HPV-16 诱导的头颈部鳞状细胞癌（HNSCC）中 DNA 甲基化调控的 microRNAs。
DNA methylation regulated microRNAs in HPV-16-induced head and neck squamous cell carcinoma (HNSCC).
机构信息
Department of Otolaryngology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India.
Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India.
出版信息
Mol Cell Biochem. 2018 Nov;448(1-2):321-333. doi: 10.1007/s11010-018-3336-6. Epub 2018 Feb 17.
INTRODUCTION
Epigenetic modifications have been reported to play an important role in regulating gene expression and these modifications become critical when they have a role in controlling another important layer of epigenetic regulation namely microRNAs. In the present study, we have identified the microRNAs that may be regulated by promoter DNA methylation and histone acetylation in Human papilloma virus-positive head and neck squamous cell carcinoma.
METHODOLOGY
HPV-negative cell line (UPCI:SCC-116) and HPV-16 +ve cell line (UPCI:SCC-090) were treated with methylation inhibitor (5-aza-2'-deoxycytidine, AZA) and acetylation inhibitor (Trichostatin-A, TSA), followed by micro-array analysis. The differentially expressed miRNAs were validated in control (n = 10), HPV-16 +ve (n = 30), and HPV -ve (n = 30) HNC, TCGA (n = 529) tissue samples, and two HPV -ve (SCC116 and Hacat) and two HPV +ve (SCC090 and SiHa) cell lines. Methylation-specific PCR (MSP) and chromatin immunoprecipitation assay (CHIP) were performed to validate their regulation. In silico and in vitro analyses of identified miRNAs were done to study putative pathways they target and their possible role in carcinogenesis.
RESULTS
Among 10 miRNAs specifically up-regulated in microarray analysis of AZA-treated SCC090 cells, we observed significantly decreased expression of hsa-miR-181c-5p, hsa-miR-132-5p, hsa-miR-658 in HPV +ve HNC cohort, TCGA tissue samples, and cell lines as compared to their HPV -ve counterpart, and their promoter region also possesses CpG islands. MSP and analysis of TCGA data (MethHC) revealed increased frequency of methylation at the promoter of hsa-miR-132-5p that is negatively correlated with its expression. In TSA-treated SCC090 cells, out of 7 miRNAs, two namely Hsa-miR-129-2-3p and Hsa-miR-449a were found to be up-regulated as compared to HPV -ve cells. However, the levels of enrichment by anti-acetyl-H3 and anti-acetyl-H4 were significantly low in cell lines compared to respective controls and both were up-regulated in HPV +ve compared to HPV -ve TCGA tissue samples. In silico analysis revealed hsa-miR-132-5p targeted canonical β-catenin/wnt pathway and modulation of down-stream genes of the pathway was observed on over-expression/inhibition of hsa-miR-132-5p.
CONCLUSION
This study suggests the role of epigenetic modifications in regulating expression of miRNAs in HPV +ve HNSCC.
简介
已报道表观遗传修饰在调控基因表达中发挥重要作用，当它们在控制另一个重要的表观遗传调控层面即 microRNAs 方面发挥作用时，这些修饰变得至关重要。在本研究中，我们鉴定了可能受 HPV 阳性头颈部鳞状细胞癌中启动子 DNA 甲基化和组蛋白乙酰化调控的 microRNAs。
方法
用甲基化抑制剂（5-氮杂-2'-脱氧胞苷，AZA）和乙酰化抑制剂（曲古抑菌素-A，TSA）处理 HPV 阴性细胞系（UPCI：SCC-116）和 HPV-16+ve 细胞系（UPCI：SCC-090），随后进行微阵列分析。在 HPV-16+ve（n=30）和 HPV-ve（n=30）HNC、TCGA（n=529）组织样本以及两个 HPV-ve（SCC116 和 Hacat）和两个 HPV+ve（SCC090 和 SiHa）细胞系中验证差异表达的 miRNAs。进行甲基化特异性 PCR（MSP）和染色质免疫沉淀分析（CHIP）以验证其调控作用。对鉴定出的 miRNAs 进行体外和体外分析，以研究其潜在的靶途径及其在致癌作用中的可能作用。
结果
在 AZA 处理的 SCC090 细胞的微阵列分析中，有 10 个 miRNAs 特异性上调，我们观察到 HPV+ve HNC 队列、TCGA 组织样本和细胞系中 hsa-miR-181c-5p、hsa-miR-132-5p 和 hsa-miR-658 的表达明显降低，与 HPV-ve 对照相比，其启动子区域也存在 CpG 岛。MSP 和 TCGA 数据分析（MethHC）显示 hsa-miR-132-5p 启动子的甲基化频率增加，这与它的表达呈负相关。在 TSA 处理的 SCC090 细胞中，在 7 个 miRNA 中，有 2 个即 Hsa-miR-129-2-3p 和 Hsa-miR-449a 与 HPV-ve 细胞相比被发现上调。然而，与相应的对照相比，细胞系中抗乙酰化 H3 和抗乙酰化 H4 的富集水平明显较低，并且两者在 HPV+ve 与 HPV-ve TCGA 组织样本相比均上调。计算分析显示 hsa-miR-132-5p 靶向经典的β-catenin/wnt 通路，并且下调 hsa-miR-132-5p 的表达可以观察到该通路下游基因的调节。
结论
本研究提示表观遗传修饰在 HPV+ve HNSCC 中调控 microRNAs 表达中的作用。
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