Abstract: Objective To investigate the role of glutamate⁃cysteine ligase catalytic subunit (GCLC) promoter methylation in the malignant transformation of hepatocyte induced by microcystin⁃LR (MCLR), as well as to assess the intervention effects of the DNA methylation inhibitor 5⁃aza⁃2'⁃deoxycytidine (5⁃aza). Methods A malignant transformation model was established by subjecting WRL68 cells to chronic exposure to 10 nmol/L MCLR until passage 25. Four experimental groups were constituted: the control group, the MCLR⁃exposed group, the 5⁃aza intervention group, and the MCLR plus 5⁃aza intervention group. The methylation status of the  GCLC gene promoter was assessed using Agena MassArray mass spectrometry⁃based nucleic acid analysis technology. Protein expression levels of GCLC and DNA methyltransferases (DNMTs) were determined via Western blot. Cellular proliferation, invasion, and migration capabilities were detected via Colony formation assay, soft agar assay, and Transwell assays, respectively. Additionally, glutamate⁃cysteine ligase (GCL) activity, glutathione (GSH) content, and 8⁃hydroxydeoxyguanosine (8⁃OHdG) levels were quantified using corresponding reagent kits. Results During the MCLR⁃induced malignant transformation of WRL68 cells, there was a progressive increase in the methylation level of the GCLC promoter, accompanied by a gradual decrease in both mRNA and protein expression levels (all  P<0.05). At passage 25 in the MCLR⁃exposed group, there was a significant elevation in the protein levels of DNMT⁃1, DNMT⁃3a, and DNMT⁃3b, while GCL activity and GSH content were markedly reduced, and 8⁃OHdG levels were significantly elevated (all P<0.05). Intervention with 5⁃aza at passage 25 in the MCLR⁃exposed group resulted in a reduction of GCLC promoter methylation levels, significantly upregulating GCLC mRNA and protein expression, enhanced GCL activity and GSH content, and reduced 8⁃OHdG levels (all P<0.05). Furthermore, 5⁃aza attenuated the migration, invasion, and cell colony formation capabilities of the MCLR⁃exposed group (all P<0.05). Conclusions GCLC promoter methylation contributes to the MCLR⁃induced malignant transformation of WRL68 cells. 5⁃aza may inhibit this malignant transformation by downregulating DNMTs expression, suppressing hypermethylation of the GCLC promoter, reactivating the expression of the silenced GCLC gene, and restoring its tumor⁃suppressive function.

Key words: Glutamate?cysteine ligase catalytic subunit, DNA methylation, 5?aza?2'?deoxycytidine, Microcystin?LR, Hepatocyte  malignant transformation