miR-7 overexpression promotes HaCaT cell proliferation and entry to the S phase of the cell cycle.

<p>(A) 5×10³ cells of independent miR-7 or pcDNA overexpressing clones were plated onto 24 well culture plates and counted every 24 hours during four consecutive days. The percentage of cells entering to S phase of the cell cycle was determined by BrdU incorporation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103987#s4" target="_blank">materials and methods</a>. BrdU+ cells were visualized under UV microscopy (B), cells were counted and the results plotted (C). Results represent the mean of three independent experiments performed with five or six pcDNA, miR-7 or miR-7+KLF4 overexpressing independent clones. ***<i>p</i><0.001 and **<i>p</i><0.01 <i>vs.</i> pcDNA, ###<i>p</i><0.001 <i>vs.</i> miR-7+KLF4.</p

KLF4 3′ UTR contains two evolutionary conserved binding sites that mediate the interaction with miR-7.

<p>(A) Schematic representation of the human KLF4 mRNA indicating the relative positions of the two identified miR-7 binding sites denoted as Seed 1 (S1) and Seed 2 (S2) located respectively, at positions 66–72 nt and 574–580 nt relative to the beginning (1915 nt) of the human KLF4 3′ UTR. Both S1 and S2 are phylogenetically conserved as shown in sequence alignments of different organisms. S1 is conserved in mammals and presents a thermodynamic stability (ΔΔG) of −5.72 kcal/mol while; S2 is conserved in all vertebrates and possesses a ΔΔG of −11.47 kcal/mol. (B) HEK-293 and A549 cells were co-transfected with 200 ng of pc/miR7, pc/miR145, pc/miR881 or empty vector (pcDNA) together with 100 ng of the wt KLF4 3′ UTR (KLF4), the mutated version of KLF4 3′ UTR for the second miR-7 binding site (KLF4-Mut) or empty vector (CHECK2). Luciferase activity was determined 48 hours post-transfection and relative luciferase units were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103987#s4" target="_blank">materials and methods</a>. Results represent the mean of at least six independent experiments. ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 <i>vs.</i> pcDNA.</p

CD43 expression confers tumoral fitness to human tumor-derived cells.

<p>A549 lung (<b>A</b>), CasKi cervix (<b>B</b>) or DLD-1 colon (<b>C</b>) tumor cells containing the empty pSuper (pSup) vector or expressing the CD43 specific RNAi (RNAi) were cultured to confluence, the monolayer was then wounded and healing was evaluated (upper panel). Cells were also cultured in soft agar as indicated in material and methods, after three weeks colonies were counted (middle panel). Cells (1X10⁶ for A549, 3X10⁶ for CasKi or DLD-1) were injected subcutaneously into nu/nu mice; one month later, animals were sacrificed and tumor weight was evaluated (lower panel). Data represents the average ± SD of four independent experiments performed with four independent pSup or RNAi clones for each cell line. *p < 0.05, **p < 0.01 vs pSup.</p

MiR-7 Promotes Epithelial Cell Transformation by Targeting the Tumor Suppressor KLF4

<div><p>MicroRNAs (miRNAs) are endogenous small non-coding RNAs that have a pivotal role in the post-transcriptional regulation of gene expression and their misregulation is common in different types of cancer. Although it has been shown that miR-7 plays an oncogenic role in different cellular contexts, the molecular mechanisms by which miR-7 promotes cell transformation are not well understood. Here we show that the transcription factor KLF4 is a direct target of miR-7 and present experimental evidence indicating that the regulation of KLF4 by miR-7 has functional implications in epithelial cell transformation. Stable overexpression of miR-7 into lung and skin epithelial cells enhanced cell proliferation, cell migration and tumor formation. Alteration of these cellular functions by miR-7 resulted from misregulation of KLF4 target genes involved in cell cycle control. miR-7-induced tumors showed decreased p21 and increased Cyclin D levels. Taken together, these findings indicate that miR-7 acts as an oncomiR in epithelial cells in part by directly regulating KLF4 expression. Thus, we conclude that miR-7 acts as an oncomiR in the epithelial cellular context, where through the negative regulation of KLF4-dependent signaling pathways, miR-7 promotes cellular transformation and tumor growth.</p></div

miR-7 overexpression promotes colony formation <i>in vitro</i> and tumor formation <i>in vivo</i> through KLF4 inhibition and regulating Cyclin D and p21 protein levels.

<p>(A) 1×10⁵ cells of three independent clones of A549 cells stably expressing pcDNA, miR-7 or miR-7+KLF4 were plated in a soft agar matrix and formed colonies were counted. (B) 3×10⁶ cells of three independent pcDNA, miR-7 or miR-7+KLF4 overexpressing A549 clones were subcutaneously injected into nude mice and after one-month post-implantation, mice were sacrificed. (C) Tumors were dissected and their mass was determined. (D) The relative expression of miR-7 on the tumors was determined by qPCR. (E) Tumors were analyzed for protein levels of KLF4, Cyclin D and p21 by Western blot assays using specific antibodies. ERK2 protein was used as loading control. The relative expression of each protein was calculated by dividing its densitometric signal by the ERK2 signal. Data were normalized considering the value of pcDNA transfected cells-derived tumors as 100%. Data represent the mean of three independent experiments using three (7 tumors), four (12 tumors) or three (8 tumors) independent miR-7, pcDNA or miR-7+KLF4 overexpressing clones, respectively. ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 <i>vs.</i> pcDNA.</p

miR-7 overexpression promotes cell cycle progression.

