miR-30家族靶向作用FANCF调控乳腺癌细胞药物敏感性的分子机制

It's verified that DNA damage and repair function play an important role in chemotherapy resistance, and there are potential correlation between the FA/BRCA pathway and NER/HR/TLS pathway in both DNA repair function and chemotherapy resistance. In the pre-test, we observed that FANCF silence could significantly increase the sensitivity of the chemotherapeutic drugs by blocking DNA repair function which were mediated through FA/BRCA pathway, inducing apoptosis and inhibiting BCRP expression However, it is still unclear whether regulation of the chemotherapy drug sensitivity by FANCF was related to DNA damage repair function which was mediated by NER/HR/TLS pathway. Meanwhile, luciferase reporter gene assays analysis confirmed that miR-30 family had binding site in FANCF mRNA. Hence, we intend to target FANCF by miR-30 family to study the function of FANCF regulating the chemotherapy sensitivity, as well as study the changes of HR/TLS/NER pathway in the DNA damage and repair process in order to explore the mechanism of FANCF increasing chemotherapy sensitivity. The study will provide a new target for the reverse of breast cancer chemotherapy resistance.

DNA损伤与修复功能在肿瘤细胞药物敏感性调控中具有重要作用。研究证实FA/BRCA通路与NER/ HR/ TLS通路共同参与DNA损伤修复及药物敏感性调控。课题组研究发现，乳腺癌细胞中FA/BRCA通路关键因子FANCF沉默后，通过阻滞FA/BRCA通路促进DNA损伤、诱导凋亡和抑制BCRP表达等增加化疗药物敏感性，但FANCF调控药物敏感性是否与NER/ HR/ TLS通路介导的DNA损伤修复功能异常相关？有待阐明。本项目拟以Luc报告基因分析验证的、与FANCF具有结合作用的miR-30家族为切入点，通过miR-30家族有效靶向沉默FANCF，研究其对FANCF表达和功能以及乳腺癌药物敏感性的影响，检测NER/ HR/ TLS 等DNA损伤修复通路功能变化，阐明FANCF调控乳腺癌药物敏感性的分子机制，为发现干预乳腺癌耐药作用新靶点、研究增加药物敏感性新方式与作用机制提供理论与实验依据

本项目拟研究miR-30家族调控乳腺癌细胞DNA损伤修复相关因子表达与功能的影响及机制。通过Targetscan生物分析系统理论预测，发现DNA损伤修复关键因子FANCF 和REV1mRNA-3’UTR区存在与miR-30家族的“种子区”，即5’端2～8或1～7核苷酸区的关键结合位点；进一步通过荧光素酶（Luciferase , Luc）报告基因分析发现miR-30c与FANCF和REV1mRNA-3’UTR有结合作用；诱导乳腺癌细胞MCF-7和MDA-MB-231中miR-30c过表达后可下调FANCF和REV1表达，促进乳腺癌细胞CDDP引起的DNA损伤；为了考察miR-30c上游调控因子，通过Gene Regulation生物信息学预测到转录因子p53与miR-30c启动子存在结合区域。CHIP实验证实p53与内源性miR-30c上游启动子区在体内有直接结合。荧光素酶报告基因分析进一步验证p53可直接与miR-30c上游启动子区结合，结合位点主要位于miR-30c转录位点上游800bp,783bp,376bp,173bp。应用基因毒性药物阿霉素激活p53、p53cDNA诱导p53高表达以及p53特异性激动剂Nutlin-3均可促进MCF-7（p53野生型）中miR-30c表达增加，但是对MDA-MB-231（p53突变型）细胞中miR-30c表达无明显影响，反之，p53沉默后可抑制MCF-7中miR-30c表达；MCF-7细胞ADR处理及转染p53cDNA后，REV1和FANCF的表达均被抑制；MCF-7细胞中miR-30c抑制后可拮抗p53对REV1和FANCF表达的抑制作用，说明p53可通过激活miR-30c的转录引起REV1,FANCF表达的下降。上述研究结果提示， p53/miR-30c/REV1,FANCF通路是调控DNA损伤修复的一个新机制。