【作者】 龙慧民；

【导师】 严春寅；

【作者基本信息】 苏州大学 ， 泌尿外科， 2009， 博士

【摘要】 背景和目的:前列腺癌是男性泌尿系最常见的肿瘤之一,而针对晚期前列腺癌的有效治疗方法极为有限,特别是当激素依赖型前列腺癌(ADPC)转化为非激素依赖型前列腺癌(AIPC)后临床缺乏有效的治疗方法,而且预后较差。临床上通常采用化疗,但疗效不满意,而且由于多数AIPC患者年龄均>65岁,难以承受常规剂量的化疗及疗程。表皮生长因子受体(EGFR)是表皮生长因子基因(erbB)家族的一员,是一种分子量170kDa的跨膜酪氨酸激酶受体,由膜外结合区域、跨膜转换区域和胞质反应区域三部分组成。其中胞内部分具有酪氨酸激酶活性和ATP底物磷酸化结合部位。EGFR广泛存在于许多病理和生理性上皮组织,其与特异性配体EGF或TGF-α结合后转化为二聚体,同时, EGFR酪氨酸激酶区的自身磷酸化后被活化,细胞内蛋白分子磷酸化,最终将信息传到胞核内,启动与细胞增殖、迁移、粘附、侵入、凋亡、终止以及耐药性增强等有关的基因转录表达。50-90%的非激素依赖型前列腺癌都出现EGFR的过表达,而且EGFR的过表达与ADPC转化为AIPC密切相关,前列腺癌组织在雄激素剥夺后对EGF所产生的生长促进作用非常敏感,全面阻断酪氨酸激酶信号传导系统来治疗晚期AIPC这一思路受到重视,于是,抑制生长因子受体,特别是EGFR的表达或发挥作用成为治疗AIPC极有希望的治疗策略。尽管人们在应用EGFR单克隆抗体和小分子酪氨酸激酶抑制剂治疗肿瘤方面取得了较大的进步,但应用于二期临床治疗AIPC效果却不太满意,通过抑制EGFR表达来治疗肿瘤的方法需要改进。RNA干扰是用与目的基因mRNA相同序列的21-23nt双链小分子RNA来特异性高效率的抑制目的基因蛋白表达,有研究表明,带有shRNA表达框架的载体可以取得特异性使靶基因沉默的作用。前列腺在解剖上具有容易到达,适于反复穿刺注射携带shRNA表达框架的病毒载体进行靶向治疗的优点。而且存在特异性瘤标,易于随访疗效。本研究以雄激素非依赖性前列腺癌细胞系PC-3作为研究对象,应用携带针对EGFR基因的shRNA片段的慢病毒颗粒pshRNA-GFP LVEGFRRNAi来处理,了解其对EGFR基因的抑制效果;同时,在体内外实验中,探讨pshRNA-GFP LVEGFRRNAi是否存在抑制雄激素非依赖性前列腺癌细胞的作用,以及探讨pshRNA-GFP LVEGFRRNAi与紫杉醇或Gefitinib联合应用是否存在抗肿瘤的协同作用。方法:1、针对EGFR基因的三个亚型同源区设计siRNA序列以及siRNA表达载体的构建。2、使用构建好的siRNA表达载体转染HT1080细胞(人成纤维肉瘤细胞),进行最有效siRNA序列的筛选;3、使用最有效的siRNA序列对应的表达载体进行慢病毒颗粒pshRNA-GFP LVEGFRRNAi的包装和生产;4、使用高质量携带目的片段(siRNA)的病毒颗粒pshRNA-GFP LVEGFRRNAi感染宿主细胞(PC-3),检测RNAi效果。5、使用pshRNA-GFP LVEGFRRNAi治疗非激素依赖型前列腺癌荷瘤裸鼠,观察对肿瘤抑制效果,并检测EGFR、MAPK、Akt、PKC、p-MAPK、p-Akt等蛋白表达情况。6、使用pshRNA-GFP LVEGFRRNAi治疗非激素依赖型前列腺癌荷瘤裸鼠,并应用紫杉醇、Gefitinib进行药物刺激实验,观察是否存在抑制肿瘤的协同效应。结果:1、针对EGFR基因成功设计了siRNA序列并构建了siRNA表达载体,而且siRNA表达载体测序正确。2、成功的在HT1080细胞(人成纤维肉瘤细胞)中进行了转染实验,转染实验后24小时,转染效率达到了70%,且Realtime PCR检测显示成功的抑制了目的基因EGFR的表达,其中所设计的2号序列的RNAi效果最好,对目的基因EGFR的表达抑制效率达到了90%以上。3、使用慢病毒包装质粒混合物和siRNA表达载体质粒DNA共转染293T细胞,生产出高质量的携带siRNA片段的慢病毒颗粒pshRNA-GFP LVEGFRRNAi。4、体外研究中应用pshRNA-GFP LVEGFRRNAi感染PC-3细胞,并应用RealTime-PCR法测得对EGFR基因抑制比对照组下降90%(1-0.00851/0.08818), Western Blot法测得为88.9%(1-0.05396/0.48455),传代2～3代后仍保持90%左右的抑制率。MTT法检测PC-3 EGFR-RNAi稳转细胞株和正常的PC-3细胞相比,EGFR基因的低表达明显减慢了细胞的增殖速度。5、对于PC-3细胞的抑制生长,RNAi的作用效果和Gefitinib(5mg/L)刺激效果相仿,但是二者共同作用时,效果明显强于二者中的任何一种。6、PC-3 EGFR-RNAi稳转细胞株,相当于使用Gefitinib(5mg/L)刺激PC-3, EGFR、Akt、MAPK、PKC、p-MAPK、p-Akt蛋白的表达量均有不同程度的下调,而且下调程度相对较大。7、应用携带针对EGFR的siRNA片段的慢病毒颗粒pshRNA-GFP LVEGFRRNAi静脉注射后裸鼠瘤体的生长受到了一定的抑制。8、使用EGFR-RNAi结合应用紫杉醇、Gefitinib进行药物刺激实验,发现结合紫杉醇治疗其肿瘤抑制率达到56.8%,结合Gefitinib治疗组其肿瘤抑制率也达到54.0%;与对照组相比RNAi结合Gefitinib以及紫杉醇组抑瘤率达62.5%(P<0.01),而且RNAi组以及RNAi+Gefitinib组EGFR以及Akt下调较为明显,但MAPK下调并不明显。结论:携带针对EGFR的siRNA片段的慢病毒颗粒pshRNA-GFP LVEGFRRNAi可以下调EGFR基因的mRNA表达,并通过降低细胞增殖活性和增加细胞凋亡达到抑制PC-3细胞生长的作用,而且这一技术与紫杉醇及Gefitinib有协同作用,有望成为对非激素依赖型前列腺癌的一种有效辅助治疗方法。更多还原

【Abstract】 Background and objective:Prostate cancer is one of the most common type of cancer in men; however, therapeutic options are limited in advantage stage. In particular, for patients at high risk of progression to more advanced PCs or those diagnosed in the late stages with metastatic and hormone-refractory prostate cancers (HRPCs), The current chemotherapeutic treatments against the metastatic HRPCs generally show no significant, or in case of docetaxel-based regimens, modest beneficial effects for improving the overall survival rate and outcome of patients in the clinics, and ultimately result in the death of patients. The median patient age in the recent multi-center trials of first-line chemotherapy for androgen-independent prostate cancer (AIPC) trials was >65 years, Older patients