First Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
Genes Chromosomes Cancer. 2014 Feb;53(2):168-76. doi: 10.1002/gcc.22128. Epub 2013 Nov 5.
Proximal type epithelioid sarcoma shares similarities with malignant rhabdoid tumor, including the lack of nuclear immunoreactivity of SMARCB1. Biallelic mutation of SMARCB1 has been convincingly established as the cause of loss of protein expression in rhabdoid tumor, but the cause in epithelioid sarcoma remains unknown. In our previous work, we demonstrated that DNA hypermethylation and post-translational modification mechanisms were not involved. In this current work, we explored the hypothesis that miRNAs regulate SMARCB1 gene expression in epithelioid sarcomas. In silico target prediction analysis revealed eight candidate miRNAs, and quantitative PCR-in 32 formalin-fixed, paraffin-embedded tumor samples comprising 30 epithelioid sarcomas and two malignant rhabdoid tumors-demonstrated significant (P < 0.001) overexpression of four miRNAs in epithelioid sarcomas: miR-206, miR-381, miR-671-5p, and miR-765. Two human tumors (fibrosarcoma and colon adenocarcinoma) and a normal cell line (human dermal fibroblast) with retained SMARCB1 expression were cultured for miRNA transient transfection (electroporation) experiments. SMARCB1 mRNA expression was analyzed by quantitative real-time PCR and immunostaining of SMARCB1 was performed to examine the effect of miRNAs transfections on both RNA and protein levels. Only three of the overexpressed miRNAs (miR-206, miR-381, and miR-671-5p) could silence the SMARCB1 mRNA expression in cell cultures; most effectively miR-206. Transfection of miR-206, miR-381, miR-671-5p, and some combination of them also eliminated SMARCB1 nuclear staining, demonstrating a strong effect on not only mRNA but also protein levels. Our results suggest loss of SMARCB1 protein expression in epithelioid sarcoma is due to the epigenetic mechanism of gene silencing by oncomiRs.
近端型上皮样肉瘤与恶性横纹肌样肿瘤具有相似性，包括缺乏 SMARCB1 的核免疫反应性。SMARCB1 的双等位基因突变已被令人信服地确立为横纹肌样肿瘤中蛋白表达缺失的原因，但在上皮样肉瘤中原因尚不清楚。在我们之前的工作中，我们证明了 DNA 高甲基化和翻译后修饰机制不参与其中。在本研究中，我们探讨了 miRNA 调节上皮样肉瘤中 SMARCB1 基因表达的假说。计算机预测分析显示了 8 个候选 miRNA，定量 PCR-在 32 个福尔马林固定、石蜡包埋的肿瘤样本中，包括 30 个上皮样肉瘤和 2 个恶性横纹肌样肿瘤-显示在上皮样肉瘤中，有 4 个 miRNA 显著（P<0.001）过表达：miR-206、miR-381、miR-671-5p 和 miR-765。两种具有保留 SMARCB1 表达的人类肿瘤（纤维肉瘤和结肠腺癌）和一个正常细胞系（人真皮成纤维细胞）进行 miRNA 瞬时转染（电穿孔）实验。通过定量实时 PCR 分析 SMARCB1 mRNA 表达，并进行 SMARCB1 免疫染色，以检验 miRNA 转染对 RNA 和蛋白水平的影响。只有三种过表达的 miRNA（miR-206、miR-381 和 miR-671-5p）可以在细胞培养物中沉默 SMARCB1 mRNA 表达；miR-206 最有效。miR-206、miR-381、miR-671-5p 的转染以及它们的一些组合也消除了 SMARCB1 的核染色，这表明不仅对 mRNA 而且对蛋白质水平都有很强的影响。我们的结果表明，上皮样肉瘤中 SMARCB1 蛋白表达的缺失是由于癌基因 miRNA 沉默基因的表观遗传机制所致。
