Expression Of G9A In Hepatocellular Carcinoma And Its Effect On Cell Proliferation And Autophagy In Hepatocellular Carcinoma Cells

Objective:G9A, the primary histone methyltransferase (HMTase) for histone H3 lysine 9, has been reported to be over-expressed in many types of cancers including hepatocellular carcinoma (HCC). Suppressing its expression or activity could repress proliferation and induce autophagy related cell death in many cancer cells. However, the correlation between G9A, cell proliferation and autophagy has not yet been reported in HCC. Therefore, this research explores the effects of G9A inhibition on HCC cells.Methods:We analyzed the expression of G9A in public available microarray datasets which included GSE14520 (n=488), GSE6764 (n=75), GSE14323 (n=124), GSE50579 (n=80). JAVA program for GSEA was used to analyze the potential genes that are influenced by G9A high expression. Huh7 and HepG2 were treated with G9A's specific inhibitor (BIX-01294) at different time points. Cell morphology changes were then observed under light microscopy and CCK8 assay was used for cell viability analysis. Flow cytometry was then performed to assess cell cycle distribution and AnnexinV/PI double staining was performed to verify cell apoptosis activation. In addition, immunofluorescence was used for LC3B punctual observation and western blotting was applied to detect the expression of cell cycle and autophagy related proteins. Furthermore, clone-formation assay was used to verify the G9A functions related to proliferation. Using immune deficiency mice as a model, the tumor size, volume and weight were analyzed to evaluate tumorigenic ability after treatment with BIX-01294 at different concentration.Results:G9A expression level was increased in HCC tumor tissues, and was significantly associated with serum AFP level in HCC patients, which could serve as a potential prognostic indicator for patients with multinodular HCC. The CCK8 assay results indicated that cell viability was dramatically decreased by BIX-01294 treatment in both Huh7 and HepG2 cell lines. Gene set differences in G9A high vs. low expressing patients from GSEA indicated that G9A regulated gene sets mainly associated with cell proliferation and cell cycle. Inhibition of G9A activity suppressed proliferation and induced G1 arrest in HCC cells, while autophagy-related cell death was induced in Huh7. The expression level of microtubule-associated protein light chain 3 beta (LC3B) was increased after treatment with 5?M BIX-01294 within 24 hours in both HCC cell lines. Importantly, linear-regression analysis indicated an inverse correlation between the expression of G9A and LC3B in HCC tumor samples in GSE14520 dataset. Using clone-formation assay we proved that, after G9A inhibition by BIX-01294, the clone-formation ability was deceased, which further proves that BIX-01294 could suppress HCC proliferation ability. We postulate that G9A regulates HCC proliferation by mediating histone methylation. In vivo experiments using SCID mice model showed that the tumor size and volume were decreased when treated with 1.0 mg/ml and 2.0 mg/ml of BIX-01294, and the weight of the mice was not significantly affected by BIX-01294 treatment.Conclusions:In summary, G9A could influence proliferation and induce autophagy related cell death, hence promoting HCC progression. Our results suggest that G9A could be used as potential prognosis marker and therapeutic target, which could provide new clues for G9A inhibitor in clinical diagnosis and treatment.