中miR206(U=21.00, P=0.012), miR-133b (U=26.00, P=0.030)和miR-382(U=24.00, P=0.021)表达水平均显著性上调(Mann-Whitney检验)。MiR-365, miR-34c, miR-20a, miR-29b, miR-30e, miR-183, miR-331和miR-195在两组大鼠个体血清中表达水平未见显著性差异(P>0.05)。同时,我们对3个有显著性差异的miRNA (miR-206, miR-133b和miR-382)进一步分析发现,在大鼠个体血清中miR-206和miR-133b的表达水平具有明显的相关性,Spearman秩相关检验显示二者的表达水平呈明显的正相关,Spearman相关系数r=0.758,95%可信区间为0.339-0.926,P=0.003。
(4)NNK诱导大鼠肺癌发生不同阶段血清miR-206和miR-133b表达分析相对于对照组血清,NNK处理组血清中miR-206和miR-133b表达水平在第1、5、10、20、40、60和80周均上调,miR-206上调倍数分别为1.43±0.11(t=-6.744, P=0.003);1.59±0.32(t=-3.215, P=0.032);2.17±0.35(t=-5.735, P=0.029);4.82±0.88(t=-7.497, P=0.017);3.23±0.99(t=-3.876, P=0.018);2.41±0.15(t=-16.250, P=0.004):1.85±0.12(t=-12.035, P=0.007):miR-133b上调倍数分别为1.08±0.10(t=-1.485, P=0.276);1.45±0.08(t=-9.208, P=0.011);2.12±0.25(t=-7.774, P=0.016);2.68±0.03(t=-97.887, P<0.001);2.33±0.17(t=-13.238, P<0.001);1.82±0.16(t=-8.953, P=0.001);1.30±0.16(t=-3.156, P=0.034)。可见在20周前大鼠血清中miR-206和miR-133b(?)目对表达水平呈上升趋势,在20周达高峰,之后其表达水平又逐渐下降。到第95周,相对于对照组血清,NNK处理组血清中miR-206和miR-133b表达水平下调,其下调倍数分别为2.09±0.64(t=5.769, P=0.004),2.78±0.30(t=26.813, P=0.001)。(5)大鼠肺组织及肺肿瘤中miR-206和miR-133b表达分析在9对NNK处理组肺肿瘤及其对应的正常肺组织中,相对于正常肺组织,在配对的肺肿瘤组织中miR-206和miR-133b均出现低水平表达,且差异均具有统计学意义(配对t检验)。同时我们检测了7只对照组大鼠肺组织中miR-206和：miR-133b的表达水平,采用单因素方差分析检验各组间的统计学差异,三组间miR-206(F=16.376, P<0.001)和miR-133b (F=11.001, P<0.001)表达水平具有显著性差异。如图3-16C和D所示,与对照组正常肺组织相比,NNK处理组中正常肺组织miR-206(P=0.001)和miR-133b(P=0.019)显著性低表达；在NNK处理组肺肿瘤组织中miR-206(p<0.001)和miR-133b (p<0.001)显著低表达。相对于NNK处理组正常肺组织,NNK处理组肺肿瘤组织中miR-206(P=0.048)和miR-133b (P=0.031)也显著低表达。
(6)肺癌细胞系中miR-206和miR-133b表达分析相对16HBE细胞,肺癌细胞系A549, QG56, H446, H1299,95-D和16HBE-T细胞中miR-206表达水平分别下调28.341±3.435,97.638±16.922,33.999±11.882,33.055±16.138,23.424±3.899和31.688±12.306倍；相对于16HBE细胞,肺癌细胞系A549, QG56, H446, H1299,95-D和16HBE-T细胞中miR-133b(图3-12B)表达水平分别下调1.793±0.410,3.926±2.014,2.894±2.135,3.768±0.734,2.176±0.710和2.652±1.186倍。
(7)肺癌和健康人血清中miR-206和miR-133b表达分析与健康人血清相比,肺癌患者血清中miR-206(图3-13A)和miR-133b(图3-13B)显著性低表达,差异具有统计学意义(miR206-U=61.00, miR133b-U=80.00, P<0.001, Mann-Whitney检验)。miR-206和miR-133b的ROC曲线分析结果显示miR-206和miR-133b ROC曲线下面积(Areas under curve, AUC)分别为0.9024(95%CI=0.8125-0.9923)和0.8720(95%CI=0.7734-0.9706)。(8)通过靶基因预测,人类miR-206和miR133b分别预测有7201和5314个靶基因,其中2577个基因是他们共同的靶基因。通过功能预测,我们发现miR-206参与了6个KEGG信号通路,包括VEGF信号通路、P53信号通路、烟酸和烟酰胺的代谢、囊泡转运的SNARE相互作用、谷胱甘肽代谢和嗅觉传导；；miR-133b参与了2个KEGG信号通路,包括Notch信号通路和脂质代谢。其中,P53信号通路和Notch信号通路与癌症发生密切相关。
结论：
(1)NNK处理第60周大鼠血清进行小分子RNA Solexa测序分析,结果.表明NNK处理可诱导血清miRNA表达发生改变。
