KHDRBS家族对前列腺癌细胞表型及雄激素受体的作用研究

发布时间：2018-07-06 17:12

本文选题：前列腺癌 + 去势抵抗性前列腺癌　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究背景前列腺癌是男性生殖系统常见的恶性肿瘤,其发生发展与抑癌基因突变、DNA修复基因失活及原癌基因异常激活密切相关,有多种信号通路参与。基因的选择性剪接调控在前列腺癌中发挥重要作用,对于前列腺癌至关重要的雄激素受体(androgen receptor,AR)受剪接调控能够产生多种异构体,其中AR-V7和AR-V567es参与前列腺癌耐受内分泌治疗并进展为去势抵抗前列腺癌的过程。研究发现RNA结合蛋白KHDRBS1能够协助剪接小体识别AR外显子3b并完成剪接最终形成AR-V7,而且KHDRBS1异常促进包括前列腺癌在内的多种恶性肿瘤进展并与临床分期、病理分级和患者预后等临床指标正相关。KHDRBS1基因有两个同源且高度保守的亚家族成员,KHDRBS2和3,二者与KHDRBS1在RNA结合功能域KH域有70%～80%的氨基酸序列一致,并且与KHDRBS1存在交互作用,同时与乳腺癌、肾癌等也有关联。但KHDRBS2和3在前列腺癌中的作用研究仍属空白。本课题中我们对KHDRBS家族在PCa进展中的作用,特别是KHDRBS2和3对前列腺癌细胞的作用以及与AR及AR剪接异构体的关系进行相关探讨,以增加对KHDRBS家族的认识,丰富前列腺癌发生进展的分子机制研究。研究目的1.明确KHDRBS家族基因在前列腺癌细胞系中的表达水平;2.明确KHDRBS2和3在前列腺癌细胞增殖、迁移、侵袭及细胞周期中的作用;3.明确KHDRBS2和3间的相互关系;4.明确KHDRBS家族与AR、AR-V7和AR-V567es的关系。研究方法和结果一、KHDRBS家族在前列腺癌中的生物信息学分析及在前列腺癌细胞系中的表达验证方法:1)应用cBioPortal在线工具对来自于TCGA等11个研究计划的前列腺癌样本集合的数据进行分析,总结KHDRBS家族基因在样本集合中基因改变的比例、表达水平以及与总生存及无病生存期的关系;2)应用Real-time PCR检测前列腺癌细胞系22RV1、LnCap、DU145和PC3细胞mRNA表达水平。结果:1)基于cBioPortal调用的11个样本集合,与KHDRBS1相比,KHDRBS2和3在PCa样本集合中的基因改变发生更为广泛,并且KHDRBS3能够影响样本集合中患者的总生存,提示KHDRBS2和3可能与PCa发生发展相关。2)样本均为去势抵抗性前列腺癌及去势抵抗性神经内分泌前列腺癌的NEPC样本集合中,KHDRBS1,2和3基因发生改变的样本比例最高,提示KHDRBS家族可能与去势抵抗性前列腺癌进展有关。3)K KHDRBS1在22RV1、LnCap、DU145和PC3中均有表达。22RV1细胞中KHDRBS2高表达,KHDRBS3低表达;而PC3细胞中KHDRBS2低表达,KHDRBS3高表达。提示二者的表达具有细胞类型特异性。二、KHDRBS2和3对前列腺癌细胞的作用方法:1)应用瞬时转染过表达及慢病毒转染敲减改变KHDRBS2和3在22RV1和PC3细胞中的表达水平,并应用Muse细胞仪、Transwell法及CCK-8法和体内成瘤实验分别检测细胞的细胞周期、迁移、侵袭及体外和体内的增殖能力。2)同前法改变KHDRBS2和3在22RV1和PC3细胞中的表达水平,应用Real-time PCR及Western-Blot检测对应细胞中KHDRBS2和3的变化,明确KHDRBS2和3在前列腺癌细胞中的关系。结果:1)瞬时过表达能够成功构建高表达KHDRBS2和3的PC3和22RV1细胞模型;2)慢病毒转染敲减KHDRBS2或3能够成功构建低表达KHDRBS2的22RV1或低表达KHDRBS3的PC3细胞模型;3)敲减KHDRBS2能够抑制22RV1细胞的增殖能力,而敲减KHDRBS3能够抑制PC3细胞的增殖能力;4)过表达或敲减KHDRBS2后KHDRBS3在mRNA和蛋白水平的表达发生降低或增高改变,而过表达或敲减KHDRBS3之后KHDRBS2的表达水平也发生降低或增高,但改变KHDRBS2或3的表达对KHDRBS 1的表达水平则无影响。三、KHDRBS家族与雄激素受体的关系方法:1)应用 Real-time PCR 检测 22RV1、LnCap、DU145 和 PC3 细胞 AR-FL、AR-V7和AR-V567es的表达水平。2)应用Real-time PCR检测有无雄激素及AR拮抗剂的条件下,22RV1细胞AR-FL、AR-V7和AR-V567es及22RV1和PC3细胞中KHDRBS1,2和3的表达水平。3)应用Real-time PCR检测过表达KHDRBS2和3及敲减KHDRBS2的22RV1细胞AR-FL、AR-V7和AR-V567es的表达水平。4)在有无雄激素的条件下,应用Real-time PCR检测过表达KHDRBS2和3的22RV1细胞中KHDRBS2和3的mRNA表达变化以及敲减KHDRBS2的22RV1细胞中KHDRBS2、KHDRBS3、AR 和 AR-V7 的 mRNA 表达变化。结果:1)22RV1细胞中AR-FL、AR-V7和AR-V567es高表达;而在PC3细胞中三种AR异构体均低表达。2)无雄激素条件下22RV1细胞中AR、AR-V7和AR-V567es及KHDRBS1和2表达水平较正常培养组显著升高,具有统计学意义,KHDRBS3虽有升高趋势,但无统计学意义;在PC3中,同样处理对于KHDRBS家族基因的表达无影响;3)敲减KHDRBS2可显著降低22RV1细胞中AR-V7的表达水平,P值为0.0168。4)过表达KHDRBS2和3以及敲减KHDRBS2的22RV1细胞,无论是否添加雄激素,KHDRBS2、KHDRBS3、AR和AR-V7的mRNA表达变化与未做过表达和敲减的22RV1细胞对雄激素的反应一致。研究结论1.cBioPortal分析前列腺癌样本提示KHDRBS2和3可能在前列腺癌及去势抵抗性前列腺癌发生进展中具有重要作用。2.在22RV1和PC3细胞,KHDRBS2和3的表达具有细胞类型特异性并与AR及其异构体表达表现出相关性。3.分别敲减KHDRBS2或3能够抑制22RV1或PC3细胞的增殖能力。4.在22RV1和PC3细胞中,KHDRBS2和3呈现交互调节的关系。5.去除雄激素导致的KHDRBS1和2表达上调可能是由AR-FL介导的。敲减KHDRBS2可以抑制22RV1细胞AR-V7的表达。KHDRBS1和2可能是AR-FL与AR-V7之间的调控因素。