<p>Values are means ± s.d.; h, hours. n = 3,</p><p>**<i>p</i><0.01,</p><p>*<i>p</i><0.05 <i>vs.</i> pcDNA values.</p><p>miR-7 overexpression promotes cell cycle progression.</p

CD43 signaling cooperates with oncogenic signals to promote cell transformation.

<p>In epithelial cells, upon interaction with putative ligand(s) present on the cell surface of neighboring cells or in the extracellular matrix, CD43 activates the PI3K/AKT pathway that results in the inhibition of the Hippo pathway by a mechanism involving Merlin phosphorylation and degradation, thus favoring cell survival and proliferation. Depending of the cellular context, signaling from intracellular CD43 located either on membrane vesicles or the nuclear membrane may also contribute to cellular transformation. Nonetheless, this might not be sufficient to overcome the anti-proliferative and death effects resulting from the activation of the ARF-p53 pathway also induced by CD43 [24]. However, in transformed cells with an impaired p53 pathway resulting from oncogenic signals like those provided by the E6 oncoprotein from HPV16, CD43 signaling promotes cells proliferation and tumor formation. </p

miR-7 overexpression induces wound-healing and migration capacities of HaCaT and A549 cells.

<p>Wound-healing assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103987#s4" target="_blank">materials and methods</a>. HaCaT (A, left panel) or A549 cells (C, left panel) transfected with the pcDNA vector, expressing miR-7 only or both miR-7 and KLF4 (miR-7+KLF4) were photographed at the indicated times. The percentage of the wound-healed area at these time points was quantified using the TScratch software and plotted as the average of all the analyzed clones (A, C right panel). Migration assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103987#s4" target="_blank">materials and methods</a>. Sixteen hours after plating, cells were photographed (B, D upper panel). The number of HaCaT (B lower panel) or A549 cells (D lower panel) transfected with the pcDNA vector, expressing miR-7 only or both miR-7 and KLF4 (miR-7+KLF4) was quantified and plotted. Data represents the average of three independent experiments performed with at least three independent clones of each analyzed condition. ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 <i>vs.</i> pcDNA.</p

miR-7 overexpression promotes A549 cell proliferation through negatively regulating KLF4.

<p>(A) 2×10⁴ cells of pcDNA, miR-7 and miR-7+KLF4 overexpressing clones were plated in 24 well culture plates. Cells were counted every 24 hours during four consecutive days. (B) Once A549 cells reached confluence (T = 0), cell numbers were determined each 24 hours during two consecutive days. Fresh medium was added 24 hours post-confluence. Results represent the mean of six independent experiments performed with five or six pcDNA, miR-7 or miR-7+KLF4 overexpressing independent clones. ***<i>p</i><0.001, *<i>p</i><0.05 <i>vs.</i> pcDNA. (C) Protein levels of KLF4 target genes products. pcDNA and miR-7 expressing A549 cells were harvested at 0, 24 and 48 hours post-arrest, total cell extracts were prepared and Cyclin D and p27 protein levels were determined by Western blot analysis. ERK2 protein was used as loading control. Numbers denote the protein levels as the percentage of each condition relative to the pcDNA cells at 0 hours post-arrest. A representative Western blot of three independent experiments is shown.</p

CD43 signaling cooperates with oncogenic signals to promote cell transformation.

<p>NIH-3T3-hEGFR (<b>A</b>) or E6/E7 transgenic mouse fibroblasts (<b>C</b>) carrying the pFNeo empty vector (pFNeo), expressing wild-type CD43 (Wt) or CD43 lacking the intracellular domain (ΔIC) were grown to confluence; the monolayer was then wounded (t=0) and healing was evaluated by light microscopy at the indicated time points. NIH-3T3-hEGFR fibroblasts were grown in soft agar as described in material and methods; after three weeks, colonies were counted (<b>B</b>). E6 transgenic mouse fibroblasts stably transfected with the indicated constructs were grown to confluence for three weeks; foci were stained with Giemsa and counted (<b>D</b>). 3X10⁶ E6/E7 fibroblasts stably transfected were injected subcutaneously into nu/nu mice; one month later, animals were sacrificed and tumor mass was weighed (<b>E</b>). Data shown are representative of at least four independent experiments performed with at least four independent pFNeo, Wt or ΔIC clones from each cell line. Graphs represent the average cell number ± SD of three independent experiments using at least 3 independent clones. *p < 0.05 vs pFNeo.</p