【作者】 王涛；

【导师】 赵善超；

【作者基本信息】 南方医科大学 ， 外科学（泌尿外）（专业学位）， 2024， 博士

【摘要】 研究背景和目的前列腺癌(Prostate Cancer,PCa)早期表现没有特异性,患者就诊时多已经进展到晚期。无缺口同系物1(Notchless homolog 1,NLE1)编码含有WD-repeat的蛋白家族成员,参与细胞骨架组装、细胞分裂、转录调节等。NLE1在结肠癌、肺癌、黑色素瘤等多种肿瘤中表达上调,可促进肿瘤细胞增殖、迁移、抑制细胞凋亡等。但NLE1在前列腺癌中的研究报道甚少,本研究将深入探究NLE1在前列腺癌组织和细胞中的表达、功能、机制,以阐明其在前列腺癌进展中的作用。研究内容和方法1、免疫组化分析前列腺癌组织芯片(含原发前列腺癌组织80例、癌旁组织8例及正常前列腺组织8例)中NLE1表达,评估组化评分与前列腺癌临床病理参数的相关性。2、通过慢病毒载体转染,稳定干扰NLE1表达的PCa细胞株DU145、PC3,以及过表达NLE1的PCa细胞株DU145构建成功,通过细胞增殖、划痕愈合实验、Transwell细胞迁移实验、裸鼠皮下移植瘤模型等实验在体内、体外水平分别探究其对PCa细胞增殖和迁移的影响。3、Affymetrix表达谱芯片检测、筛选NLE1下游效应分子,利用实时定量聚合酶链反应和蛋白质印迹测定验证下游效应分子CD44的表达水平。4、免疫共沉淀验证NLE1与CD44之间的互作关系。构建稳定敲低CD44的PCa细胞株DU145,通过细胞增殖、划痕愈合实验、Transwell细胞迁移实验等探究其对PCa细胞增殖和迁移的影响。5、人类细胞凋亡抗体阵列(Human Apoptosis Antibody Array)检测沉默NLE1后凋亡相关蛋白的表达变化。研究结果免疫组化显示前列腺癌组织中NLE1的表达较癌旁及正常前列腺组织明显升高(P<0.05);细胞表型实验证实干扰DU145及PC3中NLE1的表达后,细胞的增殖、迁移能力显著受抑制,细胞周期阻滞于G2/M期;裸鼠皮下成瘤实验证实干扰NLE1能够显著抑制移植瘤的生长速度和成瘤重量、体积;小动物活体成像数据分析显示干扰NLE1组荧光表达量明显降低(P<0.05);定量PCR结果显示,PC3感染慢病毒后,相对于对照组,干扰NLE1组中表达下调的基因有CD44、UAP1、MAT2A、SLC25A24、PLAU 和 CLEC2D(P<0.05);Western Blot证实干扰NLE1组CD44、PLAU、MAT2A蛋白表达下调。CO-IP证实NLE1蛋白与CD44蛋白之间相互结合。细胞表型实验证实在DU145中干扰CD44的表达后,细胞的增殖、迁移能力明显下降,凋亡细胞数量明显增多。机制方面,干扰NLE1后PI3K/AKT通路中p-Akt的表达明显下调,Bax、Fas表达上调,而Survivin的表达下调。研究结论1.NLE1在前列腺癌中为促癌基因,高表达NLE1促进前列腺癌的进展。2.细胞实验及动物实验证实了干扰NLE1能够抑制前列腺癌细胞的增殖、减弱前列腺癌细胞的迁移能力、诱导细胞周期阻滞。3.NLE1蛋白通过与CD44蛋白互作,发挥促进前列腺癌进展的作用,给精准治疗前列腺癌提供了新的潜在分子靶点。更多还原

