Tumor-suppressor Pdcd4 inhibits transformation and invasion and is downregulated in cancers. So far, it has not been studied as to whether miRNAs, suppressing target expression by binding to the 3'-UTR, regulate Pdcd4 or invasion. The present study was conducted to investigate the regulation of Pdcd4, and invasion/intra-vasation, by miRNAs. A bioinformatics search revealed a conserved target-site for miR-21 within the Pdcd4-3'-UTR at 228-249 nt. In 10 colorectal cell lines, an inverse correlation of miR-21 and Pdcd4-protein was observed. Transfection of Colo206f-cells with miR-21 significantly suppressed a luciferase-reporter containing the Pdcd4-3'-UTR, whereas transfection of RKO with anti-miR-21 increased activity of this construct. This was abolished when a construct mutated at the miR-21/nt228-249 target site was used instead. Anti-miR-21-transfected RKO cells showed an increase of Pdcd4-protein and reduced invasion. Moreover, these cells showed reduced intra-vasation and lung metastasis in a chicken-embryo-metastasis assay. In contrast, overexpression of miR-21 in Colo206f significantly reduced Pdcd4-protein amounts and increased invasion, while Pdcd4-mRNA was unaltered. Resected normal/tumor tissues of 22 colorectal cancer patients demonstrated an inverse correlation between miR-21 and Pdcd4-protein. This is the first study to show that Pdcd4 is negatively regulated by miR-21. Furthermore, it is the first report to demonstrate that miR-21 induces invasion/intravasation/metastasis.

译文

:肿瘤抑制因子Pdcd4抑制转化和侵袭,并在癌症中下调。到目前为止,尚未研究miRNA是否通过与3'-UTR结合来抑制靶标表达,调节Pdcd4或入侵。进行本研究以研究miRNA对Pdcd4的调控以及侵袭/血管内侵袭。生物信息学搜索显示,Pdcd4-3'-UTR中miR-21的保守靶位点为228-249 nt。在10个结直肠细胞系中,观察到miR-21与Pdcd4-蛋白呈负相关。用miR-21转染Colo206f细胞可显着抑制含有Pdcd4-3'-UTR的荧光素酶报告基因,而用抗miR-21转染RKO可提高该构建体的活性。当使用在miR-21 / nt228-249靶位点突变的构建体代替时,这种情况就被取消了。抗miR-21转染的RKO细胞显示Pdcd4蛋白增加,侵袭减少。此外,在鸡胚转移试验中,这些细胞显示出减少的血管内转移和肺转移。相反,在Colo206f中miR-21的过表达显着减少了Pdcd4蛋白的含量并增加了侵袭,而Pdcd4-mRNA却没有改变。切除的22例结直肠癌患者的正常/肿瘤组织显示miR-21与Pdcd4-蛋白呈负相关。这是第一项显示Pdcd4受miR-21负调控的研究。此外,这是第一个证明miR-21诱导侵袭/浸润/转移的报告。

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