To investigate the mechanism of circZKSCAN1 inhibiting the invasion and metastasis of hepatocellular carcinoma (HCC) cells.

HepG2 cells which differentially expressed circZKSCAN1 gene were utilized in this experiment. The migration and invasion capability of HCC cells in the OE group (HepG2 overexpressing circZKSCAN1), SH-1 and SH-2 groups (HepG2 silencing circZKSCAN1) and NC group (negative control) was assessed by colony formation assay, scratch test and Transwell chamber test, respectively. The binding sites of enriched RNA-binding protein (RBP) in circZKSCAN1 were identified by bioinformatics analysis. The target RBP was verified by RNA immunoprecipitation (RIP) and RNA pull-down experiment. The relationship between the target RBP and survival of HCC was analyzed. The number of colony formation, migration and number of membrane-penetrating cells among 4 groups were analyzed by single-factor analysis of variance and LSD-t test. The enrichment multiples between two groups were compared by t test. Survival analysis was performed by Kaplan-Meier method and Log-rank test.

Colony formation assay showed that the number of colony formation in the NC, OE, SH-1 and SH-2 groups were 10.5±1.1, 4.7±0.8, 21.7±1.8 and 22.6±2.6, respectively. Compared with the NC group, the number of colony formation in the OE group was significantly reduced, whereas those in the SH-1 and SH-2 groups were significantly increased (LSD-t=-7.386, 9.196, 7.424; P<0.05). Scratch test demonstrated that compared with the NC group, the 24- and 48-h migration ability in the OE group was significantly decreased, whereas those in the SH-1 and SH-2 groups were significantly increased. Transwell chamber test found that the number of migrating cells in the NC, OE, SH-1 and SH-2 groups was 28.5±4.3, 14.3±2.6, 47.2±7.7 and 49.6±8.0, respectively. Compared with the NC group, the number of migrating cells in the OE group was significantly reduced, while those in the SH-1 and SH-2 groups were significantly elevated (LSD-t=-4.895, 3.673, 3.474; P<0.05). Transwell chamber test showed that the number of membrane-penetrating cells in the NC, OE, SH-1 and SH-2 groups was 21.3±2.1, 7.6±1.5, 38.6±5.8 and 41.3±7.1, respectively. Compared with the NC group, the number of membrane-penetrating cells in the OE group was significantly reduced, whereas those in the SH-1 and SH-2 groups were significantly increased (LSD-t=-9.195, 4.858, 4.679; P<0.05). Bioinformatics analysis revealed that enriched RBP-binding sites in circZKSCAN1 consisted of EIF4A3, FMR1, hnRNPC and U2AF65, etc. RIP experiment indicated that circZKSCAN1 could significantly enrich intracellular EIF4A3. Compared with the NC group, the enrichment multiple in the OE group was (7.82±0.25) times (t=1.057, P<0.05). RNA pull-down experiment showed that EIF4A3 could bind with circZKSCAN1. Online website (http://gepia.cancer-pku.cn) analysis demonstrated that compared with the adjacent tissues, EIF4A3 gene was highly expressed in HCC tissues, mainly in HCC of advanced stage (F=7.72, P<0.05). However, the overall survival rate of HCC patients with high expression level of EIF4A3 gene was even worse (HR=1.90, P<0.05).

EIF4A3 is the target RBP of circZKSCAN1. circZKSCAN1 may suppress the invasion and metastasis of HCC cells by binding with EIF4A3.