Mechanisms of G0/G1 phase arrest induced by longevity-assurance homologue 2 in Hepa1-6 hepatoma cells

Abstract

Objective To investigate the effects of longevity-assurance homologue gene 2 (Lass2) on the cell cycle ofmouse hepatic carcinoma cell line Hepa1-6 and the related mechanism. Methods Lass2 recombinant eukaryotic expression vector has been successfully constructed in the early stage, and were transfected in Hepa1-6 hepatoma cell line 48 h by liposome 2000. The cell cycle of each group (negative control, empty load, experimental group) was detected by flow cytometry (PI). The relative expression levels of Lass2, Cyclin D1, nuclear factor-κB (NF-κB) p65 mRNA and protein were detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting. The phosphorylation of NF-κB p65 protein were detected by Western blotting; In order to further verify and clarify the mechanism, the C57BL/6J mouse hepatic carcinoma in situ model was constructed by injecting Hepa1-6 hepatic carcinoma cell suspension directly into the liver parenchyma of C57BL/6J mice. The naked plasmid was injected into the tail vein separately by hydrodynamics, and the total RNA and total protein in liver tissue of each group were extracted after transfection. The relative expression levels of Lass2, Cyclin D1, NF-κB p65 mRNA and protein in mice liver were detected by FQ-PCR and Western blotting. The phosphorylation of NF-κB p65 protein were detected by Western blotting. Results The relative expression levels of Lass2 mRNA and protein in Hepa1-6 hepatoma cells were significantly higher than those in negative control group and blank control group (F=1 486.895, all P=0.000; protein: F=55.964, all P=0.000), and the percentage of cell cycle G0/G1 phase increased significantly (F=239.867, all P=0.000), significantly decreased in G2/M phase and S phase (FG2/M=13.462, PG2/M=0.003, 0.005; FS=46.207, all P=0.000), at the same time, the levels of NF-κB p65, CyclinD1 mRNA and protein in the experimental group were decreased (mRNA: FNF-κB p65=29.993, FCyclin D1=113.260, all P=0.000; protein: FNF-κB p65=17.679, P=0.003, 0.002; FCyclin D1=176.357, all P=0.000), the phosphorylation of NF-κB p65 were significantly downregulated (FNF-κB p-p65=33.817, P=0.000). Conclusion Lass2 gene blocks the cell cycle of Hepa1-6 cells in G0/G1 phase, and its mechanism may be related to inhibits NF-κB p65 activation and expression and down-regulation of Cyclin D1 transcription and protein expression. Key words: Longevity-assurance homologue gene 2; Hepatocellular carcinoma; Cell cycle; Nuclear factor-κB p65; Cyclin D1