The human colorectal carcinoma cell line HCT116 and the human lung adenocarcinoma cell line A549 were sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA). HCT116 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 50 U/ml penicillin/streptomycin and 2.0 g/l sodium bicarbonate. A549 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 50 U/ml penicillin/streptomycin and 3.7 g/l sodium bicarbonate. All the cells were maintained in a 37°C humidified incubator with 5% CO₂.

HCT116 cells were seeded for assays in 12-well plates at 300,000 cells/well and in 96-well plates at 10,000 cells/well. A549 cells were seeded for assays in 12-well plates at 200,000 cells/well and in 96-well plates at 5,000 cells/well. All transfection experiments were performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The amount of plasmid and volume of Lipofectamine 2000 were optimized to achieve 70–80% transfection efficiency for all functional and molecular characterizations.

The rabbit polyclonal anti-NF2 (dilution 1:1,200; cat. no. PA5-35316) and mouse monoclonal anti-N-cadherin (dilution 1:1,500; cat. no. MA5-15633) antibodies were obtained from Invitrogen (Thermo Fisher Scientific, Inc.). The rabbit polyclonal anti-E-cadherin (dilution 1:7,500; cat. no. 07-697) and mouse monoclonal anti-GAPDH (dilution 1:1,500; cat. no. CB1001) antibodies were obtained from EMD Millipore (Burlington, MA, USA). The rabbit polyclonal anti-vimentin antibody (dilution 1:700; cat. no. SAB4503083) was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The goat anti-mouse IgG (H+L) (dilution 1:10,000; cat. no. 31430) and goat anti-rabbit IgG (dilution 1:5,000 cat. no. 31460) secondary antibodies conjugated with horseradish peroxidase were obtained from Invitrogen (Thermo Fisher Scientific, Inc.).

The 3′UTR of human NF2 isoform I (NM_000268.3) and the pre-miR-92a-1 gene (NR_029508.1) were amplified in a polymerase chain reaction (PCR) reaction mixture containing a final concentration of 1X PCR buffer (Titanium^® Taq PCR buffer; Clontech Laboratories, Inc., Mountain View, CA, USA), 0.125 µM of each deoxynucleoside triphosphate (dNTPs) (Promega Corporation, Madison, WI, USA), 2 µM each of the forward and reverse primers, 1X Taq polymerase (Titanium^® Taq polymerase; Clontech Laboratories, Inc.) and wild-type human genomic DNA template available in the Disease Molecular Biology and Epigenetics Laboratory of the National Institute of Molecular Biology and Biotechnology (DMBEL-NIMBB). The cycling conditions were as follows: Initial denaturation at 94°C for 5 min, followed by 25–30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec, with a final extension step at 72°C for 10 min.

The wild-type NF2−3′UTR fragment was cloned into the pmirGLO Dual-Luciferase miRNA target expression vector (Promega Corporation), downstream of the firefly luciferase gene. A mutant version of the NF2−3′UTR was generated by site-directed mutagenesis of the wild-type NF2−3′UTR in pmirGLO, in which a CA>GT substitution within the miR-92a-3p seed sequence-binding site (5′-GTGCAAT-3′) was introduced through overlap extension PCR. The pre-miR-92a-1 amplicon was amplified using primers targeting the flanking genomic DNA template 173 bp upstream and 176 bp downstream of the pre-miRNA sequence (Chr. 13q31.3; NC_000013.11 region: 91351141.91351567) and was cloned into the mammalian microRNA expression vector pmR-ZsGreen1 (Clontech Laboratories, Inc.). Mutation of the shared MRE of the miR-92a family resulted in the removal of its members (miR-92a, miR-92b, miR-25, miR-32, miR-363 and miR-367) from the list of miRNAs predicted to bind to the mutant 792-bp fragment of NF2−3′UTR, as analyzed using the MirTarget custom prediction function of miRDB (12) (Table I). No new unintended miRNA binding sites were introduced to the mutant NF2−3′UTR. The primer sequences are listed in Table II.

For direct quantification of mature miR-92a-3p in control and transfected HCT116 and A549 cells, QuantiGene miRNA Assay (Affymetrix; Thermo Fisher Scientific, Inc.) was used. QuantiGene Singleplex miRNA probes were obtained from the pre-designed probes offered by eBioscience (Thermo Fisher Scientific, Inc.). The custom probe for miR-92a-3p (5′-UAUUGCACUUGUCCCGGCCUGU-3′) had previously been functionally validated for sensitivity and specificity for mature miR-92a-3p in human samples and had also been evaluated for cross-reactivity towards closely related miRNA family members. Cultured HCT116 and A549 cells were subjected to the assay according to the manufacturer's instructions. The luminescent signal generated was measured using a plate-reading luminometer (FLUOstar Omega microplate reader; BMG Labtech, Cary, NC, USA). Triplicates of cell lysates per setup of untransfected cells (maintained in media supplemented with 10 and 0.5% serum) and pmR-ZsGreen1-miR-92a-transfected cells were subjected to the assay. Average signals-background of samples were interpolated in a standard curve generated using serial dilutions of synthetic miR-92a-3p-positive control to calculate the average mature miR-92a-3p expression copy number in each experimental setup.

