The proto-oncogene TWIST1 is regulated by microRNAs.

Upregulation of the proto-oncogene Twist1 is highly correlated with acquired drug resistance and poor prognosis in human cancers. Altered expression of this multifunctional transcription factor is also associated with inherited skeletal malformations. The mammalian Twist1 3'UTRs are highly conserved and contain a number of potential regulatory elements including miRNA target sites. We analyzed the translational regulation of TWIST1 using luciferase reporter assays in a variety of cell lines. Among several miRNAs tested, miR-145a-5p, miR-151-5p and a combination of miR-145a-5p + miR-151-5p and miR-151-5p + miR-337-3p were able to significantly repress Twist1 translation. This phenomena was confirmed with both exogenous and endogenous miRNAs and was dependent on the presence of the predicted target sites in the 3'UTR. Furthermore, the repression was sensitive to LNA-modified miRNA antagonists and resulted in decreased migratory potential of murine embryonic fibroblast cells. Understanding the in vivo mechanisms of this oncogene's regulation might open up a possibility for therapeutic interference by gene specific cancer therapies

Conservation of microRNA target sites in selected amniotes and the presence in <i>Twist2</i> 3′UTR.

<p>MicroRNAs underlined were tested in this study.</p

MicroRNAs 151-5p and 337-3p reduce the mobility of murine embryonic fibroblast cells.

<p>Dot-plot superposed with the box-plot of the predicted probabilities of cell migration for observations from miR-151-5p + miR-337-3p (red) and scrambled control (blue) treated cells (<i>p</i>-value 0.000135). Note the presence of miR-151-5p + miR-337-3p treated cells with extreme low estimates of probability of cell migration.</p

MicroRNA target sites are necessary for the repression of <i>Twist1</i> 3′UTR reporter.

<p>MiRNAs of interest were co-transfected with either wt or the corresponding mutant of psiCheck2-Twist1-3′UTR reporter into H1299 cells. Firefly luciferase activities were measured after 48 h, normalized to <i>Renilla</i> luciferase activities and subsequently to the respective wt+miRNA. Statistical significance was calculated by using Student's t-test and by comparing the mutant 3′UTR values with the wt 3′UTR reporter.</p

Endogenous microRNAs reduce the activity of <i>Twist1</i> 3′UTR reporter.

<p>NIH-3T3 (A) and C3H/10T1/2 (B) cells were transfected with wt or mutant psiCheck2-Twist1-3′UTR reporter only. Endogenous miRNAs in NIH-3T3 (C) and C3H/10T1/2 (D) cells were inhibited by co-transfection of 50 nM miRCURY LNA microRNA inhibitors and wt psiCheck2-Twist1-3′UTR reporter. Firefly luciferase activities were measured after 48 h, normalized to <i>Renilla</i> luciferase activities and subsequently to the wt 3′UTR (A-B) or wt+anti-Scrambled (C-D). Statistical significance was calculated by using Student's t-test and by comparing the mutant <i>Twist1</i> 3′UTR reporters or miRNA specific antagonists with the wt 3′UTR reporter or a scrambled control (*), respectively.</p

MicroRNAs repress the <i>Twist1</i> 3′UTR reporter.

<p>H1299 cells were transfected with the indicated miRNA, pRluc-N2 and pGL3-Twist1-3′UTR Firefly luciferase reporter or the empty pGL3-control vector and analyzed after 48 h. Firefly luciferase activities were normalized to the <i>Renilla</i> luciferase activities which served as internal standards, averages of triplicates were calculated and results were normalized to empty pGL3-control vector. The dashed line indicates the unrepressed expression level of the reporter (0,176; calculated from the average of two negative controls (*), miR-485 and miR-609). Statistical significance of miRNA effects was calculated by comparison with this average using Student's t-test.</p

Pairwise comparison of <i>Twist1</i> and <i>Twist2</i> mRNA sequence domains of three selected amniotes with the corresponding human sequence. Numbers represent% sequence identity.

1<p>CDS: coding sequence; ²UTR: untranslated region.</p

MicroRNAs lead to translational inhibition of <i>Twist1</i> 3′UTR reporter.

<p>(A) H1299 cells were co-transfected with wt psiCheck2-Twist1-3′UTR reporter and 20 nM synthetic precursor miRNAs. (B) H1299 cells stably expressing the psiCheck2-Twist1-3′UTR reporter were transfected with 5 nM synthetic precursor miRNAs. <i>Renilla</i> luciferase activity was measured after 48 h and normalized to the Firefly luciferase activity. <i>Renilla</i> luciferase mRNA levels were measured by qPCR, normalized to Firefly luciferase mRNA levels and subsequently to cells transfected with the negative control.</p

MicroRNA expression levels in different mouse cell lines.

<p>Endogenous miRNA expression levels in NIH-3T3, C3H/10T1/2 and C₂C₁₂ cells were measured by qPCR using TaqMan probes, and normalized to U6 snRNA. Due to the high expression of miR-145a-5p, the relative values are presented on a logarithmic scale.</p