Circular RNA circRASSF5 Functions as an Anti-Oncogenic Factor in Hepatocellular Carcinoma by Acting as a Competitive Endogenous RNA Through Sponging miR-331-3p

Zhao Zhou,1,2,* Xiaohan Cui,1,2,* Peng Gao,2 Xudong Zhang,2 Chunfu Zhu,2 Beicheng Sun1 1Department of Hepatobiliary Surgery of Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing, People’s Republic of China; 2The Affiliated Changzhou NO.2 People’s Hospital of Nanjing Medical University, Changzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Chunfu Zhu, The Affiliated Changzhou NO.2 People’s Hospital of Nanjing Medical University, Changzhou, People’s Republic of China, Email zcfmlm@njmu.edu.cn Beicheng Sun, Department of Hepatobiliary Surgery of Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing, People’s Republic of China, Email sunbc@nju.edu.cnObjective: Recently, emerging studies have validated that circular RNAs participate in multiple biological progresses in various human malignant tumors, including hepatocellular carcinoma (HCC). However, until now, the elucidated mechanism of circular RNAs is only the tip of the iceberg. In this study, we firstly identify a novel circular RNA circRASSF5 (the only circular RNA derived from the RASSF5 gene), and attempt to investigate its biological function and underlying mechanism in HCC.Methods: qRT-PCR, Western blotting and IHC were applied to detect the expression of related genes. CCK-8 assay, EdU staining, wound healing and transwell assays were used to investigate HCC proliferation, migration and invasion abilities. Animal model studies were included to investigate the function of circRASSF5 in HCC tumorigenesis and metastasis. RNA pull-down assay, luciferase reporter assay and FISH (fluorescence in situ hybridization) assay were performed to explore the potential biological mechanism underlying circRASSF5 function in HCC.Results: CircRASSF5 is obviously downregulated in both HCC tissues and cell lines. Low level of circRASSF5 is negatively associated with larger tumor size, severe vascular invasion, more portal vein tumor embolus and unfavorable prognosis. Loss-of-function assay reveals that circRASSF5 remarkably impedes the growth and metastasis of HCC cells in vitro and in vivo. Mechanistically, circRASSF5 directly interacts with miR-331-3p as a sponge, and then enhances the expression of PH domain and leucine-rich repeat protein phosphatase (PHLPP), thus restraining the progression of HCC cells.Conclusion: Altogether, we validate that circRASSF5 is a tumor suppressor in HCC, which competitively sponges with miR-331-3p and then enhances the tumor inhibitory effect of PHLPP, indicating the potential application value of circRASSF5 for HCC diagnosis and clinical treatment.Keywords: circRASSF5, miR-331-3p, PHLPP, HCC