β-hTERT激活衰老炎症因子促结肠癌转移微环境形成的机制研究

Metastatic niche is a supportive microenvironment secondary organ induced by disseminated cancer cells. It is the key factor of tumor colonization. β-hTERT is one of the four major RNA splicing variants of human telomerase reverse transcriptase. We previously discovered that Colon cancer cells expressingβ-hTERT retains a sphere-shape growth phenotype in vitro, as well as enhanced liver and lung metastasis colonization of circulating tumor cells, with a unknown mechanism. Our preliminary data shows that β-hTERT expression activates colon cancer secret IL-1,6,8,TNF-α and CXCL1, which belong to a Senescence Associated Secretory Phenotype(SASP) that closely related to metastatic niche. Meanwhile, our data indicated that Poly ADP-ribose polymerase 1(PARP1) may be a vital mediator ofβ-hTERT activating SASP factors. We propose to further investigate the clinical correlation between β-hTERT, PARP1, SASP and colon cancer metastasis. Applying a primary tissue culture technique Conditionally Reprogrammed Cells(CRC), and other up to date molecular engineering techniques, we plan to study the detailed mechanism of β-hTERT-PARP1-SASP pathway. We would also explore the therapeutic potential of peptide carrying β-hTERT antisense DNA to prevent metastasis of colon cancer. Our research will provide substantial insight for the colon cancer metastasis regulation and therapeutic alternatives.

转移微环境是肿瘤细胞播散到靶器官后诱导的支持性微环境，是转移细胞成瘤的关键因素。β-hTERT是端粒酶逆转录酶RNA的长链非编码剪接体。前期发现结肠癌高表达β-hTERT促进细胞体外呈球状生长，且增强循环肿瘤细胞在肝肺定殖，但机制不清。预实验提示β-hTERT高表达激活肠癌细胞体外分泌IL-1、6、8、TNF-α和CXCL1，后者属于一类衰老相关炎症因子（SASP），与转移微环境密切相关。同时发现DNA修复蛋白PARP1可能是介导β-hTERT调控SASP的重要中间分子。本项目拟在动物和病例水平进一步证实β-hTERT、PARP1和SASP的表达与肠癌转移的临床相关性，并采用原代细胞CRC技术及相关分子生物学方法深入研究β-hTERT-PARP1-SASP通路的调控机制，以及转导肽负载β-hTERT反义DNA对结肠癌的抗转移疗效。本研究有望为转移性结肠癌提供理论依据和治疗靶点。

本项目在前期工作基础上，拟在动物和病例水平进一步证实β-hTERT、PARP1和SASP的表达与肠癌转移的临床相关性，并采用原代细胞CRC技术及相关分子生物学方法深入研究β-hTERT-PARP1-SASP通路的调控机制，以及转导肽负载β-hTERT反义DNA对结肠癌的抗转移疗效。在项目研究工作中，我们完成了以下实验内容及科学发现：（1）在临床病例组织中发现 PARP1 的 mRNA 水平和β-hTERT 水平呈正相关，而患者术后至转移的存活时间与前两者呈反相关，证实β-hTERT 和 PARP1 促进肠癌转移的表型。CBP 与β -hTERT 的结合与患者术后转移发生时间呈明显负相关。完成β-hTERT 与 PARP1调控区的结合机制及参与转录激活的辅助因子， 以及 PARP1、 macroH2A1.1 和 CBP的相互作用机制研究。发现结肠癌细胞高表达的β-hTERT 与 PARP1基因调控区形成 RNA:DNA 三链结构介导转录因子结合而启动其表达。上调的PARP1 与macroH2A1.1 相互作用经组蛋白乙酰化途径激活 SASP 因子分泌（相关成果投稿Cancer Research中，已修回）。（2）设计β-hTERT 的小干扰 RNA 并开发一种黑磷纳米片层，负载siRNA 并成功对动物模型进行了在体治疗。这部分研究结果已于2018年在线发表在ACS Applied Material & Interfaces（IF 8.096）。