【作者】 冯艳铭；

【导师】 吴逸明；

【作者基本信息】 郑州大学 ， 流行病与卫生统计学， 2008， 博士

【摘要】 肺癌是常见的恶性肿瘤,被认为是对人类健康和生命威胁最大的恶性肿瘤之一。全球每年新增120万病例,中国每年有60万人死于肺癌,在中国肺癌5年生存率仅8.9%,非小细胞肺癌占肺癌的85%以上,而非小细胞肺癌中85%以上又都属中晚期肺癌而失去根治性手术治疗的机会。肺癌的发生是环境因素和遗传因素共同作用的结果,多种基因在肺癌的发生和发展中均起到了非常重要的作用,蛋白组学的发展使得在非小细胞肺癌（Non-small celllung cancer,NSCLC）发现了许多高表达和低表达蛋白,亲环素A（Cyclophilin A,CyPA）就是众多相关蛋白之一。CyPA是本课题组应用双向电泳技术（2-DE）在肺腺鳞癌组织中发现的特异性高表达的差异蛋白,CyPA是功能相关的蛋白家族成员之一,具有肽基脯氨基顺反异构酶活性,能催化细胞内蛋白质的折迭、装配和运输,起分子伴侣的作用。新近研究表明,CyPA参与了许多癌的发生和发展过程,其促进癌细胞生长的作用在胰腺癌研究中,取得了很有说服力的结果。RNA干扰（RNAi）技术具有快速、高效、易行的特点,在基因功能研究、基因治疗方面显示了巨大的前景。慢病毒载体介导的短发夹RNA（shRNA）表达载体能感染非分裂细胞和终末分化细胞,并且在感染后整合到宿主细胞的基因组,进行长时间的稳定表达,适合体内实验研究。研究目的1.假设特异性地沉默CyPA基因可能是抑制非小细胞肺癌生长的有效方法,为此,本课题首先构建慢病毒介导的CyPA siRNA系统。2.本课题旨在研究CyPA在NSCLC发生中的作用,以寻求其作为基因治疗新靶点的依据。因此,采用慢病毒介导的CyPA siRNA,分别在体内体外水平研究CyPA基因沉默对非小细胞肺癌细胞生长的抑制作用,对已筛选的肺癌相关蛋白CyPA的基因进行生物学功能研究。材料与方法1.CyPA mRNA和蛋白在NSCLC细胞和组织的表达水平1.1采用双链嵌合荧光染料SYBR GreenⅠ荧光定量PCR技术,设计特异性引物,提取NSCLC A549、H1299及人正常支气管上皮细胞BEAS-2B细胞的总RNA,反转录获得cDNA进行PCR,低熔点琼脂糖凝胶回收纯化PCR扩增产物,取1μl的纯化产物,作10倍系列稀释共9个梯度,取后6个稀释梯度的模板做标准曲线,分别建立内对照β-actin和目的基因CyPA定量标准曲线。检测A549、H1299及BEAS-2B细胞CyPAmRNA的表达水平,以BEAS-2B细胞作为对照,用2^-ΔΔCt计算CyPAmRNA相对表达量,通过PCR产物的熔解曲线评价其特异性。1.2收集45例NSCLC手术病人切除的肺癌组织,22例对应的癌旁组织,33例对应的正常肺组织制成组织芯片,其中鳞癌25例,腺癌20例;按TNM分期标准Ⅰ+Ⅱ期27例,Ⅲ期18例,用SP免疫组化方法和组织芯片技术,观察CyPA在NSCLC癌组织、癌旁组织及正常组织中的表达和分布情况以及与临床病理指标的关系。2.构建并筛选慢病毒介导的CyPA siRNA有效靶点和制备病毒颗粒2.1首先设计并合成4对CyPA基因siRNA靶序列寡核苷酸,退火形成双链DNA,与经HpaⅠ和XhoⅠ酶切后的pGCL-GFP载体连接产生Lv-shCyPA慢病毒质粒载体,PCR及测序筛选鉴定阳性克隆。2.2将阳性克隆Lv-shCyPA慢病毒质粒载体与pHelper1.0和pHelper2.0质粒共转染293T细胞,包装制备5个假包装病毒颗粒（Lv-shCyPA）并感染NSCLC A549、H1299细胞,Western Blot分析CyPA蛋白表达水平,筛选CyPA siRNA有效靶点并进行该靶点高滴度病毒包装,命名为Lv-shCyPA。2.3待A549、H1299细胞生长至50%～70%融合度时,将Lv-shCyPA分别按照MOI值为2、5、10、20、40感染A549细胞、H1299,根据表达荧光的细胞百分数优化MOI值。3.慢病毒介导的CyPA siRNA对A549、H1299细胞生长的体外研究3.1待A549、H1299细胞生长至50%～70%融合度时,按照优化的MOI值,加入适量的病毒,每孔细胞加Polyberne（5μg/ml）,正常培养传代4～5次,以GFP为报告基因镜下筛选GFP强表达的克隆,分离培养,建立了CyPA稳定沉默的细胞系A549/Lv-shCyPA、H1299/Lv-shCyPA。3.2将Lv-shCyPA分别按照A549细胞（MOI=20）、H1299（MOI=5）感染NSCLC A549、H1299细胞,感染第5天提取细胞总RNA,采用荧光定量RT-PCR检测CyPA mRNA表达水平。3.3取对数生长期的CyPA稳定沉默的细胞系A549/Lv-shCyPA、H1299/Lv-shCyPA,胰酶消化后重悬成2×10⁴/ml的细胞悬液,每孔接种100μl于96孔板,分别在培养1d、2d、3d、4d、5d、6d时终止培养,MTT法检测细胞增殖情况。3.4将Lv-shCyPA按照A549细胞（MOI=20）、H1299（MOI=5）感染A549、H1299细胞,用不含EDTA的0.25%胰酶消化感染第5天的细胞培养物,4℃70%乙醇固定细胞,加1mlPI（50μg/ml）染色,4℃避光30min,进行细胞周期及凋亡检测。3.5阴性对照病毒颗粒按上述步骤进行平行实验,未感染病毒的A549、H1299分别作为实验对照组。4.慢病毒介导的CyPA siRNA对A549、H1299细胞裸鼠移植瘤生长的作用4.1 A549/Lv-shCyPA、H1299/Lv-shCyPA细胞悬液（5×10⁷/ml）,注射于裸鼠协腹皮下,每组5～6只裸鼠,注射体积为100μl/只。4.2裸鼠成瘤后每四天测量1次移植瘤体积,从注射起测量到第38d,肿瘤体积=（长×宽²）×0.5,根据移植瘤体积绘制移植瘤生长曲线。4.3阴性对照病毒颗粒感染的A549、H1299按上述步骤进行致瘤,未感染病毒的A549、H1299分别作为实验对照组。5.统计学处理利用SPSS12.0软件对数据进行统计分析,采用t检验对不同感染条件下细胞CyPA蛋白、mRNA、细胞增殖率、细胞周期分布、凋亡率以及裸鼠移植瘤瘤体、重量与对照组比较;秩和检验比较不同组织CyPA蛋白表达水平的差异,以α=0.05为检验水准。结果1.CyPA mRNA和蛋白在NSCLC细胞和组织的表达水平1.1荧光定量RT-PCR检测A549、H1299、BEAS-2B细胞中CyPA mRNA的表达水平,结果显示:肺腺癌细胞A549、H1299中CyPA mRNA呈高表达,其表达水平与BEAS-2B细胞相比差异均有统计学意义（P＜0.05）。