Abstract: Objective The aim of this experiment was to investigate the molecular mechanism by which regulating synaptic membrane exocytosis 3 (RIMS3) mediated lenvatinib resistance in hepatocellular carcinoma (HCC) cells by regulation of γ-aminobutyric acid (GABA) secretion in vitro. Methods Lenvatinib-resistant Hep3B-LR and MHCC97H-LR were established by using a concentration gradient induction in Hep3B and MHCC97H cells. Bioinformatic analysis was employed to screen differentially expressed genes from transcriptome sequencing data of resistant and wild-type cell lines, as well as from external drug resistance datasets (GSE186191 and GSE211850). Cox proportional hazards regression analysis and survival analysis were applied to evaluate the impact of key genes on the survival and prognosis of patients with HCC. The IC₅₀ values were determined by CCK-8 assay, and the resistance index was calculated. siRNAs targeting RIMS3 were synthesized, and its mRNA and protein expression were detected by qRT-PCR and Western blotting, respectively. GABA levels in the cell culture supernatant was measured by ELISA. Results Resistance indices of Hep3B-LR and MHCC97H-LR were 3.3 and 3.1, respectively, greater compared to wild type; 29 drug resistance-related differentially expressed genes were identified, among which RIMS3 was significantly upregulated in resistant cells (P<0.01); RIMS3 served as an independent risk factor, and its high expression was associated with poor prognosis compared to HCC patients with low RIMS3 expression (P<0.05); targeted silencing RIMS3 (siRIMS3-1 and siRIMS3-2) significantly reduced the IC₅₀ values of resistant cells (Hep3B-LR decreased from 40.3 μM to 23.9 μM and 19.1 μM; MHCC97H-LR decreased from 20.2 μM to 8.3 μM and 12.3 μM); GABA levels in the supernatant of Hep3B-LR and MHCC97H-LR cells increased by (1.6±0.3)-fold and (1.4±0.2)-fold, respectively (P<0.05) compared to in wild cells; targeted silencing RIMS3 reduced GABA levels in the supernatant of Hep3B-LR cells by (0.3±0.1)-fold and (0.3±0.1)-fold (P<0.01), and in MHCC97H-LR cells by (0.5±0.2)-fold and (0.5±0.1)-fold (P<0.01); addition of exogenous GABA increased the IC₅₀ value of Hep3B-LR from 18.6 μM to 30.5 μM and that of MHCC97H-LR from 9.7 μM to 18.9 μM as compared with RIMS3-silenced resistant cells. Conclusion RIMS3 mediates lenvatinib resistance in HCC cells, maybe by regulating GABA secretion, and targeting RIMS3 might represent a novel strategy for reversing lenvatinib resistance.

Key words: Hep3B cells, MHCC97H cells, Lenvatinib, Drug resistance, Regulating synaptic membrane exocytosis 3, γ-aminobutyric acid, In vitro