2种酮化合物诱导HepG2细胞发生细胞自噬研究

Research on autophagy induced by two xanthone compounds in HepG2 cells

探究2种■酮化合物1-羟基-2,3,4,8-四甲氧基■酮(简称Fr15)和1-羟基-2,3,4,6-四甲氧基■酮(简称Fr17)对肝癌细胞HepG2的增殖抑制作用,结合转录组学进一步研究其增殖抑制的作用机制。通过细胞计数法检测2种■酮化合物Fr15和Fr17(0,0.03,0.15,0.3 mmoL·L^(-1))对HepG2细胞增殖的影响;流式细胞仪检测2种化合物对HepG2细胞周期的影响;荧光显微镜观察细胞中自噬体形成的数量变化;Western blot法检测细胞中自噬标志性蛋白SQSTM 1(p62)及微管相关蛋白1轻链3Ⅰ/Ⅱ(LC3Ⅰ/Ⅱ)的表达情况;对照组与实验组通过RNA-seq转录组学测序技术,分析差异表达基因;qRT-PCR法验证测序差异表达基因对比结果。结果发现细胞经化合物Fr15和Fr17处理后,随着药物浓度和时间的增加HepG2细胞的增殖受到了抑制,流式细胞仪检测发现化合物Fr15和Fr17对HepG2细胞周期影响不大。荧光显微镜表明随着化合物Fr15和Fr17浓度的增加,细胞中自噬体数量逐渐变多。Western blot结果表明加药后的细胞p62蛋白表达下降,同时LC3Ⅱ蛋白表达明显升高。RNA测序结果分析Fr15和Fr17,分别得到26 102个及52 351个差异表达基因。KEGG分析发现化合物处理后对细胞自噬信号通路影响较大。qRT-PCR验证6个与自噬相关的表达上调基因,其变化趋势与测序结果一致,6个基因均出现上调趋势。2种■酮化合物Fr15和Fr17可能通过诱导HepG2细胞发生细胞自噬来抑制其增殖。

To investigate the inhibitory effects of two xanthone compounds,1-hydroxy-2,3,4,8-4 methoxy xanthone(here in after referred to as Fr15)and 1-hydroxy-2,3,4,6-4 methoxy xanthone(here in after referred to as Fr17),on the proliferation of hepatocellular carcinoma cells HepG2,and to further investigate their mechanism in combination with transcriptomics.Cell counting was used to detect the effects of two kinds of xanthone compounds Fr15 and Fr17(0,0.03,0.15,0.3 mmoL·L-1)on the proliferation of HepG2 cells;the effects of the two compounds Fr15 and Fr17 on HepG2 cell cycle were detected by flow cytometry;the changes of autophagosomes count in cells were observed under fluorescence microscope;the expression of autophagy marker proteins autophagy marker proteins SQSTM 1(p62)and microtubule associated protein 1 light chain 3Ⅰ/Ⅱ(LC3Ⅰ/Ⅱ)in the cells was detected by Western blot;the differentially expressed genes between the control group and the experimental group were analyzed by RNA-seq transcriptome sequencing;qRT-PCR was used to verify the differentially expressed genes in sequencing.The results showed that compounds Fr15 and Fr17 inhibited the proliferation of HepG2 cells with the increase of drug concentration and time.Flow cytometry showed that compounds Fr15 and Fr17 had little effect on HepG2 cell cycle.Fluorescence microscopy results showed that the number of autophagosomes in cells increased with the increase of drug concentration.Western blot showed that the expression of p62 protein was decreased and the expression of LC3-Ⅱprotein was significantly increased after drug addition.The results of RNA sequencing showed that 26102 and 52351 differentially expressed genes were obtained in Fr15 and Fr17 respectively.Analysis of KEGG showed that drug treatment had a great effect on autophagy pathway.qRT-PCR verified that 6 up-regulated genes were related to autophagy,and their trend was consis-tent with sequencing results,where all 6 genes showed an up-regulated trend.Two xanthone compounds Fr15 and Fr17 may inhibit proliferation of HepG2 cells by inducing autophagy.