【Abstract】 Background and purposeThe early manifestations of prostate cancer are nonspecific.Patients are often at an advanced stage by the time they present.Notchless homolog 1(NLE1)encodes members of a family of proteins containing WD-repeat that are involved in a variety of cellular functions,such as cytoskeletal assembly,cell division,transcriptional regulation.Previous studies have reported that NLE1 is up-regulated in a variety of tumors,including colon cancer,lung cancer,melanoma and other tumors,which can promote the occurrence and development of tumors by promoting cancer cell proliferation,cancer cell migration,and inhibiting cell apoptosis.But studies of NLEl in prostate cancer have been poorly reported.This project will explore the expression,function and mechanism of NLE1 in prostate cancer tissues and cells,in order to clarify its role in prostate cancer progression and metastasis.Methods1.The expression level of NLEl in prostate cancer microarray(including 80 cases of primary prostate cancer,8 cases of para-cancerous tissue and 8 cases of normal prostate tissue)was analyzed by immunohistochemical study,and the connection between NLE1 expression level and clinicopathological parameters of prostate cancer was investigated.2.Prostate cancer cell lines DU 145 and PC3 with stable interference of NLE1 expression,and prostate cancer cell line DU 145 with overexpression of NLE1 were constructed by lentiviral vector transfection.The influence on the proliferation and migration of prostate cancer cells were detected in vivo and in vitro through cell proliferation,scratch healing experiment,transwell migration assay,and subcutaneous tumor formation in nude mice.3.Affymetrix expression profile chip was used to detect and screen downstream effector molecules of NLE1,and Quantitative real-time polymerase chain reaction,qRT-PCR and Western blot were used to verify the expression level of downstream effector CD44.4.Co-Immunoprecipitation(CO-IP)experiment was used to further verify the interaction between NLE1 and CD44.A stable CD44 knockdown prostate cancer cell line was constructed,and its effects on the proliferation and migration of prostate cancer cells were investigated by cell proliferation,scratch healing and Transwell cell migration experiments.5.The Human Apoptosis Antibody Array was designed to explore the changes in the expression of apoptotic proteins after the silencing of NLE1.ResultsImmunohistochemical staining of prostate cancer tissue microarray showed that NLE1 score in prostate cancer tissue was obviously higher than which in peripheral and normal prostate tissue(P<0.05).The proliferation,and migration abilities of PC3 and DU145 cells were obviously decreased after silencing NLE1 expression,and the cell cycle was arrested in G2/M phase.Mice xenograft model indicated that shNLE1 could obviously decrease the tumor size and growth rate of DU145 xenograft tumors.Small animal in vivo imaging data analysis showed that compared with shCtrl group,fluorescent expression quantity shNLEl group was obviously reduced(P<0.05).Quantitative PCR results showed that after lentivirus infection in PC3 cells,compared with shCtrl group,down-regulated genes in shNLE1 group were CD44、UAP1、MAT2A、SLC25A24、PLAU and CLEC2D(P<0.05);Western Blot results showed that compared with the shCtrl group,the shNLE1 group had significantly decreased expression of CD44、PLAU and MAT2A.The interaction between NLE1 protein and CD44 protein was confirmed by co-immunoprecipitation(CO-IP)assay.Cell function assays indiacated that the proliferation and migration ability of DU145 were significantly decreased after silencing the expression of CD44,and the number of apoptotic cells increased significantly.Mechanistically,we found that the expression of p-Akt,the core molecule of PI3K/AKT pathway,was significantly down-regulated after shNLEl treatment,while the expression of apoptosis-related proteins Bax and Fas were up-regulated,and Survivin was obviously down-regulated.Conclusion1.NLE1 is an oncogenic gene in prostate cancer,and high expression of NLE1 promotes the progression of prostate cancer.2.NLE1 knockdown significantly hinders proliferation,inhibit the migration of prostate cancer cells,and induce cell cycle arrest in vivo and in vitro functional experiments.3.The interaction between NLE1 protein and CD44 protein plays a role in promoting the progression of prostate cancer,which provides potential new molecular targets for the precision treatment of prostate cancer.更多还原