环状RNA circ-FOXO3在三阴性乳腺癌侵袭转移中的作用及机制研究

Triple-negative breast cancer (TNBC) displayed a highly invasive phenotype, and lack of therapeutic targets for effective treatment, and have a relatively poor outcome. Circular RNAs (circRNAs) are a novel class of non-coding RNA characterized by the presence of a covalent bond linking the 3’ and 5’ ends. Recently studies suggested that circRNA is associated with cancer development by regulating gene expression. In our previous studies, we examined the differential expression profile of circRNAs between TNBC tissues and adjacent non-cancerous tissues with microarray, and found that circ-FOXO3 was markedly down-regulated in TNBC tissues, whereas overexpression of circ-FOXO3 significantly suppressed TNBC cell invasion and metastasis. Therefore, our present proposal will verify the inhibition effects of circ-FOXO3 on TNBC cell invasion and metastasis, investigate pivotal molecular event of circ-FOXO3-mediated inhibition of TNBC cell invasion and metastasis, clarify the role and mechanism of circ-FOXO3 on epithelial-mesenchymal transformation and metabolic reprogramming, clarify the molecular mechanism of down-regulation of circ-FOXO3 in TNBC, and explore the pathological significance of circ-FOXO3 in TNBC cancer metastasis. The results will provide experimental and theoretical basis for circ-FOXO3 serving as therapeutic target for TNBC.

三阴性乳腺癌（TNBC）具有高度的侵袭性、且缺乏有效的治疗靶点及治疗手段，预后极差。环状RNA（circRNA）是新近发现的一类具有闭合环状结构的非编码RNA，具有重要的基因表达调控功能，与肿瘤的发生发展有密切关联。我们前期采用高通量circRNA芯片分析发现circ-FOXO3在TNBC显著下调表达，且过表达circ-FOXO能抑制TNBC侵袭转移。据此，本项目拟在前期工作的基础上，从分子、细胞、裸鼠以及临床标本水平，进一步明确circ-FOXO3抑制TNBC侵袭转移的的作用；揭示circ-FOXO3抑制TNBC侵袭转移作用中的关键分子事件，解析circ-FOXO3抑制上皮-间质转化、代谢重编程的作用及机制；阐明circ-FOXO3在TNBC下调表达的分子机制；探究circ-FOXO3在TNBC转移中的病理意义，为以circ-FOXO3作为TNBC治疗靶点提供实验基础和理论依据。

三阴性乳腺癌（TNBC）具有高度的侵袭性、且缺乏有效的治疗靶点及治疗手段，预后极差。环状RNA（circRNA）是新近发现的一类具有闭合环状结构的非编码RNA，具有重要的基因表达调控功能，与肿瘤的发生发展有密切关联。本研究采用高通量测序分析分析TNBC组织与配对癌旁组织circRNA差异表达谱，共筛选出差异表达至少两倍以上且具有统计学意义的circRNA共有105种，其中Circ-FOXO3a在三阴性乳腺癌组织中显著下调表达。进一步通过三阴性乳腺癌临床组织标本及细胞系证实circ-FOXO3a在TNBC组织及细胞中显著下调表达。通过构建circ-FOXO3a稳定表达的三阴性乳腺癌细胞株，分别从分子、细胞、裸鼠水平证实了circ-FOXO3a能够诱导三阴性乳腺癌细胞G2期阻滞、抑制细胞增殖、上皮-间质转化以及侵袭转移。采用RNA pull down-质谱分析发现circ-FOXO3a能够与组蛋白甲基转移酶WHSC1蛋白结合，并抑制WHSC1表达及入核，且下调组蛋白H3K36me2甲基化水平。通过构建WHSC1稳定敲除的三阴性乳腺癌细胞株，证实了WHSC1能够促进三阴性乳腺癌细胞上皮-间质转化及侵袭转移。功能回复实验证实circ-FOXO3a通过抑制WHSC1的表达及功能下调转录因子Snail、Zeb的表达，从而抑制三阴性乳腺癌细胞上皮-间质转化及侵袭转移。分别采用荧光原位杂交技术、免疫组化技术检测circ-FOXO3a、WHSC1在三阴性乳腺癌临床组织中的表达情况，进一步证实circ-FOXO3a表达与WHSC1表达呈负相关，且circ-FOXO3a表达与三阴性乳腺癌淋巴结转移负相关。本研究为以circ-FOXO3a/WHSC1作为三阴性乳腺癌临床诊治的新靶点提供实验基础和理论依据。