【作者】 张博雅；

【导师】 罗军；

【作者基本信息】 天津医科大学 ， 外科学 泌尿外， 2021， 博士

【摘要】 前列腺癌(Prostate cancer,PCa)在全世界范围内的发病率逐年递增,近年来成为威胁男性健康的重要疾病之一。去势抵抗性前列腺癌(Castration-resistant prostate cancer,CRPC)是其中高度恶性的进展期前列腺癌,对雄激素剥夺疗法(Androgen deprivation therapy,ADT)具有抵抗力。大部分前列腺癌导致的患者死亡也均发生在去势抵抗性前列腺癌阶段。去势抵抗性前列腺癌进展的重要机制之一是其雄激素受体(Androgen receptor,AR)或雄激素受体剪接变体(Androgen-receptor splicing variants,AR-Vs)的持续异常调节,因此探寻如何逆转或抑制雄激素受体或其剪接变异体在去势抵抗性前列腺癌中的持续活跃状态,对前列腺癌的治疗至关重要。目前,诸如比卡鲁胺(Bicalutamide),恩杂鲁胺(Enzalutamide)以及阿比特龙(Abiraterone)等针对雄激素受体信号通路的药物被证明能够有效的延长患者的总体生存率(Overall survival,OS)以及无病生存率(Disease-free survival,DFS)。以这些药物为主的雄激素剥夺疗法,虽然能够有效的降低患者PSA,抑制疾病进展,但有效期往往只有18-24个月。在此之后,肿瘤通常会表现出对于这些药物的耐药性,并且疾病的进展加速,因此,寻找新的治疗靶点变得尤为重要。同时目前的药物多仅限于雄激素受体或者雄激素受体剪切变异体,并无同时抑制两者的方式,单一的抑制在前列腺癌的治疗中效果有限,因此探寻能够同时抑制雄激素受体以及其剪切变异体的治疗方式也显得迫在眉睫。本研究旨在探索近年来新发现的长链非编码RNA KDM4A-AS1作为去势抵抗性前列腺癌的促癌基因,通过抑制雄激素受体AR及其剪切变异体AR-V7和AR-V567es促进去势抵抗性前列腺癌进展的机制,同时针对这项机制探索KDM4A-AS1在去势抵抗性前列腺癌恩杂鲁胺耐药中所起到的作用,并评估其作为治疗靶点的价值。为去势抵抗性前列腺癌的治疗创新提供理论支持。目的:1:在体内外探明KDM4A-AS1在去势抵抗性前列腺癌中的作用机制。2:探明KDM4A-AS1与恩杂鲁胺耐药之间的关联,验证靶向KDM4A-AS1在体内、体外水平治疗前列腺癌的效果。方法:1:通过RNAScope、高通量微阵列芯片以及qPCR的方法确定KDM4A-AS1在不同阶段前列腺癌患者以及前列腺癌细胞系中的表达,并分析其特点。2:通过体外慢病毒敲低KDM4A-AS1,利用Transwell、克隆形成以及MTT等功能学实验确定敲低KDM4A-AS1对前列腺癌的影响。3:通过生物信息学预测、ITRAQ质谱分析、RIP、RNA-Pulldown、免疫组化、Western-Blot等方法验证KDM4A-AS1-USP14-AR/AR-Vs复合体的存在,并明确KDM4A-AS1通过该复合体对雄激素受体以及其剪切变异体的调控。4:构建耐药细胞系以及相应的裸鼠皮下移植瘤模型,通过测量小鼠肿瘤体积、生长速率,并利用RNAScope、q PCR、Western-Blot以及免疫组化在体内水平证实靶向KDM4A-AS1对前列腺癌的治疗效果。结果:1:KDM4A-AS1作为前列腺恶性肿瘤的促进因子,在去势抵抗性前列腺癌细胞系LNCa P-AI、C4-2以及临床去势抵抗性前列腺癌患者组织中的表达显著上调;2:通过在LNCa P-AI以及C4-2细胞系中使用包含靶向抑制KDM4A-AS1序列的短发夹RNA,我们发现KDM4A-AS1的耗竭显着降低了LNCa P-AI以及C4-2细胞活力、细胞增殖能力,细胞体外迁移能力和体内肿瘤生长速度;3:我们发现通过结合雄激素受体蛋白的N端转录激活区域(AR-NTD),长链非编码RNA KDM4A-AS1显著的增强了去泛素化酶USP14-雄激素受体或雄激素受体剪切变异体复合物的稳定性,并促进了雄激素受体或雄激素受体剪切变异体的去泛素化作用,从而保护其免受E3泛素连接酶MDM2介导的泛素-蛋白酶体途径降解的影响;4:成功构建耐药细胞系以及裸鼠模型,发现单纯靶向抑制KDM4A-AS1可以显著的抑制肿瘤的生长,而通过联合使用靶向KDM4A-AS1的短发夹RNA以及恩杂鲁胺,可以显著的恢复肿瘤对恩杂鲁胺的敏感性,使肿瘤细胞被杀伤。结论:1:KDM4A-AS1在去势抵抗性前列腺癌中显著上调。2:KDM4A-AS1通过稳定USP14-AR/AR-Vs复合体促进去势抵抗性前列腺癌的进展与恩杂鲁胺的耐药,靶向KDM4A-AS1可以显著的在体内外恢复耐药细胞对恩杂鲁胺的敏感性,对于临床前列腺癌患者预后的提升具有极大的潜力。更多还原