For the endogenous dual-Luciferase assays, cells were seeded in 96-well plates and transfected with 200 ng of empty pmirGLO vector (Promega Corporation) or pmirGLO-NF2−3′UTR-wild-type. For the miRNA co-transfection Dual-Luciferase assays, cells were co-transfected with 20 ng of empty pmirGLO vector, pmirGLO-NF2−3′UTR-wild-type, or pmirGLO-NF2−3′UTR-mut92a along with 200 ng of empty pmR-ZsGreen1 vector (Clontech Laboratories, Inc.) or pmR-ZsGreen1-miR-92a construct. Luciferase activity was determined at 48 h post-transfection using the Dual-Luciferase^® Reporter Assay system (Promega Corporation) and the GloMax 20/20 luminometer (Promega Corporation) following the manufacturer's instructions. Data are expressed as mean values of normalized firefly relative luciferase units (RLUs) per setup. Raw firefly RLUs were first normalized against internal control Renilla luciferase RLUs per well before normalizing against values obtained for the empty vector control setup.

HCT116 cells were co-transfected with pmR-ZsGreen1-miR-92a and a target protector oligonucleotide specific to the conserved MRE of hsa-miR-92a-3p within the 3′UTR of the NF2 gene. The oligonucleotide was designed using Qiagen's miRNA target protector design tool (https://www.qiagen.com/ph/shop/genes-and-pathways/custom-products/custom-assayproducts/custom-mirna-products/#target-protector) using the RefSeq ID of NF2 transcript variant 1 (NM_000268) as a reference template. An effective concentration of 100 nM miR-92a target protector co-transfected with pmR-ZsGreen1-miR-92a (final concentration, 2 ng/µl) was determined through preliminary transfection optimization experiments. The efficiency of miRNA inhibition by the target protector was measured using reverse transcription-quantitative PCR (RT-qPCR) or western blotting of lysates from transfected cells vs. control setups.

To facilitate specific knockdown of the NF2 gene and directly observe its functional consequences, cells were transfected with a 21-nt siRNA oligonucleotide (Qiagen Sciences, Inc., Germantown, MD, USA; Hs_NF2_7; 5′-CACCGTGAGGATCGTCACCAT-3′) directed against all known transcript variants of human NF2. The effective concentration of Hs_NF2_7 siRNA oligo was determined to be 5 nM using preliminary transfection optimization experiments with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The efficiency of siRNA knockdown of NF2 was assessed using RT-qPCR or western blotting of lysates from transfected cells vs. negative control setups.

HCT116 cells seeded in 12-well plates were transfected after 24 h with 2 µg of empty pmR-ZsGreen1 or pmR-ZsGreen1-miR92a construct. The cells were harvested at 48 h post-transfection and total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First-Strand cDNA was generated from 2 µg total RNA per setup using M-MLV Reverse Transcriptase (Promega Corpor-ation) and oligo-dT primers, according to the manufacturer's protocol. The First-Strand cDNA samples were then used as templates for semi-RT-qPCR and RT-qPCR with SYBR^® Select Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.) using the relative quantitation method. For semi-RT-qPCR, densitometric analysis of the digitized band intensities on the agarose gels were performed using GelQuant.NET software (v1.8.2) provided by biochemlabsolutions.com. Quantified NF2 gene expression in triplicate experiments was normalized against GAPDH gene expression. For RT-qPCR, total NF2 mRNA was normalized against GAPDH mRNA measured per sample. Mean values of triplicates per setup were obtained, and the fold-change of NF2 expression relative to the empty vector control was calculated. The primers used for RT-qPCR are summarized in Table II.

HCT116 cells were seeded and transfected in 12-well plates as described for RT-qPCR. Cells were harvested 48 h post-transfection and total protein was extracted using radioimmunoprecipitation lysis buffer [(150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris (pH 8.0)] supplemented with protease inhibitors [1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EDTA and 10 µM E64 (Roche Diagnostics GmbH, Mannheim, Germany)]. For polyacrylamide gel electrophoresis, 30 µg of total protein per setup quantified through the BCA method was loaded into 4–15% Mini-PROTEAN^® TGX Stain-Free™ Protein Gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% w/v whey protein in Tris-buffered saline with 0.1% v/v Tween-20 (TBST) for 1 h at room temperature, probed overnight with the primary antibodies aforementioned, washed with TBST, and incubated with the appropriate secondary antibodies for 1 h at room temperature. Signals were developed with enhanced chemiluminescence substrate and imaged using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometric analysis of digitized band intensities was performed using GelQuant.NET software (v1.8.2) provided by biochemlabsolutions.com. Gene expression levels were normalized against GAPDH expression.

