miR-1193和DNA-PKcs协同致死脑胶质瘤细胞分子机制研究

Glioblastoma is one of the highest fatality tumors. So far, it still lacks effective treatments. In our previous studies, transfection of antisense nucleic acid against miR-1193 can induce the apoptosis of M059J cells, which is DNA-PKcs deficient. However, the mechanisms remain completely unknown. YY1AP1, which promotes YY1 expression, was predicted to be a potential target gene of miR-1193 using computational algorithms. We have published that the inhibitory function of YY1 on FEN1 and the role of FEN1 in double-stranded DNA break (DSB) repair pathways. Therefore, we hypothesize here that down-regulation of miR-1193 would result in high-expression of YY1AP1, which in turn promote the expression of YY1, leading to the inhibition of FEN1 and the obstruction of DSB. In this case, function loss of both FEN1 and DNA-PKcs simultaneously will trigger the ATM/ATR-CHK1/2-p53 apoptosis pathway, and eventually lead to cancer cell apoptosis and death. We will try to uncover the underlying mechanism about how depletion of miR-1193 and DNA-PKcs results in specific cell death of Glioblastoma, on the levels of cellular effects, molecular mechanism and animal model. The implementation of this proposal will provide key and novel information on developing miR-1193 as potential target for invention and treatment of DNA-PKcs deficient glioblastoma.

脑胶质瘤是致死率最高的癌症之一，目前仍缺乏有效治疗手段。我们发现，在DNA-PKcs缺失的M059J中转染miR-1193反义核酸，能诱导细胞凋亡，但是其调控机制未见报道。生物信息学分析提示YY1AP1是miR-1193可能的靶基因，能促进YY1表达。我们已经证实YY1抑制FEN1表达、FEN1在DNA双链断裂（DSB）修复途径有重要作用。因此，我们提出科学假说：miR-1193下调导致靶基因YY1AP1高表达，后者促进YY1表达而抑制FEN1表达，FEN1和DNA-PKcs同时缺失，细胞DSB修复严重受阻，进而激活ATM/ATR-CHK1/2-p53通路，最终导致细胞凋亡。本项目将从细胞、分子和动物模型水平，试图揭示同时缺失miR-1193和DNA-PKcs对脑胶质瘤特异性的抑制与杀伤作用。阐明该机制将为miR-1193作为新的干预靶点，治疗DNA-PKcs缺失的脑胶质瘤提供理论依据。

脑胶质瘤是最常见的原发性脑肿瘤，具有极高的浸润性和侵袭能力，手术彻底切除困难，耐药性强，极易复发，致残率高，目前临床上缺乏有效治疗手段。因此，研究和开发治疗脑胶质瘤的新策略迫在眉睫。本项目主要研究内容是miR-1193与DNA-PKcs通过协同致死效应导致脑胶质瘤细胞凋亡的作用及其分子机制，通过Luciferase荧光报系统验证了miR-1193的作用靶点及在DNA双链断裂损伤修复调控过程和细胞存活的作用机制。联合使用miR-1193反义核酸和DNA-PKcs特异小分子抑制剂，通过克隆形成、细胞周期、增殖与凋亡检测、细胞双链断裂损伤修复能力和肿瘤细胞基因组稳定性分析等多角度、多层次揭示了同时缺失miR-1193和DNA-PKcs对脑胶质瘤细胞特异性的抑制和杀伤作用。通过研究，我们得到以下重要结论：阐明了miR-1193下调导致靶基因YY1AP1高表达，后者促进YY1表达而抑制FEN1表达，FEN1和DNA-PKcs同时缺失，细胞DNA双链断裂损伤修复严重受阻，进而激活ATR-CHK1-p53信号通路，最终导致细胞凋亡。本项目对该机制的阐明，为miR-1193作为新的干预靶点，利用协同致死效应治疗DNA-PKcs缺失的脑胶质瘤提供了理论依据。