【Abstract】 The incidence of prostate cancer(PCa)is increasing year by year,and has become one of the important diseases threatening men’s health.Castration-resistant prostate cancer(CRPC)is a highly malignant advanced prostate cancer,which is resistant to androgen deprivation therapy(ADT)and most deaths caused by prostate cancer also occur in this stage.One of the important mechanisms for the progression of CRPC is the continuous abnormal regulation of its androgen receptor(AR)or androgen-receptor splicing variants(AR-Vs).Therefore,find the way to reverse or inhibit the abnormal status of AR/AR-Vs in CRPC become important.Currently,drugs targeting the AR signaling pathway,such as Bicalutamide,Enzalutamide and Abiraterone,have been shown to be effective in prolonging the overall survival(OS)and disease-free survival(DFS).ADT based on these drugs can effectively reduce patients’ PSA and inhibit disease progression,but the effective period is often only 18-24 months.After that,tumors usually show resistance to these drugs.Therefore,finding new therapeutic targets becomes particularly important.Single inhibition has limited effect in the treatment of prostate cancer,it is urgent to find a treatment that can inhibit both AR and AR-V7.This study aims to explore the mechanism by which the newly discovered long non-coding RNA KDM4A-AS1 in recent years promotes the progression of CRPC by inhibiting the AR and its splicing variants AR-V7/AR-V567 es,and explore the role of KDM4A-AS1 in CRPC enzalutamide resistance,and evaluate its value as a therapeutic target,which may provide theoretical support for the treatment of castration-resistant prostate cancer.Objective: 1: Explore the mechanism of KDM4A-AS1 in CRPC in vivo and in vitro.2: Explore the association between KDM4A-AS1 and enzalutamide resistance,and verify the effect of targeting KDM4A-AS1 in the treatment of prostate cancer in vivo and in vitro.Methods: 1: Determine the expression of KDM4A-AS1 in prostate cancer patients and prostate cancer cell lines at different stages by RNAScope,high-throughput microarray chip and q PCR,and analyze its characteristics.2: KDM4A-AS1 was knocked down by in vitro lentivirus,and functional experiments such as Transwell,clone formation and MTT were used to determine the effect of knocking down KDM4A-AS1 on prostate cancer.3: Verify the existence of KDM4A-AS1-USP14-AR/AR-Vs complex through bioinformatics,ITRAQ mass spectrometry,RIP,RNA-Pulldown,immunohistochemistry,Western-Blot,and determine the role of KDM4A-AS1 in AR/AR-Vs regulation;4: Construct enzalutamide-resistant cell lines and the corresponding nude mouse subcutaneous xenograft model,measure the mouse tumor volume and growth rate,and use RNAScope,q PCR,Western-Blot and immunohistochemistry to confirm the effect of KDM4A-AS1 in drug resistant.Results: 1: KDM4A-AS1 was significantly up-regulated in CRPC cell lines LNCa P-AI,C4-2,and CRPC patients;2: The depletion of KDM4A-AS1 significantly reduced LNCa P-AI and C4-2 cell viability and cell proliferation.Cell migration ability in vitro and tumor growth in vivo;3: We found that by binding to the N-terminal transcriptional activation region of the androgen receptor protein(AR-NTD),KDM4A-AS1 significantly enhanced the stability of USP14-AR/AR-Vs complex,and promote the deubiquitination of AR/AR-Vs,thereby protecting it from MDM2 mediated degradation of the ubiquitin-proteasome pathway;4: Successfully constructed drug-resistant cell lines and nude mouse models,and found that simply targeting inhibition of KDM4A-AS1 can significantly inhibit tumor growth.Using short hairpin RNA targeting KDM4A-AS1 and enzalutamide can significantly restore the sensitivity of tumors to enzalutamide and kill tumor cells.Conclusion: 1: KDM4A-AS1 is significantly up-regulated in CRPC.2: KDM4A-AS1 promotes the progression of CRPC and resistance to enzalutamide by stabilizing the USP14-AR/AR-Vs complex.Targeting KDM4A-AS1 can significantly restore drug-resistant in vivo and in vitro.The sensitivity of enzalutamide has great potential for improving the prognosis of clinical prostate cancer patients.更多还原