LncRNA/VPS9D1-AS1调控TGF-β信号通路促进结直肠癌细胞增殖和迁移的作用及其调控机制研究

VPS9D1-AS1 (LncVPS) was proved to be one of long non-coding RNA with dysregulated expression in colorectal cancer (CRC). But its underlying mechanisms for promoting CRC progressions still remain to be illusive. TGF-β signaling pathway exhibited an early tumor-suppressive role, but showed a tumor-promotive role at late stage. Our research demonstrated that the expressions of LncVPS were positively associated with TGFB1 in the tissues from CRC patients. The deletion of LncVPS inhibited the proliferations and migrations of CRC cells. Moreover, LncVPS was discovered to interact with TGFB1 and form the complex to scaffold the TGFB1 proteins and enhance its expressions. LncVPS was also proved to act as competitive endogenous RNA (ceRNA) by binding miR-22 and prevent its effects to abolish the expression of TGFB1. In this project, we will conduct the experimental or clinical samples analysis to illustrate the underlying mechanisms for LncVPS scaffolding TGFB1 and serving as the ceRNA to sponge miR-22. Moreover, this projects will reveal the effects for LncVPS mediating TGF-β signaling pathway to exhibit the roles of tumor promotor and enhance the proliferations and migrations of CRC cells. Our analysis will also explore the clinical significances for LncVPS, TGF-β signaling pathway, and miR-22 by detecting their levels in the samples from CRC patients. Finally, this study will reveal the novel molecular mechanism for LncVPS driving TGF-β signaling pathway to promote the progressions of CRC. In clinical, this study will drive to find the new drugs by targeting TGF-β signaling pathway.

VPS9D1-AS1（LncVPS）是结直肠癌（CRC）中高表达的非编码RNA，其作用机制尚未明确。TGF-β信号通路在早期抑制肿瘤，晚期促进肿瘤。我们新近发现LncVPS在CRC组织中与TGFB1的表达正相关，降低LncVPS可抑制癌细胞的增殖和迁移，LncVPS即与TGFB1蛋白结合发挥Scaffold作用增强其表达，还作为ceRNA发挥Sponge作用拮抗miR-22对TGFB1表达的抑制。本研究拟结合实验模型和临床样本检测进一步阐明LncVPS影响TGFB1及miR-22调控TGF-β信号通路的分子机制，探索LncVPS与TGF-β信号通路在癌细胞增殖和迁移中的作用，明确LncVPS、TGF-β信号通路、miR-22在CRC组织中表达的临床相关性。试图揭示由LncVPS激活TGF-β信号通路使其发挥促肿瘤作用参与CRC进展的新机制，为靶向TGF-β信号通路的抗癌药物开发提供新思路。

结直肠癌细胞的异常增殖和迁移是导致预后不良的主要因素。我们前期筛选到长链非编码RNA-VPS9D1-AS1（VPS）在结直肠癌（CRC）中表达显著升高且预示患者生存期短，与TGFβ信号通路有关，但其发挥作用的机制还不清楚。本项目中我们利用RNA原位杂交联合多色免疫荧光技术在CRC组织水平证实VPS与TGFβ、TGFBR1、SMAD1/5/9正相关，VPS在肿瘤组织中的高表达还与患者TNM分期、淋巴结转移正相关，而与CD8+T细胞的浸润负相关。机制探索发现VPS作为Scaffold分子与TGFβ、TGFBR1、SMAD1/5/9结合，还作为ceRNA阻断miR-22-5p/514a-3p对TGFβ通路的抑制，VPS敲除（KO）显著抑制了CRC细胞的增殖、迁移、克隆形成能力，VPS-KO细胞经历EMT途径而形态发生改变，体内实验证实VPS-KO还抑制了移植瘤的形成。干扰素刺激基因（ISG）受VPS和TGFβ信号通路调控，其中OAS1被鉴定为受影响最显著的ISG基因，其下游调控IFNAR1分子。VPS过表达（OE）的肠癌细胞抑制了CD8+T细胞的杀伤，并降低CD8+T细胞表面IFNAR1的表达和IFNγ的释放。在动物体内，VPS-OE细胞较对照细胞形成的移植瘤中T淋巴细胞的浸润显著减少，且CD8+T细胞PD1的表达显著升高。基于以上结果，我们提出VPS可作为CRC治疗的靶点。为此，我们设计和筛选可高效抑制VPS表达的寡核苷酸（ASO）药物，在动物中证实可显著抑制肿瘤的生长。综上，本项目明确了VPS通过TGFβ信号通路促进结直肠癌增殖和迁移的分子机制，为以VPS为靶点治疗CRC奠定了理论基础。下一步我们将进一步分析VPS、TGFβ、ISG基因构成的调控网络在CRC免疫反应中的机理，并探索靶向VPS设计ASO药物的临床应用前景。