| 汪鑫,闫亮亮,安然,等.卵圆细胞恶性转化相关差异性甲基化基因的筛选及验证[J].安徽医药,2020,24(12):2359-2364. |
| 卵圆细胞恶性转化相关差异性甲基化基因的筛选及验证 |
| Bioinformatics?based screening and verification of differentially methylated genes in malignant transformation of oval cells in vitro |
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| DOI：10.3969/j.issn.1009?6469.2020.12.007 |
| 中文关键词: DNA甲基化 卵圆细胞 癌，肝细胞 基因表达调控，肿瘤 泛素特异性蛋白酶类 肿瘤抑制蛋白质类 受体，表皮生长因子 癌基因 差异性甲基化区域(DMR) 差异性甲基化基因(DMG) |
| 英文关键词: DNA methylation Oval cells Carcinoma,hepatocellular Gene expression regulation,neoplastic Ubiquitin?spe? cific proteases Tumor suppressor proteins Receptor,epidermal growth factor Oncogenes DMR DMG |
| 基金项目:安徽省自然科学基金资助项目(1608085QH182) |
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| 中文摘要: |
| 目的通过对卵圆细胞恶性转化相关差异性甲基化基因的筛选及验证，初步探索卵圆细胞发生恶性转化的表观遗传学调控机制。方法利用简化甲基化测序(Reduced representation bisulfite sequencing，RRBS)对转染肝细胞癌基因 HBV X(HBX)发生恶性转化的卵圆细胞(HBX?LE6)与正常大鼠肝卵圆细胞(LE6)进行甲基化测序，获得差异性甲基化区域(Differen? tially methylated region，DMR)与相关差异性甲基化基因(Differentially methylated gene，DMG)，通过实时定量聚合酶链反应(Real?time qPCR)、蛋白质印迹法(Western Blot)、甲基化特异性聚合酶链反应(methylation specific polymerase chain reaction， MSP)进行检测，验证甲基化对 DMG表达的调控作用。结果共筛选相关 DMR 1 434个，其中高甲基化 DMR 623个(位于启动子 128个)；低甲基化 DMR 811个(位于启动子 216个)DMG共 1 987个。挑选启动子均存在高甲基化 DMR的相关基因如泛素特异性蛋白酶 18基因(USP18)、有丝分裂原活化蛋白激，酶激酶激酶 6(MAP3K6)、 SWI/SNF染色质重塑复合物(SMARCB1)、表 |
| 英文摘要: |
| Objective To explore the epigenetic regulation mechanisms in malignant transformation of oval cells byscreening and?verifyingdifferentially methylated genes(DMGs)related to malignant transformation of oval cells.Methods Reduced representa? tion bisulfite sequencing(RRBS)was applied to detect methylation sequencing on the malignant transformation of hepatocellular carcinoma gene HBV X(HBX)?LE6 oval cells and normal rat liver LE6 oval cells.Different methylated regions(DMR)and related Differentially methylated genes(DMG)were obtained.The regulatory effect of methylation on DMG expression was detected and identified by the real?time qPCR，Western Blot andmethylation specific polymerase chain reaction(MSP).Results A total of 1 434 DMR were identified in methylation level in tumor compared with non?tumor tissues，of which 623 were hypermethylated DMRs(at 128 promoters)，811 were hypomethylated DMRs(at 216 promoters)，and 1 987 were DMGs.The genes related to hypermethylated DMR were found and verified in all selected promoters，such as ubiquitin?specific protease 18 gene(USP18)，mitogen?activated protein kinase kinase kinase 6(MAP3K6)，SWI/SNF chromatin remodeling complex(SMARCB1)，epidermal growth Factor recep? tor(EGFR)， tumor suppressor(CYLD)， ring finger protein 39(RNF39)， 16?containing zinc finger and BTB domain protein(ZBTB16)，and homeobox gene D8(HOXD8).Real?time quantitative PCR indicated mRNA levels in USP18，SMRCB1，CYLD and ZBTB16 were remarkably decreased in HBX?LE6 oval cells compared with controls in LE6 oval cells(P＜0.05).The mRNA ex? pression of USP18，SMRCB1，CYLD and ZBTB16 in LE6 and HBX?LE6 were(4.50±0.43)vs.(2.09±0.18)，(7.34±0.11)vs.(2.57± 0.29)(7.30±0.27)vs.(3.44±0.13)(7.01±0.06)vs.(1.29±0.32)，respectively.Western Blot indicated that SMARCB1 protein ex? pression，wasdecreasedinHBX?LE6ov，al cells(P<0.05).SMARCB1 protein expression in LE6 and HBX?LE6 were［(6.26±0.25) vs.(1.53±0.34)，P＜0.05］.USP18，CYLDand ZBTB16 protein levels were not statistically different among the three groups(P＞0.05).After 5?aza?2’?deoxycytidine(5?Aza?CdR)intervention on HBX?LE6 cells，MSP confirmed that the hypermethylation of SMARCB1 in HBX?LE6 was inhibited by 5?Aza?CdR，and its mRNA and protein expression levels were significantly up?regulated(P＜0.05). Conclusions These results demonstrate the significance of aberrant DNA methylation in the epigenetic regulationmechanism of oval cells.DNA hypermethylation down?regulates the tumor suppressor gene SMARCB1 in oval cells may be involvedin the process of tumorigenesis，and the mechanism needs to be further studied. |
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