A Screen for Epigenetically Silenced microRNA Genes in Gastrointestinal Stromal Tumors.
A Screen for Epigenetically Silenced microRNA Genes in Gastrointestinal Stromal Tumors.
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DOI：
10.1371/journal.pone.0133754
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Shinomura Y
中科院分区：
文献类型：
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作者：
Isosaka M;Niinuma T;Nojima M;Kai M;Yamamoto E;Maruyama R;Nobuoka T;Nishida T;Kanda T;Taguchi T;Hasegawa T;Tokino T;Hirata K;Suzuki H;Shinomura Y
microRNA (miRNA) 的失调与胃肠道间质瘤 (GIST) 有关，但其机制尚不完全清楚。在这项研究中，我们旨在探索 miRNA 基因表观遗传改变在 GIST 中的作用。 GIST-T1 细胞用 5-aza-2’-脱氧胞苷 (5-aza-dC) 和 4-苯基丁酸 (PBA) 处理，然后使用 TaqMan miRNA 阵列分析 miRNA 表达谱。然后使用亚硫酸氢盐焦磷酸测序分析 DNA 甲基化。使用 MTT 测定、伤口愈合测定、Boyden 室测定和 Matrigel 侵袭测定检查 miRNA 的功能。分析基因表达微阵列以评估 GIST-T1 细胞中异位 miRNA 表达的影响。在分析的 754 个 miRNA 中，有 61 个在用 5-aza-dC 加 PBA 处理的 GIST-T1 细胞中显着上调。其中，21个miRNA基因与上游CpG岛（CGI）相关，并且miR-34a和miR-335的CGI在GIST-T1细胞和原发性GIST标本中频繁甲基化。将miR-34a或miR-335模拟分子转染GIST-T1细胞可抑制细胞增殖，并且miR-34a还抑制GIST-T1细胞的迁移和侵袭。此外，miR-34a 下调了许多预测的靶基因，包括 PDGFRA。 GIST-T1 细胞中 RNA 干扰介导的 PDGFRA 敲低抑制了细胞增殖，表明 miR-34a 的肿瘤抑制作用至少部分是通过靶向 PDGFRA 介导的。我们的结果表明，miR-34a 和 miR-335 是 GIST 中的候选肿瘤抑制 miRNA，并且它们是 GIST 中表观遗传沉默的常见靶标。
Dysregulation of microRNA (miRNA) has been implicated in gastrointestinal stromal tumors (GISTs) but the mechanism is not fully understood. In this study, we aimed to explore the involvement of epigenetic alteration of miRNA genes in GISTs. GIST-T1 cells were treated with 5-aza-2’-deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which miRNA expression profiles were analyzed using TaqMan miRNA arrays. DNA methylation was then analyzed using bisulfite pyrosequencing. The functions of miRNAs were examined using MTT assays, wound-healing assays, Boyden chamber assays and Matrigel invasion assays. Gene expression microarrays were analyzed to assess effect of ectopic miRNA expression in GIST-T1 cells. Of the 754 miRNAs analyzed, 61 were significantly upregulated in GIST-T1 cells treated with 5-aza-dC plus PBA. Among those, 21 miRNA genes were associated with an upstream CpG island (CGI), and the CGIs of miR-34a and miR-335 were frequently methylated in GIST-T1 cells and primary GIST specimens. Transfection of miR-34a or miR-335 mimic molecules into GIST-T1 cells suppressed cell proliferation, and miR-34a also inhibited migration and invasion by GIST-T1 cells. Moreover, miR-34a downregulated a number of predicted target genes, including PDGFRA. RNA interference-mediated knockdown of PDGFRA in GIST-T1 cells suppressed cell proliferation, suggesting the tumor suppressive effect of miR-34a is mediated, at least in part, through targeting PDGFRA. Our results suggest that miR-34a and miR-335 are candidate tumor suppressive miRNAs in GISTs, and that they are frequent targets of epigenetic silencing in GISTs.