靶向高效导入藤黄酸调控膀胱癌lncRNA-UCA1-miR-34b-USP22通路研究

How to reverse recurrence and resistance to chemotherapy of bladder cancer is a highlight issue with application prospect. USP22, a new ubiquitin hydrolase, is a valuable biomarker for predicting the recurrence and metastasis of malignance and its resistance to chemotherapy. We have previously demonstrated that a novel anticancer drug- Gambogic acid, could down-regulate the expression of USP22. We further proposed and tested a new hypothesis that Gambogic acid inhibited the proliferation and reversed resistance to chemotherapy of bladder cancer cells via down-regulation of lncRNA-UCA1 and enhancing miR-34b-mediated degradation of USP22. In addition, using the bladder cancer specific monoclonal antibody-Uroplakin Ib(UPIb ) and TAT peptide penetrating the cell membrane which were prepared in our preliminary studies, we constructed an innovative bladder cancer targeted drugs-Gambogic acid-TAT-UPIb based on the antibody-drug cross-linking technology. This novel drug reduced the effective concentration of anti-tumor effect of gambogic acid greatly and enhanced the ability of targeted-tumor of gambogic acid obviously. Meanwhile, we investigated the effect of Gambogic acid-TAT-UPIb on the biological behaviors of bladder cancer cells. The successful implementation of this project will provide an efficient specific and initiative therapy for the bladder cancer.

如何逆转膀胱癌复发、迁移和化疗耐药的生物学特性是极具应用前景的热点问题。USP22 是一种新近发现的泛素水解酶，参与肿瘤的发生发展，被认为是肿瘤复发、转移和化疗耐药的“癌致死标签”。本课题组前期已证实一种新型的抗肿瘤药物—藤黄酸，能够下调膀胱癌中USP22的表达，在此基础上拟验证藤黄酸通过下调lncRNA-UCA1的表达，解除lncRNA-UCA1对miR-34b的转录抑制，促使USP22转录后降解，从而抑制膀胱癌细胞增殖、逆转膀胱癌耐药的崭新假说。此外，结合本课题组前期已制备的Uroplakin Ib(UPIb)单克隆抗体和穿膜肽TAT，运用抗体-药物交联技术制备创新性藤黄酸-穿膜肽-UPIb抗体交联药物，增加藤黄酸对肿瘤细胞的靶向性及穿透杀伤作用，观察其对膀胱癌生物学行为的影响。本课题的成功实施将为膀胱癌高效特异性主动靶向治疗提供新的思路，具有重要的理论意义和广阔的应用前景。

术后辅助化疗是预防膀胱癌复发的一种重要治疗手段。然而由于膀胱癌是一种多中心起源的肿瘤，具有易复发、转移及化疗耐药等生物学特性。而且，化疗药物的毒副作用也是影响膀胱癌预后的一个关键因素。因此，寻找一种有效，且毒副作用少的药物并阐明其作用机制成为目前膀胱癌治疗的热点之一。本课题组已证实一种新型的抗肿瘤药物—藤黄酸，能够下调膀胱癌中USP22的表达（USP22被认为是膀胱癌复发及化疗耐药的“基因标签”），在此基础上发现藤黄酸通过调控USP22的表达及其相关lncRNA-miRNA-mRNA的ceRNA网络抑制膀胱癌细胞的增殖和侵袭。此外，结合本课题组前期已制备穿膜肽TAT，运用穿膜肽-药物交联技术制备创新性藤黄酸-穿膜肽交联药物，增加了藤黄酸对肿瘤细胞的靶向性及穿透杀伤作用，促进膀胱癌细胞的凋亡。本课题实验结果发现：（1）lncRNA-UCA1在5种人膀胱癌细胞株(T24、BIU-87、EJ、T24-MMC、T24-ADM）表达上调，而miR-34b在5种人膀胱癌细胞株中的表达显著下调（2）UCA1和miR-34b与膀胱癌的肿瘤的分级、有无肌层浸润和有无复发密切相关，并且两者之前的表达具有表达相关性。（3）miR-34b通过靶向调控USP22基因的表达抑制膀胱癌细胞的增殖；（4）藤黄酸通过调控lncRNA-UCA1-miR-34b-USP22通路抑制膀胱癌细胞的增殖；(5)成功合成藤黄酸-穿膜肽(GA-TAT)偶联物，且膀胱癌细胞对GA-TAT摄取能力增强，其细胞毒性明显减弱。（6）GA-TAT能够增强GA对膀胱癌细胞的促凋亡的敏感性，其机制主要是通过ROS介导的凋亡。（7）同时GA-TAT能够激活caspase-3 和 caspase-9蛋白的活性，下调Bcl-2/Bax的比例，但此作用被ROS的活性氧抑制剂NAC所逆转。本课题成功的设计并合成了由穿膜肽Tat携带的前药导入系统，将藤黄酸靶向大剂量聚集在肿瘤组织并能高效进入肿瘤细胞发挥作用。并从机制上证明GA-TAT偶联药可通过lncRNA-UCA1-miR-34b-USP22通路抑制膀胱癌细胞的增殖，通过ROS- caspase-3/caspase-9-Bcl-2/Bax通路促进膀胱癌细胞凋亡。本课题的成功实施有望解决膀胱癌术后复发及化疗耐药这一难题，为藤黄酸在肿瘤治疗方面的临床应用奠定了理论基础。