Abstract
Gene expression is potently regulated through the action of microRNAs (miRNAs). Here, we present evidence of a miRNA regulating Hakai protein. Hakai was discovered as an E3 ubiquitin-ligase that mediates the posttranslational downregulation of E-cadherin, a major component of adherens junctions in epithelial cells and a potent tumour suppressor. Recent data have provided evidence that Hakai affects cell proliferation in an E-cadherin-independent manner, thus revealing a role for Hakai in the early stages of tumour progression. Furthermore, Hakai is highly up-regulated in human colon adenocarcinomas compared to normal tissues. However, the molecular mechanisms that regulate Hakai abundance are unknown. We identified two putative sites of miR-203 interaction on the Hakai mRNA, in its 3′-untranslated region (UTR). In several human carcinoma cell lines tested, overexpression of a miR-203 precursor (Pre-miR-203) reduced Hakai abundance, while inhibiting miR-203 by using an antisense RNA (Anti-miR-203) elevated Hakai levels. The repressive influence of miR-203 on the Hakai 3′-UTR was confirmed using heterologous reporter constructs. In keeping with Hakai's proliferative influence, Anti-miR-203 significantly increased cell number and BrdU incorporation, while Pre-miR-203 reduced these parameters. Importantly, the growth-promoting effects of anti-miR-203 required the presence of Hakai, because downregulation of Hakai by siRNA suppressed its proliferative action. Finally, in situ hybridization showed that miR-203 expression is attenuated in colon tumour tissues compared to normal colon tissues, suggesting that miR-203 could be a potential new prognostic marker and therapeutic target to explore in colon cancer. In conclusion, our findings reveal, for the first time, a post-transcriptional regulator of Hakai expression. Furthermore, by lowering Hakai abundance, miR-203 also reduces Hakai-regulated-cell division.
Highlights
Carcinoma arises from epithelial cells on which cancer cells start an uncontrolled proliferation and, in order to metastasize, some cells detach from the primary tumour, migrate and invade through tissues
Data driven algorithms rely on important discriminative features learned from data using sophisticated models [27,28,29]. miR-203 was predicted to bind to Hakai mRNA at two sites within 39-untranslated region (UTR), positions 1965 to 1977 and from 2172 to 2198 (Targetscan) (Figure 1A)
To elevate miR-203 levels, the precursor miR-203 transcript was transfected and after 48 h after transfection, Hakai expression was analyzed by reverse transcription followed by quantitative real-time PCR (RT-qPCR) and Western blotting
Summary
Carcinoma arises from epithelial cells on which cancer cells start an uncontrolled proliferation and, in order to metastasize, some cells detach from the primary tumour, migrate and invade through tissues. Downregulation of cell–cell adhesion is characterized by the loss of E-cadherin, the best protein characterized and prototype member of the classical cadherins in epithelial cells, which are potent tumour suppressors in epithelial cells [1]. In 2002, the protein Hakai was identified as the first post-translational regulator of E-cadherin stability. Many studies on the emerging biological functions of Hakai have underscored its influence on tumour progression and disease [6]. Hakai has been implicated in controlling cell migration and embryogenesis [11,12,13], and it can control cell proliferation in an E-cadherin-independent manner, further supporting a role for Hakai in early stages of tumorigenesis [14,15]. To-date, no regulators of Hakai expression have been described
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