1.2组织芯片免疫组织化学技术检测,结果显示:CyPA蛋白的阳性信号见于肺癌细胞浆、肺泡上皮细胞浆,呈棕色颗粒状,其分布以广泛性表达为主,偶见局灶性或散在性分布;正常肺组织未见或少见棕色颗粒沉着。NSCLC癌组织及癌旁组织中CyPA的表达水平明显高于正常肺组织中CyPA蛋白的表达水平,其差异有统计学意义（P＜0.05）,CyPA蛋白在正常组织、癌旁组织及肺癌组织中的阳性表达率分别为9.09%（3/33）、53.62%（12/22）、82.22%（37/45）,CyPA在正常组织、癌旁组织及肺癌组织中的表达水平依次呈递增趋势;肺腺癌与肺鳞癌组织中CyPA蛋白的表达水平差异无统计学意义（P＞0.05）;不同TNM分期的的癌组织CyPA蛋白的表达水平差异无统计学意义（P＞0.05）。2.慢病毒介导的CyPA siRNA的有效靶点有效的CyPA siRNA靶序列是5’-GTGAAAGAAGGCATGAATA-3’,根据该序列制备的CyPA siRNA假包装病毒颗粒滴度为2×10⁹TU/ml。3.慢病毒介导的CyPA siRNA对A549、H1299中CyPA基因的沉默作用Lv-shCyPA感染A549、H1299细胞,CyPA mRNA和蛋白表达水平均下降,与对照组相比,CyPA mRNA相对表达量分别为0.048和0.1452,CyPA mRNA的沉默效率分别为95.2%和85.48%,CyPA蛋白水平相对表达量分别为13.48%和14.7%,其表达抑制效率分别为86.52%和85.3%,CyPA mRNA与蛋白的表达水平具有一致性,其表达水平与对照组相比差异均有统计学意义（P＜0.05）。阴性对照A549/NC、H1299/NC组CyPA mRNA和蛋白的表达水平与对照组差异无统计学意义（P＞0.05）。4.慢病毒介导的CyPA siRNA对A549、H1299细胞生长的抑制作用MTT法分析不同条件下A549、H1299细胞的生长状态,结果显示:A549/Lv-shCyPA、H1299/Lv-shCyPA细胞生长明显平缓,感染第5d细胞增殖率下降,与对照组相比差异有统计学意义（P＜0.05）。流式细胞术分析不同条件下细胞各周期分布,结果显示:不同条件下细胞G₀-G₁期、S期细胞比例与对照组相比差异无统计学意义（P＞0.05）;A549/Lv-shCyPA、H1299/Lv-shCyPA细胞G₂-M期细胞相对减少,细胞凋亡率增加,与对照组相比差异均有统计学意义（P＜0.05）;而A549/NC、H1299/NC组G₂-M期比例、细胞凋亡率与对照组相比差异均无统计学意义（P＞0.05）。5.慢病毒介导的CyPA siRNA对A549、H1299细胞裸鼠移植瘤生长的抑制作用未感染病毒组及阴性对照病毒颗粒感染组的细胞接种裸鼠后,移植瘤在第4d出现可见肿瘤,然后生长迅速,而A549/Lv-shCyPA、H1299/Lv-shCyPA感染组可见肿瘤在接种后6d出现,移植瘤生长明显迟缓。接种后第38d,A549/Lv-shCyPA、H1299/Lv-shCyPA组移植瘤体积小、重量轻,与对照组相比差异均有统计学意义（P＜0.05）,CyPA基因沉默组移植瘤瘤体重量平均降低了77.76%和78.85%。而A549/NC、H1299/NC组细胞接种后形成的移植瘤体积、重量与对照组相比差异均无统计学意义（P＞0.05）。结论1.CyPA蛋白在正常肺组织、NSCLC癌旁组织及癌组织中的表达水平依次呈递增趋势,尤其是在癌组织中呈高表达水平;同时在NSCLC A549、H1299细胞中CyPA mRNA亦呈高表达水平,CyPA mRNA的表达水平与BEAS-2B细胞相比差异有统计学意义,提示CyPA可能参与了非小细胞肺癌的发生发展。2.筛选出有效的CyPA siRNA靶序列为5’-GTGAAAGAAGGCATGAATA-3’,制备的Lv-shCyPA经体外观察对NSCLC A549、H1299中CyPA基因的表达有沉默作用,对NSCLC A549、H1299生长有抑制作用。3.慢病毒介导的CyPA siRNA在裸鼠移植瘤实验中也显示出一定的抑瘤作用,为肺癌的基因治疗和药物治疗提供新的治疗策略。更多还原

【Abstract】 Lung cancer is considered as one of the most lethal cancers which has posed a threat to the patient’s health and life.About 1,200,000 new cases were predicted every year in the world.In China,600,000 cases were died of lung cancer annually and the overall 5-year survival remains at 8.9%.About 85%of patients with lung cancer were non-small cell lung cancer（NCSLC） and among those 85%were in advanced stage and have no opportunity to have surgical treatment.Carcinogenesis of lung is cooperated with the environmental and genetic factors.Many genes play an important roles in tumorigenesis during the progression of non-small cell lung cancer.With the development of proteomics some overor low- expression protein were found in non-small cell lung cancer tissues,One of these proteins,Cyclophilin A（CyPA）,was found to have overexpression in lung tumor tissues by two-dimension electrophoresis.CyPA is a member of the peptidyl-prolyl isomerases.CyPA also catalyzes protein folding and conformational changes which depends on its capacity as chaperon proteins. Recently,correlations of CypA with tumor pathogenesis have been studied.A possible role for CyPA in tumor cell