Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal; Master in Molecular Medicine and Oncology, Faculty of Medicine-University of Porto (FMUP), Alameda Prof. Hernâni Monteiro, Porto, Portugal.
Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal.
Transl Res. 2022 Nov;249:110-127. doi: 10.1016/j.trsl.2022.06.009. Epub 2022 Jun 11.
Clear cell renal cell carcinoma (ccRCC) is highly prone to metastasize and displays an extremely low 5-year survival rate. Not only miRNAs (miRs) are key gene expression regulators but can also be epigenetically modified. Abnormal miR expression has been linked with epithelial-mesenchymal transition (EMT)-driven ccRCC progression. MiR-30a/c-5p were found downregulated in ccRCC and associated with aggressiveness. Herein, we sought to unravel miR-30a/c-5p mechanistic role in ccRCC. RNA sequencing and genome-wide methylome data of ccRCC and normal tissue samples from The Cancer Genome Atlas database were integrated to identify candidate miRs cytosine-phosphate-guanine (CpG) loci deregulated in ccRCC. TargetScan was searched to identify miR putative targets. MiR-30a/c-5p expression and promoter methylation was evaluated in vitro, by PCR. Western blot, functional and luciferase assays were performed after cell transfection with either pre-miR, antimiR, or siRNA against twinfilin-1 (TWF1). Immunohistochemistry (IHC) was performed in ccRCC tissues. We found miR-30c-5p downregulation and aberrant promoter methylation in ccRCC tissues. In vitro studies revealed concomitant miR-30a/c-5p downregulation and increased promoter methylation, as well as a significant re-expression following decitabine treatment. Functional assays demonstrated that both miRs significantly decreased cell aggressiveness and the protein levels of EMT-promoting players, while upregulating epithelial markers, namely Claudin-1 and ZO-1. Importantly, we confirmed TWF1 as a direct target of both miRs, and its potential involvement in epithelial-mesenchymal transition/mesenchymal-epithelial transition regulation. IHC analysis revealed higher TWF1 expression in primary tissues from patients that developed metastases, after surgical treatment. Our results implicate miR-30a/c-5p in ccRCC cells' aggressiveness attenuation by directly targeting TWF1 and hampering EMT.
透明细胞肾细胞癌（ccRCC）极易转移，5 年生存率极低。miRNAs（miRs）不仅是关键的基因表达调控因子，还可以被表观遗传修饰。异常的 miR 表达与上皮-间充质转化（EMT）驱动的 ccRCC 进展有关。miR-30a/c-5p 在 ccRCC 中表达下调，并与侵袭性相关。在此，我们试图揭示 miR-30a/c-5p 在 ccRCC 中的作用机制。整合癌症基因组图谱数据库中 ccRCC 和正常组织样本的 RNA 测序和全基因组甲基化数据，以鉴定 ccRCC 中失调的候选 miR 胞嘧啶-磷酸-鸟嘌呤（CpG）位点。通过搜索 TargetScan 来鉴定 miR 的潜在靶标。通过 PCR 评估 miR-30a/c-5p 的表达和启动子甲基化。用 pre-miR、antimiR 或针对 twinfilin-1（TWF1）的 siRNA 转染细胞后，进行 Western blot、功能和荧光素酶测定。在 ccRCC 组织中进行免疫组织化学（IHC）分析。我们发现 miR-30c-5p 在 ccRCC 组织中下调和异常启动子甲基化。体外研究表明，miR-30a/c-5p 同时下调和启动子甲基化增加，以及用地西他滨处理后显著重新表达。功能测定表明，两种 miR 均显著降低细胞侵袭性和 EMT 促进因子的蛋白水平，同时上调上皮标志物，即 Claudin-1 和 ZO-1。重要的是，我们证实 TWF1 是两种 miR 的直接靶标，其可能参与 EMT/间充质上皮转化调节。IHC 分析显示，手术后转移性患者原发组织中 TWF1 的表达更高。我们的结果表明，miR-30a/c-5p 通过直接靶向 TWF1 并阻碍 EMT 来减弱 ccRCC 细胞的侵袭性。