Cells in 12-well plates were transfected 24 h after seeding with 2 µg of empty pmR-ZsGreen1 or pmR-ZsGreen1-miR-92a construct. Upon reaching >90% confluence, a thin artificial wound was created on the cell monolayer using a sterile 200-µl pipette tip. Cells were washed once with 1X phosphate-buffered saline (PBS), and maintained in the appropriate media supplemented with 10 or 2% FBS. Wound closure was monitored every 12 h by capturing three fields of view per setup at a magnification of ×40 using an Olympus IX71 inverted fluorescence microscope (Olympus Corp., Tokyo, Japan). Percent open wound area was analyzed using the TScratch software (13) and reported as the mean % open wound area per setup relative to % open wound area upon initial scratching (t=0 h).

Cells in 96-well plates were transfected 24 h after seeding with 200 ng of empty pmR-ZsGreen1 or pmR-ZsGreen1-miR-92a construct and maintained in the appropriate media supplemented with 10 or 2% FBS. The number of metabolically active cells per setup was measured at 48, 72 and 96 h post-transfection upon incubation with 10 µl of CellTiter 96^® Aqueous One Solution Cell Proliferation Assay reagent (Promega Corporation) per well, until color development. Absorbance values at 460 nm of each setup were measured with a colorimetric plate reader (FLUOstar Omega microplate reader; BMG Labtech). Cell counts were calculated from a standard curve (number of cells vs. A₄₆₀) generated using serial dilutions of an untransfected cell suspension. Mean cell counts were calculated per setup for each time-point.

Cells were seeded in 96-well plates and transfected in triplicate, as described above. HCT116 cells were incubated in RPMI-1640 media supplemented with 10 or 2% FBS alone, or with 2.5 mM sodium butyrate for induction of apoptosis. A549 cells were incubated in DMEM supplemented with 10% FBS or without FBS. Caspase-Glo^® 3/7 assay reagent (Promega Corporation) was added to each well at 24 h post-induction. The plates were incubated at ambient temperature for 2 h. Luminescence per well was measured with the FLUOstar Omega microplate reader (BMG Labtech). Mean luminescence readings per setup were calculated for each time-point and statistically analyzed.

HCT116 and A549 cells were seeded into an 8-well chamber slide (Ibidi GmbH, Martinsried, Germany) and transfected after 24 h. The cells were fixed at 48 h post-transfection with 4% paraformaldehyde for 20 min. PBS was used to wash cells between steps. Cells were permeabilized using 0.1% Triton X-100 in 1X PBS for 15 min and blocked with 1% bovine serum albumin (BSA) in PBS for 20 min. For actin staining, the cells were incubated at a 1:100 dilution of tetramethylrhodamine-conjugated phalloidin (Invitrogen; Thermo Fisher Scientific, Inc.). The nuclei were counterstained with Hoechst 33258 (1 µg/µl). Stained cells were mounted in 70% glycerol and were visualized under an Olympus IX83 inverted fluorescence microscope (Olympus Corp.), using a red fluorescent filter (λ_ex/λ_em: 490/525 nm) to visualize filamentous actin structures, a blue fluorescent filter (λ_ex/λ_em: 355/465 nm) to visualize the nuclei, and a green fluorescent filter (λ_ex/λ_em: 490/525 nm) to visualize cells transfected with pmR-ZsGreen1.

Statistical analysis of data was performed using unpaired two-tailed t-test to measure differences between two setups. Analysis of variance with post-hoc Tukey's honestly significant difference (HSD) was used to test significant differences between multiple setups. Data from all quantitative experiments are presented as the mean ± standard deviation (SD). In all tests, significance value was defined as P<0.05, P<0.01 and P<0.001.

To identify candidate miRNAs targeting the 3′UTR of the predominant NF2 isoform 1 mRNA (NM_000268.3), the key characteristics of a functional miRNA:target interaction were analyzed. Context score percentile and aggregate preferentially conserved targeting scores (P_CT) of each miRNA binding site was provided by TargetScanHuman v6.2 (http://www.targetscan.org/vert_61/) (Table III). The thermodynamic stability of miRNA:target interaction was analyzed using PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html). MirSVR scores predicting the likelihood of downregulation of NF2 by miRNAs from sequence and structure characteristics of predicted miRNA binding sites were obtained from microRNA.org. Rank and target scores of miRNAs predicted to target NF2−3′UTR were analyzed by mirDB (http://mirdb.org/miRDB/). In silico analyses identified a phylogenetically conserved MRE for hsa-miR-92a-3p within the 3′UTR sequence of wild-type NF2 (Fig. 1A). A summary of the bioinformatics analysis profiles of hsa-miR-92a-3p is presented in Table IV. Predictive scores of bioinformatics tools consistently ranked hsa-miR-92a-3p (hereinafter referred to as miR-92a) as a good potential candidate, and it was thus selected for the present study.

