TR4/LincRNA-p21通路对去势抵抗前列腺癌放疗敏感性影响及机制研究

Prostate cancer (PCa) is a rapidly increasing cause of death in senior men in China and costs a lot of money. Radiation therapy (RT) is one of the major therapies for PCa and has been proved to be safe and effective in the past decades. But when PCa develops to castration resistant stage (CRPC), it is refractory to almost all current treatments available. The Testicular Orphan Receptor 4 (TR4) is a member of the nuclear receptor superfamily that mediates various biological activities including development, spermatogenic function, and female fertility. Recent reports indicate that TR4 has a protective role against oxidative stress such as ultra violet (UV) and chemotherapy. Previous studies found that TR4 promotes invasion and chemo-resistance of PCa. This induced our interest that whether TR4 also has a protective role in the RT of PCa. Our preliminary data showed that TR4 protected PCa C4-2 cells from RT by its anti-apoptosis and pro-proliferation effects, and this phenotype linked to the inhibition of LincRNA-p21. Knocking down TR4 by siRNA could restore radiation sensitivity of PCa C4-2 cells and increase the expression of LincRNA-p21 without changing the mRNA or protein level of p53, which is the up-stream of LincRNA-p21. In this study, we will investigate the expression of TR4 and LincRNA-p21 in PCa tissues that have different radiation sensitivity. We will examine the protecting function of TR4 to RT in multiple CRPC cell lines. We will further dissect the mechanism how TR4 affect p53/LincRNA-p21 signaling pathway. Also we will use subcutaneous transplanted tumor radiation model in nude mice to further confirm our hypothesis. Our findings may provide new therapeutic target for the treatment of advanced PCa.

放射治疗是前列腺癌主要治疗手段之一，但当从激素敏感性前列腺癌发展为去势抵抗性前列腺癌时，肿瘤对各种治疗不敏感。睾丸孤核受体4 (Testicular Nuclear Receptor 4, TR4)与前列腺癌的侵袭转移、化疗耐药等密切相关。我们的初步研究发现RNA干扰沉默TR4后前列腺癌C4-2细胞放疗敏感性增加，表现为增殖能力降低、凋亡增加，但其作用机制尚不明确。本研究将检测前列腺癌临床标本中TR4与放疗敏感性的相关性；在多个去势抵抗前列腺癌细胞系中，敲减/过表达TR4，研究TR4对前列腺癌放疗后增殖、凋亡的影响；再使用TR4稳定敲减/过表达的前列腺癌细胞系建立裸鼠移植瘤模型，给予放射治疗，进一步研究TR4对放疗敏感性的影响；并探讨TR4是否通过与p53竞争性结合LincRNA-p21启动子，从而对LincRNA-p21及下游信号通路进行调控实现对前列腺癌放疗后增殖、凋亡的影响。

抑制TR4可使前列腺癌细胞放疗敏感性增加，但其作用机制尚不明确。我们进行了如下研究：(1)敲减TR4对去势抵抗前列腺癌细胞放疗敏感性的影响；(2)过表达lincRNA-p21对去势抵抗前列腺癌细胞放疗敏感性的影响；(3)检测沉默TR4所引起的放疗敏感性增加是否可部分被同时敲减lincRNA-p21所回复；(4)前列腺癌细胞沉默TR4后lincRNA-p21、BAX、NOXA表达的变化；(5)检测沉默TR4所引起的NOXA、BAX蛋白的增加增加是否可部分被同时敲减lincRNA-p21所回复；(6)检测敲减TR4引起的lincRNA-p21表达升高能否被同时敲减P53所抑制；(7)染色质免疫共沉淀及荧光素酶报告基因验证TR4与p53对lincRNA-p21启动子区的竞争性调控关系；(8)构建动物模型，体内水平验证；(9)除了TR4/lincRNA-p21通路，还研究了TR4通过miR-212影响前列腺癌放疗敏感性的机制。结果：(1)不同CRPC细胞敲减TR4后放疗效果增强；(2)CRPC细胞过表达lincRNA-p21后放疗效果增强；(3)沉默TR4所引起的放疗敏感性增加可部分被同时敲减lincRNA-p21所回复；(4)沉默TR4后lincRNA-p21、BAX、NOXA表达增加；(5)同时沉默TR4和lincRNA-p21，发现沉默所引起的NOXA、BAX的增加可部分被同时敲减lincRNA-p21所回复；(6)同时敲减TR4和P53，结果显示敲减TR4引起的lincRNA-p21表达升高能够被敲减P53所抑制；(7)染色质免疫共沉淀证实TR4与p53对lincRNA-p21启动子区的竞争性结合；(8)荧光素酶报告基因证实TR4对lincRNA-p21启动子活性的调控；(9)通过裸鼠移植瘤模型，在体内水平得到了验证。以上研究结果证实，TR4与p53对lincRNA-p21竞争性转录调控，最终引起放疗抵抗，圆满完成了课题任务。此外，我们还研究发现TR4通过促进miR-212启动子区甲基化抑制miR-212，而miR-212通过直接靶向BRCA1抑制其表达，最终影响前列腺癌放疗敏感性。另外我们研究发现一个受TR4调控的基因EZH2，通过共表达基因网络，影响前列腺癌放疗敏感性，并实验验证了共表达网络中一个基因TROAP，通过EZH2/TROAP通路，促进前列腺癌进展。