UHRF1 Induces Methylation of the TXNIP Promoter and Down-Regulates Gene Expression in Cervical Cancer.
UHRF1 Induces Methylation of the TXNIP Promoter and Down-Regulates Gene Expression in Cervical Cancer.
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DOI：
10.14348/molcells.2021.0001
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发表时间：
2021-03-31
影响因子：
3.8
通讯作者：
Choi WS
中科院分区：
文献类型：
--
作者：
Kim MJ;Lee HJ;Choi MY;Kang SS;Kim YS;Shin JK;Choi WS
肿瘤抑制基因的DNA甲基化和随之而来的下调是在癌症发展过程中对表观遗传刺激的反应。类似地，包括人乳头瘤病毒（HPV）在内的人肿瘤病毒上调和增强DNA甲基转移酶（DNMT）和组蛋白脱乙酰酶（HDAC）活性，从而降低肿瘤抑制基因（TSG）表达。含有PHD和RING指结构域1（UHRF 1）的泛素样蛋白（一种DNA甲基化的表观遗传调节因子）在HPV诱导的宫颈癌中过表达。在这里，我们研究了UHRF 1在宫颈癌中的作用，通过敲低其在HeLa细胞中的表达，使用慢病毒编码的短发夹（sh）RNA和进行cDNA微阵列。我们检测到显着升高的表达硫氧还蛋白相互作用蛋白（TXNIP），一个已知的TSG，在UHRF 1敲低细胞，该基因是高甲基化的宫颈癌组织和细胞系，如全基因组甲基化分析所示。通过免疫印迹和免疫组化进一步检测宫颈癌中UHRF 1的上调和TXNIP的降低，并通过Oncomine数据库分析证实。使用染色质免疫沉淀，我们确定了倒置的CCAAT结构域包含UHRF 1结合位点的TXNIP启动子，并证明UHRF 1敲低降低UHRF 1启动子结合，并通过该区域的去甲基化增强TXNIP表达。通过焦磷酸测序和甲基化特异性聚合酶链反应进一步证实了TXNIP启动子CpG甲基化在宫颈癌组织中。重要的是，通过siRNA或UHRF 1拮抗剂（百里醌）下调UHRF 1可诱导细胞周期停滞和凋亡，而稳定和促进UHRF 1功能的泛素特异性蛋白酶7（USP 7）可通过HPV病毒蛋白E6/E7过表达而增加。这些结果表明HPV可能通过UHRF 1介导的TXNIP启动子甲基化诱导癌变，从而表明CpG甲基化与宫颈癌之间可能存在联系。
DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.