[Abstract]:Background prostate cancer is a common malignant tumor in the male reproductive system. Its development is closely related to the mutation of the tumor suppressor gene, the inactivation of the DNA repair gene and the abnormal activation of the proto oncogene. There are a variety of signaling pathways involved. Androgen receptor (AR) is regulated by splicing and can produce a variety of isomers, in which AR-V7 and AR-V567es are involved in prostate cancer tolerance and endocrine therapy and are progressing to the process of prostatic cancer. The study found that RNA binding protein KHDRBS1 can assist splicing corpuscles to identify 3B in AR exons and complete the splicing and eventually form AR-V7, and K. HDRBS1 abnormalities promote the progression of a variety of malignant tumors, including prostate cancer, and clinical staging, pathological grading, and patient prognosis. The.KHDRBS1 gene has two homologous and highly conserved subfamily members, KHDRBS2 and 3, and two and KHDRBS1 are consistent with the amino acid sequences of 70% to 80% in the KH domain of RNA binding domain, and There is a interaction with KHDRBS1, which is also associated with breast and renal cancer. But the role of KHDRBS2 and 3 in prostate cancer is still a blank. In this subject, we discuss the role of the KHDRBS family in the progression of PCa, especially the role of KHDRBS2 and 3 on prostate cancer cells and the relationship between AR and AR splicing isomers. To increase the understanding of the KHDRBS family and to enrich the molecular mechanism of the progression of prostate cancer. Purpose 1. to clarify the expression level of the KHDRBS family in the prostate cancer cell lines; 2. to clarify the role of KHDRBS2 and 3 in the proliferation, migration, invasion and cell cycle of prostate cancer cells; 3. clear the relationship between KHDRBS2 and 3, and the correlation between the 3 and the KHDRBS2. The relationship between the KHDRBS family and the AR, AR-V7 and AR-V567es. Research methods and results 1. Bioinformatics analysis of the KHDRBS family in prostate cancer and the expression in the prostate cancer cell lines: 1) use the cBioPortal online tool to analyze the data of the collection of prostate cancer samples from the 11 research projects, such as TCGA. The ratio of gene change, expression level and relationship with total survival and disease-free survival in the collection of KHDRBS family genes; 2) detection of mRNA expression levels in prostate cancer cell lines 22RV1, LnCap, DU145 and PC3 cells using Real-time PCR. Results: 1) based on the 11 sample sets used for cBioPortal modulation, KHDRBS2 and 3 were compared to KHDRBS1, KHDRBS2 and 3 were PCa The genetic changes in the sample set occur more widely, and KHDRBS3 can affect the total survival of the patients in the sample collection, suggesting that KHDRBS2 and 3 may be associated with the development of.2 associated with the development of PCa. All of the samples are in the set of NEPC samples of the castrated and castrated neuroendocrine prostate cancer, and the KHDRBS1,2 and the 3 genes have changed. The proportion of the samples was the highest, suggesting that the KHDRBS family might be associated with the progression of prostatic cancer in.3). K KHDRBS1 expressed KHDRBS2 high expression in.22RV1 cells in 22RV1, LnCap, DU145 and PC3, while PC3 cells expressed low expression and expressed high expression. The expression of the two was specific to cell type. Two 3 methods of action on prostate cancer cells: 1) the expression level of KHDRBS2 and 3 in 22RV1 and PC3 cells was altered by