(2)通过大鼠个体血清中差异表达miRNA的实时定量分析,发现NNK处理组大鼠血清miR-206和miR-133b较对照组血清显著性高表达,而且血清miR-206和miR-133b的表达水平呈明显正相关；在大鼠肺癌组织,肺癌细胞系以及肺癌患者血清中,miR-206和miR-133b也呈现协同变化的关系,可能作为肿瘤抑制,niRNA参与NNK诱导的肺癌发生发展。
(3)通过对NNK诱导大鼠肺癌发生发展各阶段血清中miR-206和miR-133b的实时定量检测,发现NNK处理组血清在早期呈上升趋势,在20周表达水平达高峰,之后逐渐降低,最后在第95周NNK处理组血清中呈低表达。
(4)通过在大鼠肺癌组织,肺癌细胞系及有吸烟史的肺癌患者血清中进一步得以鉴定,发现血清miR-206和miR-133b是NNK诱导肺癌发生发展的潜在生物标志物。
Background:
As is known to all, cigarette smoke contains substantial amounts of lung carcinogens, including4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), polycyclic aromatic hydrocarbons (PAHs), metals and miscellaneous organic compounds etc. Of these lung carcinogens, NNK is a tobacco-specific N-nitrosamine and the most important lung carcinogen. Although many carcinogens in cigarette smoke contribute to lung cancer, the key compound NNK plays a major role in lung carcinogenesis. Many studies showed that NNK is a potent lung carcinogen in rats, mice, and hamsters. NNK induced lung tumorigenesis have remarkable organospecificity. The systemic administration of NNK to rats is a reproducible and robust method for the induction of lung tumors.
Lung cancer is one of the most common cancers and a leading cause of cancer death in the world. Many studies indicate that cigarette smoking causes most lung cancers. Despite great efforts to improve smoking cessation and the treatment of patients with lung cancer, the5years survival rate for lung cancer patients has not significantly improved over the past30years. Scarcity of diagnostic biomarkers for early stage lung cancer is the most important causes. Discovery and use of efficient biomarker for early stage lung cancer will be able to improve the5years survival rate of lung cancer.
MicroRNAs (miRNAs) are a class of small endogenous noncoding RNA (about19-25nt) that usually post-transcriptional negative regulate the expression of mRNA. Previous studies showed that miRNAs involve in a variety of biological processes including cancer development and progression, cell growth and proliferation, apoptosis etc. Increasing evidences have demonstrated that expression of miRNAs is dysregulated in a variety of cancers, such as lung cancer, pancreatic cancer, prostate cancer, breast cancer and hepatocellular carcinoma etc. MiRNAs differential expression profile can classify variety of cancers. Several recent studies indicated that miRNAs are stable and detectable in serum or plasma. These data consistently indicate that circulating miRNAs are potential biomarker for cancer.