may not tolerate standard or investigational cytotoxic chemotherapies as well as younger patients.The epidermal growth factor receptor (EGFR) , as one of the members of the EGFR/ErbB receptor family of receptor tyrosine kinases (RTKs), is a 170 kDa type I transmembrane glycoprotein containing a ligand-binding ectodomain, a single hydrophobic transmembrane region, and a cytoplasmic tail which includes a tyrosine kinase domain and docking sites for a variety of signaling effectors, At least seven ligands can bind to the extracellular part of EGFR including EGF and transforming growth factor alpha (TGFα), Ligand binding induces homo- and/or heterodimerization, thereby resulting in kinase activation and phosphorylation of tyrosines in the ErbB tail. The downstream signaling network generated by EGF-R activation is able to control cell growth, survival and migration. 50–90% of HRPC cells show an overexpression of EGFR. The human EGFR tyrosine kinases are part of a network of pathways which are implicated in the development and progression of prostate cancer, in particular as part of the progression to androgen-independent growth, EGFR plays a critical role in tumor growth, and the prostate tissue becomes more susceptible to the growth-promoting action of EGF family growth factors during androgen withdrawal, The general inhibition of tyrosine kinase signaling pathways provides therapeutic advantage against prostate cancer metastasis, Therefore, inhibiting the activation of growth factor receptors, especially EGFR, may be a promising strategy for the treatment of prostate cancer.Blockade of EGF receptor signaling pathway represents a new perspective on the development of novel and selective anticancer therapies. Although considerable progress has been made in the application of EGF receptor-targeted antibodies and small molecule tyrosinekinase inhibitors, none of these agents is curative. Phase II study was undertaken to evaluate the efficacy of tyrosinekinase inhibitors in patients with HRPC, the results were disappointing .Current therapies of its expression are still need to be improved.In RNA interference (RNAi), duplexes of 21-23nt RNAs (small interfering RNA, siRNA) corresponding to mRNA sequences of particular genes are used to efficiently inhibit the expression of the target proteins in mammalian cells. It is approved that vectors containing shRNA expressing cassette could induce gene silencing of the target gene. Prostate cancer is located in the prostate only, fit to puncture according to it’s anatomy, and has sepecific tumor MAPKs which are prone to follow-up, so prostate cancer is fit to inject the vectors containing shRNA expressing cassette for local gene targeted therapies.This study is to probe the effect of pshRNA-GFP LVEGFRRNAi on the PC-3 cell’s growth and the expression of EGFR protein, and further confirm the effect of inhibiting AIPC cells, thirdly to to investigate the efficacy and safety of pshRNA-GFP LVEGFRRNAi combined with paclitaxel and Gefitinib for the treatment of AIPC cells in vitro and in vivo.Methods: 1.The target sequences were selected according to homological region of three EGFR transcript variants, and the siRNA expression vectors were constructed. 