growth has been demonstrated by the overexpression of CyPA in human pancreatic adenocarcinoma tissues and addition of exogenous CypA significantly stimulated pancreatic cancer cell proliferation in a dose-dependent manner. The technique of RNAi is rapid,cost effective and can be easily adapted to study homo -logous gene function in a wide variety of organisms.Lentiviral vector hold great promise for gene therapeutic applications because it can mediated stable gene transfer both in dividing and non-dividing cells.In addition,the lentiviral products are integrated into the host genome ensuring siRNA stable long-term expression.Therefore Lentiviral vector is adapted to study in vivo.Objectives1.We proposed that it is a potential pathway to inhibite the growth of NSCLC by silencing CyPA gene.So,CyPA siRNA mediated by lentiviral vector was firstly constructed and screened.2.To investigate the biological functions of CyPA among the development in non-small cell lung cancer,and to seek new treatment targets.Lentiviral-vector-mediated siRNA of CyPA was used to study its inhition of A549 and H1299 in vitro and in vivo,respectively.Materials and Methods1.The expression of CyPA mRNA and protein in NSCLC cell and lung tissues1.1 Total RNA was extracted from cells using TRIZOL reagent.RT and cDNA amplification were performed according to SYBR GreenⅠreal-time PCR kit.Primer pairs were designed for CyPA andβ-actin.The products amplified were purified and serial diluted ranging from 10 to 10⁹ as a standard curve,respectively.β-actin as an internal control used to normalized the signal value of each sample.The relative quantitation of CyPA mRNA was presented as unit values of 2^-ΔΔCt.To confirm amplification specificity the PCR products were subjected to a melting curve analysis.1.2 The expressions of CyPA protein were examined in 45 lung cancer tissues,22 paired tumor-adjacent tissues and 33 normal pulmonary tissues using tissue microarray immuno-histochemistry method.The relationship of CyPA expression in NSCLC with clinic pathological characteristic was analyzed.2.Construction and screening of CyPA siRNA mediated by lentiviral vector2.1 Four target sequences were selected and the complementary DNA contained both sense and antisense oligonucleotides were designed,synthesized and cloned into pGCL-GFP vector.The recombinant lentiviral vector containing CyPA shRNA was confirmed by PCR and sequencing.2.2 The positive recombinant lentiviral vector was cotransfected with pHelper1.0 and pHelper 2.0 into 293T cells to pack lentivirus particles which was named after Lv-shCyPA. At the same time,the packed virus infected A549、H1299,the expression level of CyPA protein after infection was detected by Western Blot in order to screen the effective target of CyPA siRNA.2.3 A549 and H1299 were infected with specific or negative control packed virus （Lv-shCyPA,NC） using multiplenty of