Research on the effect and mechanism of hepatoma cells HepG2 exosome on its own methylation / 医学研究生学报

ABSTRACT

ObjectiveThe prognosis of patients with advanced liver cancer is poor and there is no effective treatment so far. This paper observed the effect of hepatoma cells HepG2 exosomes on its own methylation and attempted to explore its mechanism.MethodsThere was experimental group and control group in the research. The medium has been changed when the cells grow to 60% in a DMEM culture dish with 10% serum. Cells in the control group were cultured in serum-free DMEM medium while the experimental group were cultured in serum-free DMEM medium added 100 L (0.5mg/mL) exosomes, and the supernatant was retained after incubation for 48h. HepG2 cells were cultured and exosomes were extracted by overspeed differential centrifugation, and identified by particle size analysis, transmission electron microscopy, Western blot and other methods. The effect of exosomes of hepatocellular carcinoma cells HepG2 on cell proliferation was analyzed by scratch test. Fluorescence antibody staining was used to observe the change of automethylation level. Fluorescence quantitative PCR was used to detect mRNA expression levels of methyltransferase-related genes DNMT3A, DNMT3B, DNMT1, and apoptosis-related genes Bax and BcI2.ResultsUnder inverted fluorescence microscope, red fluorescent exosomes could be seen entering the cell, surrounding the blue fluorescent nucleus or on the nucleus, indicating that DiI entered the cell membrane or cytoplasm. The area ratio of 6 and 12 h in the experimental group [(57.25±2.06, 83.92±3.17) %] was significantly higher than that in the control group [(28.32±1.22, 40.03±1.74) %] (P<0.05). The genes expressions of Bax, DNMT3A and DNMT3B in the experimental group were higher than those in the control group (P<0.05). The expression of BcI2 was lower than that of the control group (P<0.05).ConclusionThe exosomes of hepatoma cell HepG2 can enhance DNA methylation level by changing the transcriptional expression of DNMT3A, DNMT3B and other genes to affect the expression of apoptose-related genes Bax and BcI2, and to promote the proliferation and growth capacity of hepatoma cell HepG2.