Previous several studies declared that chemical carcinogens can cause the alteration of miRNAs expression. Male F344rats continuously fed with NNK up to20weeks resulted in alteration of miRNA expression in lung tissues of rat. To our knowledge, relative study on alteration of circulating miRNAs expression in carcinogenesis induced by chemical carcinogens is not still reported. Chemical carcinogenesis usually includes four stages:initiation; progression; development and transformation. During NNK-induced lung carcinogenesis, the alteration of serum miRNAs expression is not still unclear. We hypothesized that the levels of serum miRNAs were change and associated with lung carcinogenesis induced by NNK. Previous studies showed that male F344rat is highly sensitive to NNK-induced lung carcinogenesis. The dose-response relationship indicated that administration of low dose NNK mainly induce lung carcinogenesis. Therefore, in the present study, NNK was systemically administered to rats to induce lung carcinogenesis. Using this animal model, Blood of rats were collected at1,5,10,20,40,60,80and95weeks after NNK treatment and sera of rats were isolated. Serum miRNAs differential expression profile was analyzed by small RNA solexa sequence. Using Quantitative real-time PCR (qRT-PCR), the differential expression serum miRNAs were identified in each rat individuals. Significant differential expression miRNAs (miR-206and miR-133b) were selected for further study. The expression levels of miR-206and miR-133b were identified in rat serum at different stages of NNK induced lung carcinogenesis. The potential of miR-206and miR-133b as biomarker for lung cancer was identified in rat sera, rat lung tumors, lung cancer lines and human sera, respectively. Our study will provide important data for explore the biomarker of chemical carcinogens induced lung carcinogenesis. We predicted the barget genes and functions of miR-206and miR133b by computational bioinformatic analysis. These studies will provide experimental evidences for further study on the functions of circulating miRNAs.
Objective:
(1) Male F344rats were administered with subcutaneous injections of low-dose NNK (3times weekly for20weeks), and animals were maintained at SPF animal house until the95th week to induce lung carcinogenesis.
(2) MiRNAs differential expression profile of rat serum in control and NNK treatment group was analyzed by small RNA solexa sequencing and candidate miRNAs were screened.
(3) Using Quantitative real-time PCR (qRT-PCR), the differential expression serum miRNAs were identified in each rat individuals. Significant differential expression miRNAs (miR-206and miR-133b) were selected for further study.
(4) The expression levels of miR-206and miR-133b were identified in rat serum at different stages of NNK induced lung carcinogenesis.
(5) The potential of miR-206and miR-133b as biomarker for lung cancer was identified in rat sera, rat lung tumors, lung cancer lines and human sera, respectively. Our study will provide important data for explore the biomarker of chemical carcinogens induced lung carcinogenesis.
Methods:
(1) The construction of rat model for lung cancer induced by NNK:Rats from control group and NNK treatment group were respectively administered with subcutaneous injections of0.3ml saline and NNK (1.15mg/kg,0.0055mmol/kg) in0.3ml saline (newly prepared before each injection). Each rat was given injections3times weekly for20weeks. Afterward, animals were maintained at SPF animal house until the95th week to be sacrificed.
(2) Small RNA solexa sequencing of rat serum:Equal volume serum from each rat of control or NNK treatment group at the60th week was polled together, respectively. Pooled serum samples of control and NNK treatment group were send to Beijing Genomics Institute at Shenzhen for small RNA solexa sequencing and miRNAs differential expression profile was analyzed.
(3) Candidate serum miRNAs expression analysis for rat individuals by qRT-PCR:According to miRNAs differential expression profile to select candidate miRNAs, Using Quantitative real-time PCR, The differential expression serum miRNAs were identified in each rat individuals. Significant differential expression miRNAs (miR-206and miR-133b) were selected for further study.
(4) qRT-PCR analysis of the expression of miR-206and miR-133b at different stages of NNK treatment:Using Quantitative real-time PCR, miR-16was selected as endogenous control gene and the level of miR-206and miR-133b were detected in rat sera of1,5,10,20,40,60,80and95week.
(5) The expression of miR-206and miR-133b in lung tissues of rats:Using Quantitative real-time PCR, RNUB6was selected as endogenous control gene and the level of miR-206and miR-133b were detected in lung tissues of rats.
(6) The expression of miR-206and miR-133b in lung cancer cell lines:Using Quantitative real-time PCR, RNUB6was selected as endogenous control gene and the level of miR-206and miR-133b were detected in lung cancer cell lines.
(7) The level of miR-206and miR-133b in human sera of health control and lung cancer patients:Using Quantitative real-time PCR, miR-16was selected as endogenous control gene and the level of miR-206and miR-133b were detected in human sera of health control and lung cancer patients.
