Bone Metastasis-Derived Extracellular Vesicles Fuel Hepatocellular Carcinoma Metastasis By Transferring ALKBH5-targeting MiR-3190-5p

Objective:Bone is the second leading metastatic organ for hepatocellular carcinoma(HCC).Patients with HCC and bone metastasis suffer poor quality of life and reduced survival time.The connection of bone-liver axis is beneficial to the diagnosis and treatment of HCC.Extracellular vesicles(EVs)have been widely involved in HCC formation and metastasis.However,the communication between primary HCC and bone lesions mediated by EVs remains unclear,and the possible effect of bone metastasis on the progression of HCC remains largely unknown.This study attempted to explore the effect of bone-metastasized HCC-derived EVs(BM-EVs)on HCC in situ and the specific molecular mechanism.Methods:This study used xenograft mouse models and tail vein injection of EVs to explore the role of EVs in bone metastasis of HCC progression.In vitro,transwell and wound healing assays were used to investigate the effect of BM-EVs on the motility of HCC cells.Micro RNA(miRNA)microarray,methylated RNA immunoprecipitation sequencing and RNA sequencing were performed to explore the underlying mechanism,and stable cell lines with overexpression or knockdown were constructed to verify the mechanism.This study also established an HCC-specific miRNA antagomir delivery system applying an aptamer/liposome complex.Results:Tibial-injected bone lesions secreted EVs and tail vein-injected EVs were detected in mice orthotopic liver tumors,suggesting that BM-EVs can colonize in HCC cells.Tail vein injection of EVs extracted from bone metastatic cells was found to promote in situ proliferation and extrahepatic metastasis of HCC.Furthermore,miR-3190-5p(miR-3190)was found to be upregulated in HCC bone metastatic cells and their derived EVs.In HCC tissue microarrays,high expression of miR-3190 predicted poorer prognosis and higher metastasis rate.miR-3190 can promote migration and invasion of HCC cells in vitro,and down-regulating miR-3190 in EVs eliminated the promotion effect of EVs on HCC.Through the prediction of public databases,the detection of RNA and protein levels,and luciferase reporter assays,it was confirmed that Alk B homolog 5(Alk B homolog 5,ALKBH5)is a downstream negative effector of miR-3190.ALKHB5 inhibited the motility of HCC cells in vitro,and overexpression of ALKBH5 suppressed the pro-metastasized function of EVs on HCC mediated by miR-3190.Mechanistically,ALKBH5 regulated DEPDC1 and NTSR1 in an N6-methyladenosine(m~6A)-dependent manner,and regulated FN1,CDH2,EGFR,ITGA6,ITGB4 and other genes in an m6A-independent manner,which co-activated downstream metastasis-related pathways,promoting HCC metastasis.Finally,miR-3190 antagonist-loaded liposomes with HCC affinity successfully suppressed HCC progression in mice treated with BM-EVs.Conclusions:These findings indicated that EVs derived from HCC bone metastasis initiated a pro-metastatic cascade in orthotopic HCC by delivering ALKBH5-targeting miR-3190,providing a new understanding for the EV-mediated bone-liver axis communication.Furthermore,miR-3190 is a potential therapeutic target to inhibit tumor progression in HCC patients with bone metastasis.