FKBP51调控去势抵抗性前列腺癌形成的分子信号及机制研究

发布时间：2018-05-07 04:17

本文选题：去势抵抗性前列腺癌 + FKBP51　；参考：《天津医科大学》2016年博士论文

【摘要】：目的:检测在人前列腺组织及裸鼠人前列腺癌异种种植瘤中FKBP51的表达情况,人工构建雄激素非依赖性前列腺癌细胞LNCaP-AI,并动态检测去势抵抗过程中细胞内FKBP51基因表达的变化及其在CRPC发生所起的作用,初步探究FKBP51在CRPC发生过程中上下游信号通路的改变以及长链非编码RNA在其中发挥的作用,探索前列腺癌由ADPC转变为CRPC的可能机制。方法:利用免疫组织化学技术检测人前列腺组织及裸鼠人前列腺癌异种种植瘤中FKBP51的表达;持续使用无雄激素培养基培养LNCaP细胞,人工构建雄激素非依赖性前列腺癌细胞LNCaP-AI;通过western blot动态检测前列腺癌去势抵抗过程中FKBP51基因表达的变化及其在CRPC发生所起的作用,以及其相关的NF-κB信号通路与AKT信号通路蛋白的变化;利用在线分析软件catRAPID、Lnc RNA芯片、RNA免疫共沉淀实验分析前列腺癌去势抵抗过程中长链非编码RNA的表达变化情况及其参与FKBP51作用信号通路改变的可能机制。结果:1.FKBP51在人前列腺癌组织标本及裸鼠异种种植瘤标本中均有较高表达,且其表达在前列腺癌进展为去势抵抗时明显升高。2.LNCa P前列腺癌细胞在无雄激素条件下培养1个月,细胞生长速度明显放缓且大量死亡,培养3个月后细胞生长逐渐加快,成为雄激素非依赖的LNCaP-AI细胞亚系,并对抗雄药物比卡鲁胺及MDV3100产生耐药。LNCaP-AI细胞中FKBP51表达量明显升高,且敲除FKBP51后细胞生长受到抑制。3.在去雄培养LNCaP-AI细胞系过程中,AR-v7表达量逐渐增高,且可不依赖雄激素调控KFBP51的表达。在LNCaP-AI细胞中FKBP51主要与IKK结合参与NF-κB信号通路的调节,使caspase-3蛋白表达降低,BCL-2蛋白表达增高,从而抑制前列腺癌细胞的凋亡,而其对AKT信号通路无明显作用。4.在列腺癌去势抵抗过程中,多种长链非编码RNA的表达发生明显变化,其中PCAT-1表达量升高,进一步研究发现PCT-1可以通过与FKBP51结合,封闭FKBP51与PHLPP结合的位点,阻碍PHLPP对AKT的降磷酸化作用,从而影响FKBP51对下游的信号通路的调节。结论:本实验通过构建雄激素非依赖性前列腺癌细胞模型LNCaP-AI,证实FKBP51在去势抵抗性前列腺癌的发生发展中发挥重要作用,其通过与长链非编码RNA PCAT-1共同作用,调节NF-κB及AKT信号通路,促进前列腺癌细胞的生长并抑制其凋亡,从而促进去势抵抗性前列腺癌的发生。本研究提示FKBP51有可能成为治疗去势抵抗性前列腺癌的一个新的药物靶点。
[Abstract]:Objective: to detect the expression of FKBP51 in human prostate tissues and xenoimplants of human prostate cancer in nude mice. Androgen independent prostate cancer cell line LNCaP-AIwas constructed, and the changes of FKBP51 gene expression and its role in the pathogenesis of CRPC were dynamically detected during castration resistance. To explore the changes of upstream and downstream signaling pathways of FKBP51 in the pathogenesis of CRPC and the role of long chain non-coding RNA in the process, and to explore the possible mechanism of the transition of prostate cancer from ADPC to CRPC. Methods: immunohistochemical technique was used to detect the expression of FKBP51 in human prostate tissue and xenoimplant tumor of nude mice, androgen free medium was used to culture LNCaP cells. The androgen-independent prostate cancer cell line LNCaP-AIwas constructed, and the expression of FKBP51 gene and its role in the development of CRPC were dynamically detected by western blot during castration resistance of prostate cancer. The changes of NF- 魏 B signaling pathway and AKT signal pathway protein; The expression of long chain non-coding RNA during castration resistance of prostate cancer and its possible mechanism involved in the changes of signaling pathway of FKBP51 were analyzed by using the on-line analysis software catRIDD LNC RNA microarray and co-immunoprecipitation assay. Results 1. FKBP51 was highly expressed in both human prostate cancer tissues and xenoimplants of nude mice, and the expression of FKBP51 increased significantly when prostate cancer progressed to castration resistance. 2. LNCa P prostate cancer cells were cultured without androgen for 1 month. After 3 months of culture, the cells grew faster and became androgen independent LNCaP-AI cell sublines, and the expression of FKBP51 in the anti-androgen cells was significantly higher than that in Carous amine and MDV3100 resistant. LNCaP-AI cells. The cell growth was inhibited after knockout of FKBP51. The expression of AR-v7 in LNCaP-AI cell line was increased gradually, and the expression of KFBP51 was not regulated by androgen. In LNCaP-AI cells, FKBP51 and IKK are involved in the regulation of NF- 魏 B signaling pathway, which can decrease the expression of caspase-3 protein and increase the expression of BCL-2 protein, thus inhibit the apoptosis of prostate cancer cells, but it has no obvious effect on AKT signaling pathway. During ovariectomized resistance, the expression of long chain non-coding RNA was significantly changed, and the expression of PCAT-1 was increased. It was further found that PCT-1 could block the site of FKBP51 binding to PHLPP by binding to FKBP51. Blocking the dephosphorylation of AKT by PHLPP, thus affecting the regulation of downstream signal pathway by FKBP51. Conclusion: this study demonstrated that FKBP51 plays an important role in the development of castrated resistant prostate cancer through the establishment of androgen independent prostate cancer cell line LNCaP-AII. it works together with long chain noncoding RNA PCAT-1. Regulation of NF- 魏 B and AKT signaling pathway to promote the growth of prostate cancer cells and inhibit their apoptosis, thus promoting the development of castrated resistant prostate cancer. This study suggests that FKBP51 may be a new drug target for ovariectomized prostate cancer.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

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