外泌体传递的circ-CLASP2调控乳腺癌侵袭转移的功能及机制研究

circ-CLASP2 was identified as a novel circular RNA which was highy over-expressed in high invasive breast cancer cells and exosomes. Expression level validation showed this circRNA was enriched in exosomes and tumor tissues, positively correlated with lymph node metastasis, and was able to promote the invasion and metastasis of breast cancer cells. Exosomes could transfer circ-CLASP2 to recipient cells, circ-CLASP2 transferred by exosomes participated in the regulation of invasion and metastasis of recipient cells. Bioinformatics analysis and preliminary experiment showed that circ-CLASP2 could be bind to tumor-suppressor gene miR-195. Therefore we hypothesized that circ-CLASP2 transferred by exosomes could regulate the invasion and metastasis of breast cancer cells, and the probable mechanism was playing ceRNA mechanism by binding to miR-195.We plan to clinically evaluate the relationship of cir-CLASP2 and invasion of breast cancer , prove circ-CLASP2 could promote the invasion and metastasis of breast cancer using overexpress/silence strategy both in vivo and in vitro experiments, predict the regulating downstream target of circ-CLASP2 in ceRNA mechanisms using RNA - seq, bioinformatics methods, and clarify the ceRNA molecular mechanism of competitive combination of circ-CLASP2 with miR-195 to regulate the expression of target gene using RIP ,dual-luciferase reporter gene system and rescue experiement, and clinically evaluate its correlation with the invasion and metastasis of breast cancer. There’s little reports of the function and mechanism of cirRNA in exosomes and breast cancer, this study can provide new theory and new targets for the prevention and therapy of breast cancer.

circ-CLASP2（简称CC）是前期通过芯片筛选获得的一条在高侵袭性乳腺癌细胞及外泌体中显著上调的环状RNA。验证显示CC在肿瘤组织及外泌体中富集、与淋巴结转移相关，功能上促进乳腺癌的侵袭转移。外泌体可包裹CC传递至受体细胞，且传递的CC参与了其对乳腺癌侵袭转移的调控。生物信息学分析、荧光素酶报告基因、AGO-RIP等预实验提示CC能够吸附抑癌基因miR-195发挥竞争性内源性RNA（ceRNA）作用。故提出“外泌体传递的CC吸附miR-195通过ceRNA机制促进乳腺癌侵袭转移”的科学假说。本项目拟采取过表达/沉默策略，体内外证实外泌体传递的CC促进乳腺癌侵袭转移；通过RNA-seq、信息学筛选并多层面验证下游靶标基因；设计拯救实验，阐明CC发挥ceRNA作用促进受体细胞的侵袭转移。外泌体中环状RNA在乳腺癌侵袭转移中的功能鲜有报道，本研究可为其防治提供新思路。

Circ-CLASP2是前期通过芯片筛选获得的一条在高侵袭性乳腺癌细胞及外泌体中显著上调的环状RNA，体外功能验证显示circ-CLASP与乳腺癌细胞的侵袭转移相关，通过生物信息学分析、荧光素酶报告基因、AGO-RIP等提示circ-CLASP2能够吸附miR-195通过ceRNA机制发挥作用，后通过预测软件，选取候选靶基因，并通过PCR、荧光素酶报告基因、WESTERN等试验验证了其可能影响下游BCL2、FGF2靶基因表达而发挥作用。本项目还在组织中验证了circ-CLASP2的表达，发现circ-CLASP2在乳腺癌组织中高表达，且表达水平与淋巴结转移相关。并在血液标本中亦发现乳腺癌患者表达水平较高。此外，本项目对另一妇科肿瘤卵巢癌的环状RNA表达进行了研究，通过对卵巢癌组织及正常组织测序筛选并在细胞中验证获得三条表达差异的环状RNA，并在组织中初步验证。期望通过本研究，对妇科肿瘤包括乳腺癌、卵巢癌的发生机制提供新思路，为后续治疗提供新的方向。