(8) Bioinformatic analysis:To predict the functions of miR-206and miR-133b, we predicted the target genes of miR-206and miR-133b from http://www.microrna.org/microrna/. The common target genes of miR-206and miR-133b were analyzed to find those target genes that are associated with carcinogenesis. The functions of miR-206and miR-133b are also predicted by http://acgt.cs.tau.ac.il/fame/index.html. Pathways of cancer or lung cancer were analyzed from http://www.genome.jp/kegg/pathway/to find those target genes involving in cancer pathway.
(9) Statistical analysis:Statistical analysis was performed using SPSS software (version13.0, IL, USA).Normality test was performed for data of each group using the Shapiro-Wilk normality test. For parametric comparisons, two independent samples t-test (for two independent samples), one-way analysis of variance (ANOVA) for multiple samples and paired t-test (for two paired samples) were employed respectively. For nonparametric comparisons, the Mann-Whitney test (for two nonparametric groups) and the Kruskall-Wallis test (for more than two nonparametric groups) were used respectively. Fisher's exact test was performed to compare the incidences of lung tumors between control and NNK treatment. Spearman correlation analysis was used to evaluate the correlation of two variables. In order to evaluate the predictive value of serum miRNAs, the receiver operating characteristic (ROC) curve were established for discriminating health control and patients with lung cancer. The area under the ROC curve (AUC) was calculated in order to better identify the potential of serum microRNAs as a diagnostic marker. All p-values are two-sided and less than0.05was considered statistically significant.
Results:
(1) The construction of rat model for lung cancer induced by NNK:At the95th week,19lung tumors were found in10rats of NNK treatment group, only1lung tumor was found in7rats of control group. No pathological changes were observed in lung tissues of rats of control. Most of lung tumors were moderately differentiated adenocarcinoma. Compared to control group, the incidence of lung tumor in NNK treatment group was statistical significance (Fisher's exact test, P=0.004).
(2) Serum miRNAs differential expression profile:We detected387known miRNAs of Rattus norvegicus (reference to miRBase Release13). Of these miRNAs,181miRNAs could be detected in control serum or NNK treatment serum and82differential expression miRNAs have significantly difference between control serum and NNK treatment serum. Compared with control serum,37miRNAs expression were up-regulated;45miRNAs expression were down-regulated;99miRNAs expression were equal. Of these181miRNAs,20miRNAs specifically expressed in the serum of control group;8miRNAs specifically expressed in the serum of NNK treatment group;153miRNAs commonly expressed in the serum of control and NNK treatment.
(3) Candidate serum miRNAs expression analysis for rat individuals by qRT-PCR:Compared with control serum, the expression level of miR-206(U=21.00, P=0.012), miR-133b (U=26.00, P=0.030) and miR-382(U=24.00, P=0.021) were up-regulated significantly in NNK treatment group (Mann-Whitney tests). MiR-365, miR-34c, miR-20a, miR-29b, miR-30e, miR-183, miR-331and miR-195have no significances between rat sera of control and NNK treatment group.The expression of miR-206and miR-133b was closely associated in serum of rat individuals. Spearman rank correlation analysis show that the expression of miR-206and miR-133b are positive correlation, Spearman r=0.758,95%CI=0.339-0.926, P=0.003.
(4) qRT-PCR analysis of the expression of miR-206and miR-133b at different stages of NNK treatment:Compared with control serum, the levels of miR-206and miR-133b expression were significantly up-regulated in rat serum of NNK treatment group at1,5,10,20,40,60and80weeks, respectively. Fold change up-regulated of miR-206were respectively1.43±0.11(t=-6.744, P=0.003);1.59±0.32(t=-3.215, P=0.032);2.17±0.35(t=-5.735, P=0.029);4.82±0.88(t=-7.497, P=0.017);3.23±0.99(t=-3.876, P=0.018);2.41±0.15(t=-16.250, P=0.004);1.85±0.12(t=-12.035, P=0.007). Fold change up-regulated of miR-133b were respectively1.08±0.10(t=-1.485, P=0.276);1.45±0.08(t=-9.208, P=0.011);2.12±0.25(t=-7.774, P=0.016);2.68±0.03(t=-97.887, P<0.001);2.33±0.17(t=-13.238, P<0.001);1.82±0.16(t=-8.953, P=0.001);1.30±0.16(t=-3.156, P=0.034). At95weeks after NNK treatment, miR-206and miR-133b were significantly down-regulated compared to rat serum of control. Fold change were2.09±0.64(t=5.769, P=0.004);2.78±0.30(t=26.813, P=0.001), respectively.