transient transfection and lentivirus transfection, and the cell cycle, migration, invasion and proliferation of.2 in vitro and in vivo were detected by Muse cytometer, Transwell and CCK-8 and in vivo tumorigenesis. The expression level of KHDRBS2 and 3 in 22RV1 and PC3 cells was changed by the same method. Real-time PCR and Western-Blot were used to detect the changes of KHDRBS2 and 3 in the corresponding cells, and the relationship between KHDRBS2 and 3 in prostate cancer cells. Results: 1) transient overexpression could successfully construct PC3 and 22RV1 cell models with high expression of KHDRBS2 and 3; 2) transfection of lentivirus Knockout KHDRBS2 or 3 can successfully construct a PC3 cell model with low expression of KHDRBS2 22RV1 or low expression KHDRBS3; 3) knock down KHDRBS2 can inhibit the proliferation of 22RV1 cells, while knockout KHDRBS3 can inhibit the proliferation of PC3 cells; 4) the expression of overexpression or knockout KHDRBS2 decreases or increases in mRNA and protein levels. The expression level of KHDRBS2 was also reduced or increased after overexpression or subtraction of KHDRBS3, but the expression of KHDRBS2 or 3 had no effect on the expression level of KHDRBS 1. Three, the relationship between the KHDRBS family and androgen receptor: 1) the use of Real-time PCR to detect 22RV1, LnCap, DU145 and PC3 cells, and the expression of water. In the presence of androgen and AR antagonists, 22RV1 cells AR-FL, 22RV1 cells AR-FL, AR-V7 and AR-V567es, and KHDRBS1,2 and 3 expression level.3 in 22RV1 and PC3 cells were detected by Real-time PCR. Under conditions, Real-time PCR was used to detect the mRNA expression of KHDRBS2 and 3 in KHDRBS2 and 3 22RV1 cells and the expression of KHDRBS2, KHDRBS3, AR and AR-V7 in the 22RV1 cells that knocked down KHDRBS2. The expression level of AR, AR-V7, AR-V567es, KHDRBS1 and 2 in the 22RV1 cells was significantly higher than that in the normal culture group, with statistical significance, although KHDRBS3 had a tendency to increase, but there was no statistical significance. In PC3, the same treatment had no effect on the expression of KHDRBS family gene; 3) reducing KHDRBS2 significantly reduced the AR-V7 table in 22RV1 cells. Level, P value is 0.0168.4) over expression of KHDRBS2 and 3, and KHDRBS2 knockout 22RV1 cells, no matter whether or not adding androgens, KHDRBS2, KHDRBS3, AR and AR-V7, the expression of mRNA is consistent with that of the 22RV1 cells that have not been expressed and knocked down. The progression of adenocarcinoma and castration resistant prostate cancer has an important role in the development of.2. in 22RV1 and PC3 cells, KHDRBS2 and 3 expression has cell type specificity and the expression of AR and its isomers expresses a correlation of.3., KHDRBS2 or 3 can inhibit 22RV1 or PC3 cell proliferation.4. in 22RV1 and PC3 cells, and 3 The relationship between the expression of.5. and the up-regulated expression of KHDRBS1 and 2 may be mediated by AR-FL. Knockout KHDRBS2 can inhibit the expression of AR-V7 in 22RV1 cells,.KHDRBS1 and 2 may be a regulatory factor between AR-FL and AR-V7.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

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