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为了获取和发掘α-硫辛酸作用于肝癌细胞后的转录组数据库和Bcl-2家族蛋白差异表达基因,本研究采用高通量测序技术Illumina HiSeqTM2000对BEL-7402细胞的α-硫辛酸处理组和对照组进行转录组测序,再进行系统的生物信息学分析并筛选出Bcl-2家族蛋白相关的差异表达基因。然后通过PCR及Western blotting技术检测部分基因的表达,来验证测序结果的准确性和可靠性。最后再用流式细胞仪检测α-硫辛酸对肝癌细胞系HepG2、BEL-7402凋亡率的影响。本次测序分析共获得4 671个差异表达基因,包括2 223个表达上调,2 448个表达下调。可归类到48个GO (Gene ontology)分类中,KEGG Pathway显著性富集共涉及6条途径,对差异性表达基因进一步筛选得到8个Bcl-2家族蛋白相关基因。PCR及Western blotting分析表明,在肝癌细胞HepG2、BEL-7402细胞中,α-硫辛酸处理组的Bax、Bak促凋亡基因m RNA及蛋白质水平表达均上调,与测序分析结果一致。流式细胞仪分析表明,α-硫辛酸可促进肝癌细胞系BEL-7402、HepG2凋亡,凋亡率分别为24.70%和25.40%,p值均小于0.05,差异具有统计学意义。本研究通过对α-硫辛酸作用于肝癌细胞后的转录组进行测序分析,得到了大量转录本信息,测序结果高效、准确,可为研究α-硫辛酸诱导肝癌细胞凋亡的分子机制提供重要线索。
Abstract：To obtain the transcriptome database and Bcl-2 family protein-related differentially expressed genes of BEL-7402 cells which treated with α-lipoic acid(α-LA), we use the high-throughput sequencing technology(Illumina HiSeqTM2000) to sequence the transcriptome of BEL-7402 cells' control groups and the treated groups,and we performed a systematic bioinformatics analysis and selected the differentially expressed genes related to Bcl-2 family proteins. Then we chose some genes for PCR and Western blotting analyses to verify the reliability and accuracy of sequencing results. Finally, we detected the apoptosis rate of hepatoma cell lines HepG2 and BEL-7402 treated with α-LA by flow cytometry. A total of 4 671 differentially expressed genes were obtained in this transcriptome analysis, including 2 223 up-regulated genes and 2 448 up-regulated genes, which could be classified into 48 GO classifications and 6 metabolic pathways. There were 8 Bcl-2 family protein-related genes we obtained from further analysis of differentially expressed genes. PCR and Western blotting analyses showed that the expression of Bax and Bak genes' mRNA and protein levels were up-regulated in the α-lipoic acid-treated group in liver cells HepG2 and BEL-7402. So the validation results was consistent with the sequencing results.Flow cytometry analysis showed that α-LA promote the apoptosis of BEL-7402 and HepG2 cells, the apoptosis rate were 24.70% and 25.40%, p<0.05, the differences were statistically significant. In this study, a large amount of transcript information was obtained by sequencing the transcriptome of hepatocellular carcinoma cells which treated with α-lipoic acid. The sequencing results were efficient and accurate, which provides important clues for studying the molecular mechanism of α-lipoic acid-induced apoptosis of hepatoma cells.
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基本信息:
DOI：10.13417/j.gab.039.003672
中图分类号:R735.7
引用信息:
[1]张满桥,文娅,陆菁潇,等.α-硫辛酸作用肝癌细胞的转录组分析及验证[J].基因组学与应用生物学,2020,39(08):3672-3680.DOI:10.13417/j.gab.039.003672.
基金信息:
国家自然科学基金面上项目(81672282; 81372507);; 国家科技部国际合作项目(2014DFA31050);; 深圳市发改委工程实验室组建项目(深发改[2011]1782号)共同资助
2020-08-25
2020-08-25