(5) The expression of miR-206and miR-133b in lung tumors tissues of rats: In9rat lung tumor tissues and corresponding normal lung tissues. Compared with normal lung tissues, miR-206and miR-133b was significantly low-expression in9paired lung tumor tissues (paired t-test). Meanwhile, we examined the levels of miR-206and miR-133b expression in7normal rat lung tissues of control group. Statistically significant differences were analyzed using ANOVA. Among three groups, the levels of miR-206(F=16.376, P<0.001) and miR-133b (F=11.001, P<0.001) have significantly difference. Compared with normal lung tissues of control, miR-206(P=0.001) and miR-133b(P=0.019) were low-expression significantly in normal lung tissues of NNK treatment group and lung tumor tissues of NNK treatment group (p<0.001). Compared to normal lung tissues of NNK treatment group, miR-206(P=0.048) and miR-133b (P=0.031) were low-expression significantly in paired lung tumor tissues of NNK treatment group.
(6) The expression of miR-206and miR-133b in lung cancer cell lines:The level of miR-206was significantly down-regulated28.341±3.435,97.638±16.922,33.999±11.882,33.055±16.138,23.424±3.899and31.688±12.306fold in A549, QG56, H446, H1299,95-D and16HBE-T cell relative to16HBE cell, respectively. The level of miR-133b was down-regulated1.793±0.410,3.926±2.014,2.894±2.135,3.768±0.734,2.176±0.710and2.652±1.186fold in A549, QG56, H446, H1299,95-D and16HBE-T cell relative to16HBE cell, respectively.
(7) The level of miR-206and miR-133b in human sera of health control and lung cancer patients:Compared with health controls, the expression of serum miR-206and miR-133b were significantly down-regulated in lung cancer patients (miR206-U=61.00, miR133b-U=80.00, P<0.001, Mann-Whitney test). Receiver operating characteristic (ROC) curve analyses show that the areas under curve (AUC) of serum miR-206and miR-133b were0.9024(95%CI=0.8125-0.9923) and0.8720(95%CI=0.7734-0.9706), respectively.
(8) Bioinformatic analysis:has-miR-206and has-miR-133b were predicted7201and5314target genes, respectively. And of these target genes,2577genes are common target genes of miR-206and miR-133b. By functions prediction, we found that miR-206involve in6KEGG pathways, including VEGF signaling pathway, p53signaling pathway, Nicotinate and nicotinamide metabolism, SNARE interactions in vesicular transport, Glutathione metabolism, Olfactory transduction. MiR-133b involve in2KEGG pathways, including Notch signaling pathway, Sphingolipid metabolism. Of these pathways, P53signaling pathway and notch signaling pathway are closely related to carcinogenesis.
Conclusions:
(1) Small RNA solexa sequencing of rat serum at the60th week indicate that NNK can induce the alteration of the expression of serum miRNAs.
(2) qRT-PCR analysis of differential expression miRNAs in sera of rats individuals showed that miR-206and miR-133b were significantly up-regulated in rat sera of NNK treatment group. Moreover, the expression of miR-206and miR-133b was positive correlation. The expression of miR-206and miR-133b in lung tumors and lung cancer cell lines has significantly covariant. They may involve in NNK induced lung carcinogenesis as tumor suppressor.
(3) The level of serum miR-206and miR-133b at different stages of NNK induced lung carcinogenesis indicated that alteration of serum miRNAs expression followed a two-step evolution. At early stage, the level of miR-206and miR-133b was progressively increased and the peak of expression occurs at the20th week. Afterward, the level of miRNAs was progressively decreased and low expression was observed at the95th week.
(4) By identifying the expression of miR-206and miR-133b in rat sera, rat lung tumors, lung cancer cell lines and sera of lung cancer patients with smoking history, serum miR-206and miR-133b may be potential biomarker for lung carcinogenesis induced by NNK.
引文
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