NEAT1作为ceRNA在膀胱癌侵袭转移中的作用和机制研究

The invasion and metastasis are greatly involved in the prognosis of bladder cancer. Our former study found that the expression of NEAT1 and VEGF-C were up-regulated in the bladder cancer tissues and the cell lines, while the expression of miR-101 was down-regulated . Binding sites of NEAT1 and miR-101 were found by the predicted databases and luciferase report assay. MiR-101 could inhibite the invasion and metastasis of bladder cancer by targeting VEGF-C. After overexpression of NEAT1,the expression of NEAT1 and VEGF-C were up-regulated in the bladder cancer cells, while the expression of miR-101 was down-regulated. Taken together, we assume that as a ceRNA, NEAT1 is involved in the invasion and metastasis of bladder cancer, inducing VEGF-C by competively binding to miR-101. The project is supposed to investigate the relationship between the clinicopathological data and the expression of NEAT1 in bladder cancer. The effects of NEAT1 on the development of bladder cancer is expected to explore by cells and animal experiments. The present study helps to demonstrate the role of NEAT1 as a ceRNA in the bladder cancer and clarify further the mechanisms of the invasion and metastasis of bladder cancer.

膀胱癌侵袭转移是影响膀胱癌预后的关键所在。申请人课题组的前期研究发现，NEAT1和VEGF-C在膀胱癌组织和膀胱癌细胞株中呈高表达，而miR-101则呈低表达。通过数据库预测和荧光素酶实验证明了NEAT1和miR-101具有靶向结合位点；而miR-101可靶向作用于VEGF-C，抑制膀胱癌的侵袭转移。在过表达NEAT1的实验中发现，NEAT1和VEGF-C的表达明显上调，而miRNA-101的表达则明显下降。在以上基础上，申请人提出了NEAT1作为ceRNA通过竞争性地与miR-101结合，上调VEGF-C基因的表达，参与促进膀胱癌的侵袭转移过程。本课题拟先通过回顾性研究NEAT1在膀胱癌中的表达及与临床病理资料的关系，再通过细胞实验及动物模型观察NEAT1对膀胱癌发生发展的影响，以期探讨NEAT1作为ceRNA在膀胱癌侵袭转移中的作用及机理，其结果有助于进一步阐明膀胱癌侵袭转移的机制。

项目背景：膀胱癌侵袭转移是影响膀胱癌预后的关键所在。深入探索膀胱癌侵袭转移的具体机制具有重要的研究价值。LncRNA NEAT1的表达上调在多种肿瘤中起着促进肿瘤恶性发展的作用，但在膀胱癌中的作用尚未明确。主要研究内容及结果：我们通过组织学研究发现NEAT1在膀胱癌组织中的表达明显增加。进一步细胞实验证实，NEAT1均能够明显增强膀胱癌T24细胞和5637细胞的增殖、迁移和侵袭能力。其后，我们又在膀胱癌细胞中进行了过表达NEAT1的实验，结果显示，NEAT1的mRNA表达水平明显上调，VEGF-C的蛋白和mRNA表达明显上调，而miR-101的mRNA表达水平则明显下降。为了进一步验证miR-101与NEAT1的关系，我们又在T24细胞和5637细胞中进行了过表达NEAT1的荧光素酶实验，结果显示NEAT1过表达可使miR-101-WT（wild-type野生型）荧光素酶活性显著下降，但对miR-101-MUT（mutated-type突变型）荧光素酶活性无明显改变，证明了NEAT1能够靶向结合miR-101。利用脂质体将合成的miR-101模拟物(mimic)和miR-101抑制物(inhibitor)导入已转染NEAT1过表达载体的膀胱癌T24和5637细胞株。结果发现，NEAT1能够明显增强T24细胞和5637细胞的增殖、迁移和侵袭能力，miR-101 inhibitor +NEAT1过表达组细胞的增殖、迁移和侵袭能力较NEAT1组进一步增强。相反，miR-101 mimic+NEAT1过表达组细胞的增殖、迁移和侵袭能力弱于NEAT1组。这证明了miR-101表达下调能够增强NEAT1对T24细胞和5637细胞增殖、迁移和侵袭能力的促进作用，miR-101表达上调则能够削弱NEAT1对T24细胞和5637细胞增殖、迁移和侵袭能力的促进作用。体内实验进一步证明NEAT1能够明显促进裸鼠模型膀胱癌的发生和转移。科学意义：本课题证明了NEAT1作为ceRNA通过竞争性地与miR-101结合，上调VEGF-C基因的表达，参与促进膀胱癌的侵袭转移过程，已发表学术论文3篇，为进一步阐明膀胱癌侵袭转移机制提供了新思路。