Nanoparticles Mediated circROBO1 Silencing to Inhibit Hepatocellular Carcinoma Progression by Modulating miR-130a-5p/CCNT2 Axis

Hongyu Meng,1,2,* Ruixi Li,3,* Yuankang Xie,1,* Zhaohong Mo,2 Hang Zhai,2 Guangquan Zhang,3 Guohui Liang,4 Xianjie Shi,3 Boxuan Zhou1,2 1Department of Hepatobiliary Surgery, The First Affiliated Hospital of Gannan Medical University, Ganzhou, People’s Republic of China; 2Department of Hepatobiliary Surgery, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of China; 3Department of Hepatobiliary and Pancreatic Surgery, The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, People’s Republic of China; 4School of Clinical Medicine, Henan University, Kaifeng, People’s Republic of China*These authors contributed equally to this workCorrespondence: Boxuan Zhou; Xianjie Shi, Email zhoubx3@mail2.sysu.edu.cn; shixianjie301@126.cmBackground: Circular RNAs (circRNAs) are becoming vital biomarkers and therapeutic targets for malignant tumors due to their high stability and specificity in tissues. However, biological functions of circRNAs in hepatocellular carcinoma (HCC) are still not well studied.Methods: Gene Expression Omnibus (GEO) database and qRT-PCR were used to evaluate expression of circROBO1 (hsa_circ_0066568) in HCC tissues and cell lines. CCK-8, colony formation, EdU staining, flow cytometry for cell cycle analysis, and xenograft model assays were performed to detect the circROBO1 function in vitro and in vivo. RNA pull-down, RNA immunoprecipitation (RIP), and Luciferase reporter assays were used to investigate the relationship among circROBO1, miR-130a-5p, and CCNT2. More importantly, we developed nanoparticles made from poly lactic-co-glycolic acid (PLGA) and polyethylene glycol (PEG) chains as the delivery system of si-circROBO1 and then applied them to HCC in vitro and in mice.Results: circROBO1 was obviously upregulated in HCC tissues and cell lines, and elevated circROBO1 was closely correlated with worse prognosis for HCC patients. Functionally, knocking down circROBO1 significantly suppressed HCC cells growth in vitro and in mice. Mechanistically, circROBO1 acted as a competing endogenous RNA to downregulate miR-130a-5p, leading to CCNT2 expression upregulation. Furthermore, miR-130a-5p mimic or CCNT2 knockdown reversed the role of circROBO1 overexpression on HCC cells, which demonstrated that circROBO1 promoted HCC development via miR-130a-5p/CCNT2 axis. In addition, we developed nanoparticles loaded with si-circROBO1, named as PLGA-PEG (si-circROBO1) NPs, which significantly prevented the proliferation of HCC cells, and did not exhibit apparent toxicity to major organs in vivo.Conclusion: Our findings firstly demonstrate that circROBO1 overexpression promotes HCC progression by regulating miR-130a-5p/CCNT2 axis, which may serve as an effective nanotherapeutic target for HCC treatment.Keywords: nanoparticles, hepatocellular carcinoma, circROBO1, cell proliferation, drug delivery