miR-139-5p Targets CENPM to Suppress EMT and Malignant Progression of Hepatocellular Carcinoma via Regulating the Akt/mTOR Pathway and β-Catenin Nuclear Translocation

Aihui Fan,1,* Xiaomin Zhang,1,* Hongjing Cui,2,* Xianling Gong,3 Zhanping Gou,3 Chong Yan,3 Yanfei Guan,1 Shengchun Wang,4 Jingyi Zheng,1 Wanyi Li,5 Linghui Dian,3,5 Jilun Cheng3,5,61Dongguan Key Laboratory of Stem Cell and Regenerative Tissue Engineering, School of Basic Medical Sciences, Guangdong Medical University, Dongguan, Guangdong, People’s Republic of China; 2Department of Histology and Embryology, School of Basic Medical Sciences, Guangdong Medical University, Dongguan, Guangdong, People’s Republic of China; 3Department of Traditional Chinese Medicine, School of Pharmacy, Guangdong Medical University, Dongguan, Guangdong, People’s Republic of China; 4Department of Pathology, School of Basic Medical Sciences, Guangdong Medical University, Dongguan, Guangdong, People’s Republic of China; 5Dongguan Key Laboratory of Screening and Research of Anti-Inflammatory Ingredients in Chinese Medicine, Guangdong Medical University, Dongguan, Guangdong, People’s Republic of China; 6Dongguan Branch, National Engineering Research Center for Modernization of Traditional Chinese Medicine, Dongguan, Guangdong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jilun Cheng, Department of Traditional Chinese Medicine, School of Pharmacy, Guangdong Medical University, No. 1 Xincheng Blvd, Songshan Lake National High-Tech Industrial Development Zone, Dongguan, Guangdong, 523808, People’s Republic of China, Email [email protected] Linghui Dian, Email [email protected]: This study aims to screen key miRNA/mRNA axes associated with hepatocellular carcinoma (HCC), and then conduct functional validation and mechanistic exploration, thereby offering novel clues for screening HCC-targeted therapeutic candidates.Methods: Bioinformatics analysis was performed to identify key HCC-related miRNA/mRNA axes. The direct interaction between hsa-miR-139-5p and centromere protein M (CENPM) was verified via a dual-luciferase reporter (DLR) assay. We carried out colony formation, Transwell and in vivo xenograft assays to validate the biological functions of the hsa-miR-139-5p/CENPM axis. The underlying mechanism of this axis was further explored via in vitro cellular experiments. The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort was used for prognostic evaluation of this axis.Results: Six key HCC-related miRNA/mRNA regulatory axes were screened out. The hsa-miR-139-5p/CENPM axis was selected for further validation. DLR assay confirmed that hsa-miR-139-5p directly targeted CENPM. Sole overexpression of hsa-miR-139-5p significantly downregulated CENPM protein levels, and suppressed in vitro cell colony formation, migration and invasion, as well as in vivo growth of subcutaneous HCC xenografts. Notably, simultaneous overexpression of CENPM could fully rescue the tumor-suppressive phenotypes induced by hsa-miR-139-5p overexpression. In contrast, sole overexpression of CENPM markedly promoted HCC cell malignant behaviors and subcutaneous xenograft growth, while concurrent transfection of hsa-miR-139-5p could attenuate these oncogenic effects driven by CENPM overexpression. Mechanistically, hsa-miR-139-5p restrained the activation of the Akt/mTOR pathway and inhibited nuclear translocation of β-catenin to suppress epithelial-mesenchymal transition (EMT) by targeting CENPM in HCC cells. The hsa-miR-139-5phigh/CENPMlow subgroup had significantly better survival outcomes than the hsa-miR-139-5plow/CENPMhigh subgroup.Conclusion: hsa-miR-139-5p targeted CENPM to suppress EMT and malignant progression of HCC via regulating the Akt/mTOR pathway and β-catenin nuclear translocation. The combined expression pattern of hsa-miR-139-5p and CENPM correlated significantly with HCC patient prognosis. Our findings offer new evidence for HCC prognostic stratification and targeted intervention candidate screening.Keywords: bioinformatics analysis, hepatocellular carcinoma, CENPM, hsa-miR-139-5p, Akt/mTOR pathway, EMT