DNMT1调控CRMP4启动子甲基化在预测前列腺癌GS升级的机制研究

Prostate biopsy is the gold standard of prostate cancer (PCa). The difference in PCa multifocal and inter-doctoral scores resulted in approximately 35% of patients with GS escalation after surgery, which seriously affected preoperative pathological grade and treatment options. Our team reported that CRMP4 is a suppressor gene of PCa metastasis, verified that methylation is closely related to PCa lymph node metastasis, and found 34% of 966 PCa patients with GS escalation and poor prognosis. We preliminarily demonstrated that RelA/p65 can recruit DNMT1 to participate in CRMP4 methylation using CoIP, resulting in histone H3K27 and H3K9 methylation activates CRMP4 transcription. In this study, the CRMP4 promoter will be modified by site-directed methylation, and the malignant evolution of PCa by CRMP4 via upstream E2F1/NF-κB/DNMT1 and downstream Sema3B/NP2/VEGFC pathways will be verified in vitro and in vivo. We originally proposed that CRMP4 is related to GS upgrade by CRMP4 and DNMT1-mediated PCa de novo DNA methylation, providing a reliable basis for the establishment of accurate pathological grades and GS upgrade prediction markers in patients with PCa before surgery, guiding PCa patients to "individualized" treatment.

前列腺穿刺活检是诊断前列腺癌（Prostate cancer, PCa）金标准。PCa多灶性及医生间评分差异致约35%患者在术后出现GS升级，严重影响术前病理分级及治疗方案选择。本团队报道CRMP4为PCa转移抑制基因，申请人验证其甲基化与PCa淋巴结转移密切相关，并报道966例PCa患者中34%出现GS升级且预后较差。预实验用CoIP验证RelA/p65能募集DNMT1参与CRMP4甲基化，从而导致组蛋白H3K27,H3K9甲基化使CRMP4转录激活。本研究拟采用定点甲基化对CRMP4启动子进行修饰，体内外验证CRMP4通过上游E2F1/NF-κB/DNMT1及下游Sema3B/NP2/VEGFC通路调控GS升级分子机制。原创性提出并阐明CRMP4与DNMT1如何介导PCa起始性DNA甲基化与GS升级的相关性。为PCa患者建立GS升级预测标记物提供可靠依据，指导PCa患者“个体化”治疗。

前列腺癌（Prostate cancer, PCa）在术后出现GS升级会严重影响术前病理分级及治疗方案选择。本团队前期报道966例PCa患者中34%出现GS升级且预后较差，并发现CRMP4甲基化与PCa淋巴结转移密切相关，预实验用CoIP验证RelA/p65能募集DNMT1参与CRMP4甲基化，从而导致组蛋白H3K27,H3K9甲基化使CRMP4转录激活。本研究采用定点甲基化对CRMP4启动子进行修饰，体内外验证CRMP4通过上游E2F1/NF-κB/DNMT1及下游Sema3B/NP2/VEGFC通路调控GS升级分子机制。原创性提出并阐明CRMP4与DNMT1如何介导PCa起始性DNA甲基化与GS升级的相关性。并开发了基于CRMP4启动子甲基化的分类器， 同时开展了在低危PCa多中心临床研究，在训练集中，当CRMP4分类器≥8时，预测GS升级的敏感性、特异性、AUC及HR分别为85.19%,95.24%, [AUC] 0.90[95% CI 0.86-0.95)] 和 [HR]1.81[95% CI 1.54-2.14]，在外部验证集(71.94%,95.54%, [AUC] 0.89[95% CI 0.86–0.93] 和 [HR]1.52[95% CI 1.38-1.66])和独立验证集(74.47%, 95.45%, [AUC] 0.88[95% CI 0.84–0.92]和[HR]1.54[95% CI 1.40-1.70])中得到了一致的结果，为PCa患者建立GS升级预测标记物提供可靠依据。目前已发表密切相关研究成果4项，申请发表专利2项，已获批1项。我国目前尚无相关AS研究队列，我们首次报道了中国第一个也是最大样本量的GS升级与预后相关本研究。基于本研究结果，我们拟结合中国低危PCa患者的临床特征，结合和借鉴西方国家AS的相关经验，构建首个中国人群的低危前列腺癌队列研究。用我们自主研发的甲基化检测分类器，在穿刺后建立尽可能准确的临床分期，精准预测GS升级患者，早期为患者制定合理的治疗方案，避免过度治疗和贻误病情。