调控miR-101/VEGF-C通路抑制膀胱癌淋巴道转移与降低化疗耐药的双重作用研究

Bladder cancer of advanced-stage is lack of effective treating method. There are two main reasons. One is the invasion and metastasis of tumor; the other one is the chemotherapy resistance. It will be an important milestone of the treatment for bladder cancer, if these two problems could be solved simultaneously. Our former research of ESS(30700832） shows that VEGF-C could promote the lymphatic metastasis of bladder cancer and inhibit the apoptosis of cancer cells induced by mitomycin;meanwhile，another research of HNNSF shows that mir-101 regulats expression of VEGF-C in bladder cancer.We assume that it is possible to inhibit lymphatic metastasis and reduce chemotherapy resistance of bladder cancer at the same time by regulating the pathway of mir-101/VEGF-C.The purpose of this topic is to find out whether the up-regulated expression of mir-101 could inhibit the expression of VEGF-C,and then reduce the ability of invasion and metastasis of bladder cancer as well as the chemotherapy resistance,through cell experiment , animal experiment and clinical case analysis. We are about to realize more about the function of miRNA in the process of lymphatic metastasis of bladder cancer,and furtherly provide potential new target for the control of bladder cancer cell invasion and metastasis and the chemotherapeutic sensitization.

晚期膀胱癌治疗棘手，究其原因主要在于:肿瘤向深部浸润和远处转移、化疗耐药。同时解决这两个问题，则膀胱癌的治疗水平将会明显提高。申请人所在的课题组前期国家自然科学基金课题（30700832）研究提示VEGF-C促进了膀胱癌细胞的淋巴道转移，且抑制丝裂霉素诱导膀胱癌细胞的凋亡；同时，另一省自然科学基金重点项目课题研究显示miR-101在膀胱癌中调控VEGF-C的表达。那么，我们设想：调控miR-101/VEGF-C通路是否可以同时抑制膀胱癌淋巴道转移与降低化疗耐药性呢？本课题拟先进行细胞实验，然后深入到动物实验，临床病例分析，多个角度进行深入研究，以期明确升高miR-101的表达是否可以抑制VEGF-C，是否可达到同时降低膀胱癌的浸润转移能力和化疗耐药性的目的。其结果有望深入认识miRNA在膀胱癌淋巴道转移过程中的作用，并为控制膀胱癌侵袭转移、化疗增敏提供新的靶点。

项目背景：膀胱癌是死亡率较高的恶性肿瘤，浸润转移是致死的主要原因。研究膀胱癌浸润转移机制及分子生物学调控过程，对膀胱癌的早期诊断、选择治疗方法、及判断预后有重要意义。.主要研究内容及结果：MiR-101与VEGF-C在国内外研究中被发现与多种肿瘤的发生发展存在密切联系，项目负责人预实验中荧光定量PCR检测了膀胱癌miR-101 的表达情况，结果发现在膀胱癌组织及淋巴结转移灶中，与VEGF-C蛋白水平表达谱相反，其表达明显下调，显示miR-101可能在膀胱癌中调控VEGF-C的表达及抑制了淋巴结转移。我们对其展开深入研究。首先我们运用细胞转染技术证明过表达MiR-101可明显抑制VEGF-C表达，证明MiR-101可调控VEGF-C表达。然后构建VEGF-C野生型3’UTR质粒和突变型3’UTR质粒。运用qRT-PCR, western blot,荧光素酶报告基因实验验证，证实miR-101是通过转录后水平调控VEGF-C。将VEGF-C过表达的质粒（带RFP）转入已过表达miR-101的细胞系，通过transwell试验、划痕试验证实VEGF-C的蛋白可恢复由miR-101引起的细胞迁移、侵袭能力下降。最后分别将过表达MiR-101的膀胱癌细胞株与空白对照组予不同浓度顺铂培养，顺铂敏感试验检测发现过表达MiR-101过表达细胞株抑制率较之对照组明显提高，提示顺铂化疗敏感性增强。.科学意义：本课题首次对MiR-101/VEGF-C在膀胱癌中作用机制及对化疗敏感性作用做出研究，发表SCI论文2篇，为膀胱癌发生发展机制研究及治疗提出新的思路。.关键词：膀胱癌；MiR-101；VEGF-C；化疗耐药；转移