肝癌细胞HepG2外泌体对其自身甲基化的影响及其机制探究

Research on the effect and mechanism of hepatoma cells HepG2 exosome on its own methylation

目的晚期肝癌患者预后很差,迄今为止尚无有效的治疗方法。文章观察肝癌细胞HepG2外泌体对自身甲基化水平的影响,并尝试探讨其机制。方法研究设置实验组和对照组,待细胞在10%血清的DMEM培养皿中长至60%进行换液,对照组胞用无血清的DMEM培养基培养,实验组为无血清的DMEM加入100μL(0.5 mg/mL)外泌体,孵育48 h后留取上清液。HepG2细胞培养并用超速差速离心法提取其外泌体,用粒径分析、透射电镜、Western blot等方法对其进行鉴定。通过划痕实验分析肝癌细胞HepG2细胞外泌体对细胞增殖能力的影响;荧光抗体染色观察其对自身甲基化水平的变化;荧光定量PCR检测甲基转移酶相关基因DNMT3A、DNMT3B、DNMT 1和凋亡相关基因Bax、BcI-2等mRNA表达量的水平变化。结果倒置荧光显微镜下可见红色荧光的外泌体进入了细胞内,围绕于蓝色荧光的细胞核周围或核上,表明DiI进入了细胞膜或细胞质内。实验组6、12 h面积占比[(57.25±2.06、83.92±3.17)%]较对照组[(28.32±1.22、40.03±1.74)%]明显增加(P<0.05)。实验组Bax、DNMT3A、DNMT3B基因表达高于对照组(P<0.05);而BcI-2表达低于对照组(P<0.05)。结论肝癌细胞HepG2自身外泌体可以通过改变DNMT3A、DNMT3B等基因的转录表达量来增强DNA甲基化水平从而影响凋亡相关基因Bax、BcI-2类基因的表达,进而促进肝癌细胞HepG2增殖生长能力。

Objective The 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.Methods There 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.Results Under 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).Conclusion The 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.