ABSTRACT
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.
Subject(s)
Adenocarcinoma/genetics , Adenocarcinoma/pathology , Apoptosis Regulatory Proteins/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , MicroRNAs/physiology , RNA Interference , RNA-Binding Proteins/genetics , Animals , Base Sequence , Caco-2 Cells , Chick Embryo , Down-Regulation , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , HCT116 Cells , HT29 Cells , Humans , Molecular Sequence Data , Neoplasm Invasiveness , Neoplasm Metastasis , Sequence Homology, Nucleic Acid , Tumor Cells, CulturedABSTRACT
Tumor suppressor Pdcd4 has recently been shown to inhibit invasion by activating activator protein-1 (AP-1); however, little is known of the functionally significant Pdcd4-target genes. The urokinase receptor (u-PAR) promotes invasion/metastasis, and is associated with poor cancer-patient survival. The present study was conducted (1) to investigate a role for Pdcd4 in intravasation, invasion and u-PAR regulation, and (2) to describe mechanisms by which this is achieved. Fourteen cell lines showed reciprocal expression of u-PAR/Pdcd4. Resected tumor/normal tissues of 29 colorectal cancer patients demonstrated a significant inverse correlation between Pdcd4/u-PAR. siRNA-Pdcd4-transfected GEO cells significantly increased endogenous u-PAR mRNA/protein. A u-PAR-promoter-chloramphenicol acetyl transferase (CAT)-reporter was reduced in activity with increasing Pdcd4 expression in RKO. Deletion of a putative Sp-1-binding site (-402/-350) inhibited u-PAR promoter regulation by Pdcd4, this being paralleled by a reduction of Sp1 binding to this region in pdcd4-transfected cells. Pdcd4-transfected cells showed an increase in Sp3 binding to u-PAR promoter region -152/-135, the deletion of which reduces the ability of Pdcd4 to suppress u-PAR promoter activity. Surprisingly, the u-PAR-AP-1 site was not targeted by Pdcd4. Finally, RKO cells overexpressing Pdcd4 showed an inhibition of invasion/intravasation (chicken embryo metastasis assay). These data suggest Pdcd4 as a new negative regulator of intravasation, and qas the invasion-related gene u-PAR. It is the first study to implicate Pdcd4 regulation of gene expression via Sp1/Sp3.
