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

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 1434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1987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)、表皮生长因子受体(EGFR)、肿瘤抑制因子(CYLD)、环指蛋白39(RNF39)、含16的锌指和BTB结构域蛋白(ZBTB16)、同源异型盒基因D8(HOXD8)进行验证。USP18、SMRCB1、CYLD、ZBTB16在LE6和HBX⁃LE6中的mRNA相对表达量分别为[(4.50±0.43),(2.09±0.18)]、[(7.34±0.11),(2.57±0.29)]、[(7.30±0.27),(3.44±0.13)]、[(7.01±0.06),(1.29±0.32)],与LE6相比,其在HBX⁃LE6中表达显著下调(P<0.05)。与LE6相比,SMARCB1在HBX⁃LE6中的蛋白表达明显下降[(6.26±0.25)比(1.53±0.34),P<0.05]。USP18、ZBTB1、CYLD的蛋白表达在三组间差异无统计学意义(P>0.05)。5⁃Aza⁃CdR对HBX⁃LE6细胞干预后,MSP证实SMARCB1在HBX⁃LE6中的高甲基化被5⁃Aza⁃CdR抑制,其mRNA与蛋白表达水平均显著上调(P<0.05)。结论DNA甲基化参与卵圆细胞恶性转化的表观遗传学调控机制,DNA高甲基化下调卵圆细胞中抑癌基因SMARCB1可能参与了癌变的过程,但机制仍需要进一步研究明确。

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 1434 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 1987 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 was decreased in HBX⁃LE6 oval 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 regulation mechanism of oval cells.DNA hypermethylation down⁃regulates the tumor suppressor gene SMARCB1 in oval cells may be involved in the process of tumorigenesis,and the mechanism needs to be further studied.