Chen Hong-Qiang, Zhao Ji, Li Yan, He Li-Xiong, Huang Yu-Jing, Shu Wei-Qun, Cao Jia, Liu Wen-Bin, Liu Jin-Yi
Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, PR China.
Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, PR China; College of Public Health and Management, Ningxia Medical University, Yinchuan 750004, PR China.
Toxicol Lett. 2018 Jun 1;289:42-53. doi: 10.1016/j.toxlet.2018.03.003. Epub 2018 Mar 5.
Microcystin (MC) is a cyclic heptapeptide compound which could lead to the development of hepatocellular carcinoma. However, the underlying epigenetic regulation mechanism is largely unknown. In this study, microcystin-LR (L: lysine, R: arginine, MC-LR) was used to induce the malignant transformation of human hepatocyte L02 cell line. The profile of gene expression, microRNA (miRNA) and DNA methylation were detected through high-throughput sequencing. Compared with control group, the expression of 826 genes and 187 miRNAs changed significantly in MC-LR treated group. DNA methylation sequencing analysis showed that 2592 CpG sites differentially methylated in promoter or the coding DNA sequence (CDS) of genes, while DNA methyltransferase 3 alpha (DNMT3a) and DNA methyltransferase 3 beta (DNMT3b) were dramatically up-regulated. Functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that significantly changed mRNAs and microRNAs were mainly involved in the formation of cancer, proliferation, invasion, migration and metabolism. MiRNA-mRNA network and mRNA-mRNA network analysis showed that hsa-miR-320a, hsa-miR-331-3p, hsa-miR-26a-5p, hsa-miR-196a-5p, hsa-miR-221-3p, coiled-coil domain containing 180 (CCDC180), melanoma antigen gene family member D1 (MAGED1), membrane spanning 4-domains A7 (MS4A7), hephaestin like 1 (HEPHL1), BH3 (Bcl-2 homology 3)-like motif containing, cell death inducer (BLID), matrix metallopeptidase 13 (MMP13), guanylate binding protein 5 (GBP5), adipogenesis regulatory factor (ADIRF), formin homology 2 domain containing 1 (FHDC1), protein kinase CAMP-dependent type II regulatory subunit beta (PRKAR2B), nodium leak channel, non-selective (NALCN), myosin light chain kinase 3 (MYLK3), epidermal growth factor receptor (EGFR) and zinc finger protein 704 (ZNF704) were key miRNAs and genes in the malignant transformation induced by MC-LR in L02 cells. Moreover, we found that expression of MYLK3, EGFR and ZNF704 were regulated by DNA methylation and miRNAs, and these genes affected the cell cycle and cell division. Our study suggested that characteristic gene alterations regulated by DNA methylation and miRNA could play an important role in environmental MC-LR induced hepatic carcinogenesis.
微囊藻毒素（MC）是一种环状七肽化合物，可导致肝细胞癌的发生。然而，其潜在的表观遗传调控机制在很大程度上尚不清楚。在本研究中，使用微囊藻毒素-LR（L：赖氨酸，R：精氨酸，MC-LR）诱导人肝细胞L02细胞系发生恶性转化。通过高通量测序检测基因表达、微小RNA（miRNA）和DNA甲基化谱。与对照组相比，MC-LR处理组中826个基因和187个miRNA的表达发生了显著变化。DNA甲基化测序分析表明，2592个CpG位点在基因启动子或编码DNA序列（CDS）中存在差异甲基化，而DNA甲基转移酶3α（DNMT3a）和DNA甲基转移酶3β（DNMT3b）显著上调。功能分析和京都基因与基因组百科全书（KEGG）通路分析表明，显著变化的mRNA和miRNA主要参与癌症形成、增殖、侵袭、迁移和代谢。miRNA-mRNA网络和mRNA-mRNA网络分析表明，hsa-miR-320a、hsa-miR-331-3p、hsa-miR-26a-5p、hsa-miR-196a-5p、hsa-miR-221-3p、卷曲螺旋结构域包含蛋白180（CCDC180）、黑色素瘤抗原基因家族成员D1（MAGED1）、跨膜4结构域A7（MS4A7）、类铁转运蛋白1（HEPHL1）、含BH3（Bcl-2同源结构域3）样基序的细胞死亡诱导剂（BLID）、基质金属蛋白酶13（MMP13）、鸟苷酸结合蛋白5（GBP5）、脂肪生成调节因子（ADIRF）、含formin同源结构域2的蛋白1（FHDC1）、蛋白激酶CAMP依赖性II型调节亚基β（PRKAR2B）、非选择性钠泄漏通道（NALCN）、肌球蛋白轻链激酶3（MYLK3）、表皮生长因子受体（EGFR）和锌指蛋白704（ZNF704）是MC-LR诱导L02细胞恶性转化中的关键miRNA和基因。此外，我们发现MYLK3、EGFR和ZNF704的表达受DNA甲基化和miRNA调控，且这些基因影响细胞周期和细胞分裂。我们的研究表明，DNA甲基化和miRNA调控的特征性基因改变可能在环境MC-LR诱导的肝癌发生中起重要作用。
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