2. HT-1080(a human fibrosarcoma cell line) cells were transfected with the three recombinant vectors, and interference effect was detected by RT-PCR. 3. the most effective siRNA expression vector was co-transfected with lentiviral package plasmids into 293T cells to product lentiviral particles. 4. PC-3 cells were infected by the recombinant lentivirus, and the RNA interference effect was detected. 5. The pshRNA-GFP LVEGFRRNAi was used to treat the non-hormone-dependent prostate cancer in nude mice, the inhibition effect was investigated, and the expression levels of EGFR、MAPK、Akt、PKC、p-MAPK、p-Akt were measured. 6. Nude mice burdened with prostate cancer were injected intravenously with virus supernant which contained pshRNA-GFP LVEGFRRNAi, then treated with paclitaxel or Gefitinib, the cooperative antitumor effect was investigated.Result: 1.The siRNA sequences targeted to EGFR gene were designed and the expression vectors were cloned and verified by sequencing. 2. HT1080 cells were transfected with siRNA expression vectors. 24 hours after transfection, the transfect efficiency was about 70%. Real-time PCR analysis revealed that siRNA expression vectors can inhibit the expression of EGFR. The second vector had a better interference effect, and the interference effect reach about 90%.3. The lentiviral package vectors and siRNA expression vector were co-transfected to 293T cells, and high quality pshRNA-GFP LVEGFRRNAi was obtained. 4. pshRNA-GFP LVEGFRRNAi was used to infect the PC-3 cells. Compared with control group , real-time PCR results showed that the expression of EGFR decreased 90%(1-0.00851/0.08818), and results of western blot revealed the inhibition effect was 88.9% (1-0.05396/0.48455).After three passages, the inhibition effect remained 90%. MTT measurement showed that EGFR-RNAi stable PC-3 cell line which expresses EGFR in a low level has a lower proliferation rate. 5. The inhibition effect on PC-3 cells of RNAi is similar as Gefitinib (5mg/L), and the combination of these two treatments, can inhibit the growth of PC-3 better. 6. The expression levels of EGFR、MAPK、Akt、PKC、p-MAPK、p-Akt in EGFR-RNAi stable cell line were down-regulated significantly compared with the cells treated with Gefitinib (5mg/L). 7. Intravenous injection of the pshRNA-GFP LVEGFRRNAi encoding the siRNA targeting EGFR can inhibit the growth of tumor in nude mice. 8. PC-3 were transplanted into nude mice, and treated with pshRNA-GFP LVEGFRRNAi and paclitaxel or Gefitinib. The results showed that RNAi plus paclitaxel treatment can inhibit tumor growth with 56.8% tumor suppression. The tumor growth inhibition rate of the combination of Gefitinib and RNAi was as high as 54.0%. Compared to the control group, the group of RNAi plus Gefitinib and paclitaxel showed the highest effect against tumor with tumor growth inhibition rate as much as 63.5% (P<0.01) . The EGFR and Akt expression levels of the RNAi group or the RNAi plus Gefitinib group were decreased, but the MAPK levels were not affected.Conclusion: SiRNA-mediated inhibition of EGFR gene induced by shRNA expressing pshRNA-GFP LVEGFRRNAi is capable of suppressing EGF receptor expression with significantly inhibiting cellular proliferation and tumor growth due to cell cycle arrest and apoptosis induced. There was cooperation effect between pshRNA-GFP LVEGFRRNAi, paclitaxel and Gefitinib. RNAi targeting EGFR may constitute a useful approach in the treatment of human androgen-independent prostate cancer (AIPC).更多还原