infection of 5,10,20,respectively.The infected cells were maintained in culture medium for 4-5 passages and selected the single colony with GFP strongly expressed to establish stable silencing lines.The MOI of A549 and H1299 were optimized according to GFP.3.The growth of A549 and H1299 was suppressed by lentiviral mediated CyPA siRNA in vitro3.1 A549 and H1299 cells were infected by Lv-shCyPA or NC according to the MOI of A549/20 and H1299/5,respectively.Cultured cells were collected at 5^th day,and total RNA was extracted from cultured cells.The quantity of CyPA mRNA was quantited by real-time RT-PCR.3.2 Add stable silencing lines A549/Lv-shCyPA and H1299/Lv-shCyPA 100μl into 96 well （approximately 5×10³cells/well）.After incubation for 1d,2d,3d,4d,5d,6d,respectively. MTT assay were performed at the end of the cultivation period.The growth curve of cells and proliferation rate of cells were analyzed,respectively.3.3 A549 and H1299 cells were infected by Lv-shCyPA or NC according to the MOI of A549/20 and H1299/5,respectively.At 5^th day after infected,cells were harvested and fixed in 70%ethanol.Cell cycle and apoptosis rate were analyzed using the flow cytometer.3.4 Uninfected cells of A549 and H1299 were as control groups in the study,respectively.3.5 Data from real-time PCR,Western Blot,MTT and FCM are expressed as（?）±s from at three independent groups.Significant differences were determined by paired Student t-test（two tails,paired） using SPSS12.0 software for Window XP.4.The growth of xenografts were inhibited by lentiviral mediated CyPA siRNA in vivoApproximately 1×10⁶ A549/Lv-shCyPA,A549/NC,H1299/Lv-shCyPA,H1299/NC and their parental cells were injected subcutaneously into the right flank of the mouse.Tumor growth and animal weight were monitored every 4 days starting from the 4^th day to 38^th after injection.Tumor dimensions were measured with calipers and the volume was calculated according to the equationⅤ（mm³）=ab²/2（a:length,b:width,b≤a）.RESULTS1.The expression of CyPA mRNA and protein in NSCLC cell and lung tissues1.1 The overexpression of CyPA mRNA in A549 and H1299 were significant difference compared to that in BEAS-2B by fluorescent quantitative PCR（P＜0.05）,and so,we can take A549 and H1299 as the target cell for CyPA gene silencing.1.2 The expressions of CyPA protein in NSCLC tissues and paired tumor adjacent tissues were significantly higher than that in the normal lung tissues（P＜0.05）.The positive rates of CyPA protein in the normal lung tissues,paired tumor adjacent tissues and NSCLC tissues were 9.09%（3/33）,53.62%（12/22） and 82.22%（37/45）,respectively.We can see the level expressions were increased in turn.No significant correlations were found in histological types（P＞0.05） and TNM stage（P＞0.05）.2.Construction and screening of CyPA siRNA mediated by lentiviral vectorThe effective sequence of CyPA siRNA was 5’-GTGAAAGAAGGCATGAATA-3’. Pseudotyped