Anti-Tumor Effect of Celastrol on Hepatocellular Carcinoma by the circ_SLIT3/miR-223-3p/CXCR4 Axis

BACKGROUND: Celastrol is a potential anti-tumor agent in hepatocellular carcinoma (HCC). Identifying the molecular determinants of the anti-HCC effect of celastrol is still challenging. In this study, we undertook to associate circular RNAs (circRNAs) with the anti-HCC molecular determinants of celastrol. METHODS: Cell colony formation, proliferation, migration, invasion and apoptosis were determined using the colony formation, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTS), transwell and flow cytometry assays, respectively. The levels of circRNA slit guidance ligand 3 (circ_SLIT3), miR-223-3p and C-X-C motif chemokine receptor 4 (CXCR4) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Ribonuclease R (RNase R) and actinomycin D assays were performed to assess the stability of circ_SLIT3. Targeted relationships among circ_SLIT3, miR-223-3p and CXCR4 were confirmed by the dual-luciferase reporter assay. In vivo assays were performed to detect the roles of celastrol and circ_SLIT3 on tumor growth in vivo. RESULTS: Celastrol repressed HCC cell proliferation, migration, invasion, and enhanced apoptosis in vitro and suppressed tumor growth in vivo. Celastrol down-regulated circ_SLIT3 expression in HCC cells, and celastrol exerted an anti-tumor effect on HCC in vitro and in vivo by down-regulating circ_SLIT3. Mechanistically, circ_SLIT3 directly interacted with miR-223-3p, and circ_SLIT3 controlled CXCR4 expression by sponging miR-223-3p. Moreover, miR-223-3p was involved in the celastrol/circ_SLIT3-mediated regulation on HCC progression. Furthermore, celastrol exerted the anti-HCC effect in vitro through the miR-223-3p/CXCR4 axis. CONCLUSION: Our present work first identified the circ_SLIT3/miR-223-3p/CXCR4 axis as a novel mechanism of the anti-HCC effect of celastrol, providing a new insight into the involvement of circRNAs in the anti-tumor molecular determinants of celastrol.