CirR-387与let-7d-5p竞争性调节GALNT1经EGFR/E-cadherin通路调控肝细胞癌EMT的机制研究

The metastasis of hepatocellular carcinoma (HCC) is closely associated with epithelial-mesenchymal transition (EMT). CircRNA has been reported to regulate the EMT progress by ceRNA mechanism. Our previous researches showed that over-expression of cirR-387 on HepG2 could inhibit the metastasis of HepG2 cells and the mRNA expressions of EMT-related molecules, such as MMP9 and N-cadherin. Bioinformatics analysis showed that cirR-387 might regulate GALNT1 by competition with let-7d-5p miRNA. Moreover, knockdown of GALNT1 suppressed p-EGFR and E-cadherin expressions. Therefore, we supposed that cirR-387/let-7d-5p competition mediated GALNT1 regulations might play essential roles on EMT progress via the EGFR/E-cadherin pathway. To prove this hypothesis, the IHC, qPCR, WB and Caliper IVIS Lumina II techniques are going to be applied to explore that how the cirR-387 impacts the invasion, metastasis and tumorigenic ability of HCC cells in vitro and in vivo. Gain and loss experiments WB and CHIP etc are going to be used to explore how cirR-387 and let-7d-5p regulate GALNT1 and thus to regulate EMT progress via EGFR/E-cadherin pathway, post the experiments that aim to confirm the interactions between CirR-387and let-7d-5p and between let-7d-5p and GALNT1.This project aims to explore the functional roles of cirR-387 on the regulatory role on HCC EMT progress, this may lead to a solid foundation on the mechanism exploration of circRNA in the cancer signaling pathway in future.

肝细胞癌的侵袭转移与EMT密不可分。CircRNA可通过CeRNA机制调节EMT。前期发现过表达CirR-387能抑制HepG2转移潜能及MMP9表达，并可能与let-7d-5p竞争性调节GALNT1，且敲低GALNT1可抑制p-EGFR和E-cadherin的表达。据此提出“CirR-387可能与let-7d-5p竞争性结合GALNT1经EGFR/E-cadherin信号通路调控肝细胞癌EMT”的科学假说。拟利用qPCR和WB等技术研究CirR-387对HCC体内外转移能力的影响。在确定CirR-387与let-7d-5p及let-7d-5p与GALNT1互作的基础上，采用过表达、RNAi及CHIP等技术，研究CirR-387与let-7d-5p竞争性结合GALNT1经EGFR/E-cadherin信号通路调控肝细胞癌EMT的机制，为揭示HCC的EMT机制进而寻找治疗靶点提供数据支撑。

肝细胞癌的侵袭转移与EMT密不可分。CircRNA可通过CeRNA机制调节EMT，对EMT具重要调控作用的circRNA和糖基化作用在HCC侵袭转移过程中是否存在相互调控机制，对这个科学问题的深入探究对于揭示HCC侵袭转移机制进而寻找新的抗HCC药物靶点具有重要意义。在本项目中，我们发现：①CirR-387与肝癌密切相关，且在癌组织中高表达而在癌旁组织中低表达。②过表达CirR-387能促进肝癌细胞在体外的侵袭和转移。③CirR-387主要定位在肝癌细胞的细胞质中。④CirR-387通过吸附let-7d-5p后间接调控GALNT1从而影响细胞的生物学功能。⑤GALTN1能影响肝癌细胞在体外的侵袭和转移。此外，我们发现了另外两种分子对肝癌术后复发及预后评估能力更为显著：⑥环状RNA hsa_circ_0005218通过靶向miR-31-5p/CDK1通路促进肝细胞癌早期复发。⑦circ_0008928通过miR-873-3p / PAN3轴促进HBV阳性肝细胞癌的进展。