virus were packed and concentrated.The titer of pseudotyped virus was 2×10⁹TU/ml.3.The expression of CyPA mRNA and protein were downregulated by lentiviral mediated CyPA siRNA3.1 GFP was strongly expressed in infected cell lines at 5^th day.The relative expression of CyPA mRNA in A549/Lv-shCyPA and H1299/Lv-shCyPA were 0.048 and 0.1452, respectively.The CyPA mRNA was downregulated by 95.2%and 85.48%compared to the parent cells,and the relative expression of CyPA protein were 14.78%and 13.78%compared to control groups.CyPA protein was downregulated by 86.52%and 85.3%compared to the parent cells,respectively（P＜0.05）.So,it is suggested that these results confirm consistency.3.2 A549/Lv-shCyPA and H1299/Lv-shCyPA cells grew more slowly than control groups. Proliferation rate of A549 and H1299 infected by Lv-shCyPA was significant declined at 5^th day compared to control group（P＜0.05）.Flow cytometric analysis demonstrated no significant cell cycle arrested in G₀-G₁ phase and S phase,while G₂-M phase was decreased relatively in A549/Lv-shCyPA and H1299/Lv-shCyPA compared to control groups （P＜0.05）.The apoptosis rate of A549/Lv-shCyPA and H1299/Lv-shCyPA was higher than control groups（P＜0.05）.No significant difference was found both in distribution of cell cycle and in apoptosis rate of A549/NC and H1299/NC compared to control groups （P＞0.05）.4.Xenografts growth were inhibited by lentiviral mediated CyPA siRNA in vivoVisible xenograft tumors in the parent cell groups and NC groups were readily detectable at 4^th day after implantation and grew rapidly in the next days.In contrast, visible tumors were only detectable at 6^th day after inoculated by A549/Lv-shCyPA and H1299/Lv-shCyPA.The xenograft tumors of A549/Lv-shCyPA and H1299/Lv-shCyPA cells remarkably delayed tumor growth and remained at a similarly small average size at 38^th days after inoculation compared to the control group（P＜0.05）,the mass of xenograft tumors inoculated by A549/Lv-shCyPA and H1299/Lv-shCyPA were decreased at 77.76% and 78.85%,respectively.No significant difference was found in H1299/NC and A549/NC compared to the control group（P＜0.05）.Conclusion1.It is consistent with the results performed by 2-DE method that CyPA is overexpressed in NSCLC tissues,suggesting that CyPA might serve as one of the biomarkers involved the development and progression of NSCLC.2.The sequence of CyPA siRNA was 5’-GTGAAAGAAGGCATGAATA-3’.Pseudotyped lentivirus particles containg CyPA siRNA can dowrtregulate the expression of CyPA mRNA in A549 and H1299,and also can suppress the cell growth of A549 and H1299 in our study.3.The results show that CyPA siRNA mediated by lentiviral vector can inhibite the xenograft tumor growth of A549 and H1299 in vivo.This data suggested that CyPA may have a good performance in therapies for NSCLC.Lentiviral vector system could be a potential therapeutic approach.更多还原