环状RNA-crEPSTI1促进三阴乳腺癌生长与转移的机制研究

Recent studies have shown that circular RNA could regulate microRNAs and gene expression, which is closely related with the occurrence and development of cancers. We previously confirmed that miR-200b could inhibit the growth and metastasis of triple negative breast cancer. Through a high-throughput circular RNA microarray and preliminary experiment，we found that CircRNA-crEPSTI1 was highly expressed in triple negative breast cancer cell lines and tissues. Meanwhile, its high expression was positively correlated with lymph node metastasis and prognosis. The functional experiment results showed that overexpression of crEPSTI1 could down regulate miR-200b and miR-4753 and promoted the proliferation and invasion of triple-negative breast cancer. Bioinformatics predicts that both miR-200b and miR-4753 can target SOX2. Further experiments revealed that crEPSTI1 could also bind to oncogene SRC, which can promote the expression of SOX2. Therefore, we propose a scientific hypothesis that to promote the growth and metastasis of triple negative breast cancer, crEPSTI1 rescuer the inhibition of SOX2 by absorbing miR-200b and miR-4753, and/or regulate the expression of SOX2 by direct binding to SRC. In this project, CRISPR / Cas9 and animal imaging techniques will be used to elucidate the mechanism. Ultimately, this project could provide a new theoretical basis for pathogenesis researches and new therapeutic targets for triple negative breast cancer.

环状RNA调控miRNA和基因的表达，与肿瘤的发生发展密切相关。申请者前期证实miR-200b具有抗肿瘤作用，可抑制三阴乳腺癌的生长与转移。本项目通过环状RNA芯片和预实验发现，crEPSTI1在三阴乳腺癌中表达升高，过表达crEPSTI1可下调miR-200b和miR-4753，促进三阴乳腺癌细胞增殖与侵袭。生物信息学预测SOX2是miR-200b和miR-4753的共同靶基因；进一步实验发现crEPSTI1可与SRC结合，文献证实SRC可促进SOX2表达。由此我们提出科学假说：crEPSTI1可通过吸附miR-200b和miR-4753，解除miRNA对SOX2的作用，或/和直接结合SRC调控SOX2的表达，进而促进三阴乳腺癌生长与转移。本项目拟采用CRISPR/Cas9和动物成像等技术阐明crEPSTI1上、下游分子的调控机制，为三阴乳腺癌生长与转移的研究提供新的理论依据和治疗靶点。

肿瘤防治是目前全球性最为严峻的公共卫生问题之一。蛋白编码基因的遗传密码子在理解突变如何驱动肿瘤发生发展方面起到了重要的作用。但当人类基因组计划完成时，人们发现编码基因组仅占全部基因序列的不到2％，大量的基因序列属于非编码基因，这一重大发现彻底颠覆了人们以往对于基因组的认识。本项目首先通过对3对人三阴乳腺癌肿瘤组织及其配对的正常乳腺上皮组织进行环状RNA基因芯片筛选三阴乳腺癌的环状RNA差异表达谱。挑选其中在三阴乳腺癌中表达量最高的circEPSTI1进行功能研究。探讨circEPSTI1对三阴乳腺癌的增殖，克隆形成和凋亡的影响。根据MREs分析和荧光素酶报告基因检测，探讨circEPSTI1可能作为miRNAs海绵，并且在裸鼠皮下成瘤实验中来验证我们的发现。通过原位杂交以及免疫组化评估了240例三阴乳腺癌患者肿瘤组织中的circEPSTI1以及相关基因的蛋白表达水平，探讨circEPSTI1与三阴性乳腺癌患者临床病理因素以及预后的关系。结果提示1.circEPSTI1在三阴乳腺癌细胞以及组织中高表达，具有促进三阴乳腺癌恶性生物学行为的功能；2.circEPSTI1可通过竞争性吸附miR-4753与miR-6809，调控BCL11A的表达；3.在三阴乳腺癌患者当中，circEPSTI1高表达与肿瘤大小、淋巴结转移情况以及TNM分期相关，并且提示患者的预后不良。我们进一步通过qRT-PCR证实了上调的circKIF4A的表达。探讨其在三阴乳腺癌进展中的功能以及研究circKIF4A对miRNA及其靶基因的调节作用及其潜在调节机制。结果提示circKIF4A显著上调，并与三阴乳腺癌较差的存活率呈正相关。抑制circKIF4A可以抑制三阴乳腺癌细胞增殖和迁移。荧光素酶报告基因测定和RNA免疫沉淀测定显示circKIF4A和KIF4A可以与miR-375结合，并且circKIF4A通过海绵miR-375调节KIF4A的表达。circKIF4A可以作为三阴乳腺癌的预后生物标志物和治疗靶标。