外泌体运载的hsa_circ_0000549介导miR-7调控EGFR通路促进乳腺癌放疗抵抗的机制研究

Radiotherapy resistance has always been a conundrum in the treatment of breast cancer. However, the molecular mechanisms remain obscure. Our previous study confirmed that hsa_circ_0000549 was significantly up-regulated in the blood exosomes of radiotherapy resistant breast cancer patients. Our resent investigations further showed that hsa_circ_0000549 could adsorb miR-7, and miR-7 can affect the radiotherapy resistance of breast cancer by regulating the EGFR pathway. Integrating preliminary experimental results, we proposed the novel mechanism of hsa_circ_0000549 promoting radiotherapy resistance in breast cancer: hsa_circ_0000549 delivered by exosomes, through binding with miR-7, weakens the inhibition of miR-7 on EGFR and up-regulates the expression of EGFR, thereby leading to the activation of EGFR pathway and further cascade activation of downstream related signaling molecules. EGFR enters the nucleus and is involved in repairing DNA damage, thereby promoting radiotherapy resistance in breast cancer. Based on a series of experiments at molecular, cellular, animal and clinical levels, our project intends to elucidate the molecular mechanism of radiotherapy resistance in breast cancer focusing on hsa_circ_0000549→miR-7→EGFR pathway regulatory network, aiming to provide novel ideas and theoretical basis for the intervention of radiotherapy resistance in breast cancer.

放疗抵抗一直是乳腺癌治疗的难题，但机制未明。我们的前期研究证实：hsa_circ_0000549在放疗抵抗乳腺癌患者的血液外泌体中显著上调，进一步研究发现hsa_circ_0000549可吸附miR-7，而miR-7可通过调控EGFR通路影响乳腺癌的放疗抵抗。综合前期预实验结果，我们提出hsa_circ_0000549促进乳腺癌放疗抵抗的新机制：外泌体运载的hsa_circ_0000549通过与miR-7结合，减弱miR-7对EGFR的抑制而上调EGFR的表达，进而EGFR通路活化，激活下游相关信号分子，诱导EGFR入核参与DNA损伤修复，从而促进乳腺癌的放疗抵抗。本项目将通过分子、细胞、动物和临床等不同层面的一系列实验，力求阐明以hsa_circ_0000549→miR-7→EGFR通路为核心的乳腺癌放疗抵抗分子机制，旨在为防治乳腺癌放疗抵抗提供新的思路和理论依据。

乳腺癌是女性最常见的恶性肿瘤，放射治疗是乳腺癌的主要治疗手段之一。然而，由于患者的放射敏感性存在差异，部分患者经放疗后会出现放疗抵抗现象，严重影响放疗效果和预后，最终导致肿瘤复发和转移。因此，放疗抵抗一直是乳腺癌治疗的难题，但机制未明。本项目围绕“外泌体运载的hsa_circ_0000549介导miR-7调控EGFR通路促进乳腺癌放疗抵抗”进行深入研究， 取得了一系列原创性研究成果：证实了申报书中关于“外泌体运载的hsa_circ_0000549介导miR-7调控EGFR通路促进乳腺癌放疗抵抗”的科学假设。本课题采用高通量circRNA芯片技术，分别检测和比较了放疗抵抗与放疗敏感的乳腺癌患者血液外泌体中circRNA的差异，并通过定量RT-PCR技术筛选和验证，发现hsa_circ_0000549的表达水平与乳腺癌的放疗抵抗相关。进一步实验证实放疗抵抗组血液外泌体中hsa_circ_0000549的表达显著高于放疗敏感组，且高表达hsa_circ_0000549的患者预后更差，强烈提示hsa_circ_0000549在乳腺癌放疗抵抗中扮演重要角色。体外实验中，我们发现沉默乳腺癌细胞系中hsa_circ_0000549的表达，明显提高了乳腺癌细胞对放疗的敏感性。在动物实验中，我们通过建立雌性裸鼠（NU/NU）乳腺癌异种移植模型，证实了hsa_circ_0000549促进乳腺癌细胞放疗抵抗。在此基础上，我们通过circBase和miRecords预测了hsa_circ_0000549的靶点，发现其与miR-7有共同的结合位点，并进一步利用核酸转染技术和双荧光素酶报告基因系统等相关实验证实miR-7是hsa_circ_0000549的靶点。沉默hsa_circ_0000549可上调miR-7、下调p-EGFR和p-AKT的表达，阐明了hsa_circ_0000549促进乳腺癌放疗抵抗的新机制。本研究为乳腺癌的放疗提供了新的治疗靶点和思路，对指导临床患者的放疗方案制定、改善疗效与预后均具有重要意义。