Department of Frontier Surgery, Chiba University Graduate School of Medicine, Chiba, Japan.
Int J Oncol. 2012 Sep;41(3):985-94. doi: 10.3892/ijo.2012.1537. Epub 2012 Jun 28.
The aim of this study was to determine whether histone acetylation regulates tumor suppressive microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC) and to identify genes which are regulated by these miRNAs. We identified a miRNA that was highly upregulated in an ESCC cell line by cyclic hydroxamic acid-containing peptide 31 (CHAP31), one of the histone deacetylase inhibitors (HDACIs), using a miRNA array analysis. miR-375 was strongly upregulated by CHAP31 treatment in an ESCC cell line. The expression levels of the most upregulated miRNA, miR-375 were analyzed by quantitative real-time PCR in human ESCC specimens. The tumor suppressive function of miR-375 was revealed by restoration of miR-375 in ESCC cell lines. We performed a microarray analysis to identify target genes of miR-375. The mRNA and protein expression levels of these genes were verified in ESCC clinical specimens. LDHB and AEG-1/MTDH were detected as miR‑375-targeted genes. The restoration of miR-375 suppressed the expression of LDHB and AEG-1/MTDH. The ESCC clinical specimens exhibited a high level of LDHB expression at both the mRNA and protein levels. A loss-of-function assay using a siRNA analysis was performed to examine the oncogenic function of the gene. Knockdown of LDHB by RNAi showed a tumor suppressive function in the ESCC cells. The correlation between gene expression and clinicopathological features was investigated by immunohistochemistry for 94 cases of ESCC. The positive staining of LDHB correlated significantly with lymph node metastasis and tumor stage. It also had a tendency to be associated with a poor prognosis. Our results indicate that HDACIs upregulate miRNAs, at least some of which act as tumor suppressors. LDHB, which is regulated by the tumor suppressive miR-375, may therefore act as an oncogene in ESCC.
本研究旨在确定组蛋白乙酰化是否调节食管鳞状细胞癌（ESCC）中的肿瘤抑制性 microRNAs（miRNAs），并鉴定受这些 miRNAs 调控的基因。我们使用 miRNA 阵列分析鉴定了一种在 ESCC 细胞系中由组蛋白去乙酰化酶抑制剂（HDACI）之一的环羟肟酸肽 31（CHAP31）高度上调的 miRNA。miR-375 在 ESCC 细胞系中经 CHAP31 处理后强烈上调。在人 ESCC 标本中通过定量实时 PCR 分析最上调 miRNA，miR-375 的表达水平。在 ESCC 细胞系中恢复 miR-375 揭示了 miR-375 的肿瘤抑制功能。我们进行了微阵列分析以鉴定 miR-375 的靶基因。在 ESCC 临床标本中验证了这些基因的 mRNA 和蛋白表达水平。LDHB 和 AEG-1/MTDH 被检测为 miR-375 靶向基因。恢复 miR-375 抑制了 LDHB 和 AEG-1/MTDH 的表达。ESCC 临床标本在 mRNA 和蛋白水平上均表现出高水平的 LDHB 表达。使用 siRNA 分析进行的失活功能测定用于检查基因的致癌功能。RNAi 敲低 LDHB 在 ESCC 细胞中显示出肿瘤抑制功能。通过免疫组织化学对 94 例 ESCC 进行了基因表达与临床病理特征之间相关性的研究。LDHB 的阳性染色与淋巴结转移和肿瘤分期显著相关。它也有与不良预后相关的趋势。我们的结果表明，HDACI 上调 miRNAs，至少其中一些作为肿瘤抑制因子起作用。LDHB 受肿瘤抑制 miR-375 调节，因此可能在 ESCC 中作为癌基因起作用。