To test for endogenous regulation of NF2 via its 3′UTR, a 792-bp segment of the NF2−3′UTR encompassing nucleotides 26–818 after the stop codon was cloned into the miRNA target expression vector pmirGLO (Promega Corporation) (Fig. 1A). Dual-Luciferase assays revealed a significant decrease in normalized firefly RLUs in HCT116 (Fig. 1B) and A549 (Fig. 1C) cells overexpressing wild-type NF2−3′UTR compared with cells transfected with the vector only, suggesting that NF2 is endogenously regulated via its 3′UTR in both cell lines. Endogenous expression and significant overexpression of mature miR-92a from the pmR-ZsGreen1-miR-92a construct in HCT116 cells (Fig. 1D) and A549 cells (Fig. 1E) was verified and measured using the QuantiGene miRNA assay as described in Materials and methods. Co-transfection Dual-Luciferase assay results demonstrated a dose-dependent decrease in luciferase reporter expression corresponding to co-transfection of increasing amounts of miR-92a expression construct with wild-type NF2−3′UTR in both HCT116 (Fig. 1F) and A549 cells (Fig. 1G) under serum-depleted conditions.

To confirm the specificity of the interaction of miR-92a with its predicted binding site within the cloned NF2−3′UTR segment, dual-luciferase experiments were conducted using an NF2−3′UTR expression construct with mutated miR-92a MRE (Fig. 1H). Luciferase readouts from cells transiently overexpressing miR-92a and co-transfected with the mutant NF2−3′UTR were comparable to readouts from the empty vector controls in both HCT116 (Fig. 1I) and A549 cells (Fig. 1J), revealing that downregulation of luciferase reporter expression by miR-92a was reversed by destruction of its MRE within the NF2−3′UTR. Overall, these results demonstrated that miR-92a downregulated NF2 via its 3′UTR, in part by directly interacting with its conserved MRE within the NF2−3′UTR sequence.

MicroRNAs can regulate a gene either by causing mRNA degradation and/or by translational repression. Semi-RT-qPCR results revealed significant downregulation of NF2 mRNA expression in HCT116 cells overexpressing miR-92a compared with cells transfected with empty vector under serum-deprived conditions (Fig. 2A), which corroborated with the results obtained in parallel RT-qPCR experiments (Fig. 2B). Western blot analyses of transfected HCT116 cells also revealed markedly lower Merlin protein expression levels in cells transiently overexpressing miR-92a vs. the empty vector control (Fig. 2C). These results suggest that miR-92a can downregulate NF2 expression at both the mRNA and protein level, either through transcript degradation alone, or through translational repression as well.

Typically, miRNAs target multiple mRNAs within the cell via partial seed sequence complementarity with their respective 3′UTRs. To demonstrate that the regulatory effects of miR-92a overexpression on NF2 mRNA and protein expression is due to direct interaction of miR-92a with NF2−3′UTR, a target protector oligonucleotide (TP-92a) was used to specifically block the conserved MRE of miR-92a within the NF2−3′UTR. HCT116 cells transfected with miR-92a alone or co-transfected with a negative control target protector (TPneg) displayed significantly downregulated NF2 mRNA expression compared with control cells transfected with empty vector and TPneg. This negative regulatory effect was abolished upon co-transfection of miR-92a with TP-92a (Fig. 2D). Similarly, co-transfection of miR-92a with TP-92a rescued the expression of Merlin, which was downregulated in cells transfected with miR-92a alone or co-transfected with TPneg (Fig. 2E). Overall, these results demonstrated that miR-92a downregulates NF2 mRNA and protein expression specifically by binding to its conserved MRE within the NF2−3′UTR sequence.

Merlin is known to be involved in contact-mediated inhibition of proliferation (2,3). To determine whether upregulation of miR-92a can override this function of Merlin, proliferation rates were assessed in HCT116 and A549 cells transfected with miR-92a expression construct. Under serum-depleted conditions (2%), HCT116 (Fig. 3A) and A549 (Fig. 3B) cells overexpressing miR-92a exhibited a significant increase in the number of viable cells between 72 and 96 h post-transfection, at which point the cells were observed to reach semi-confluence (>90% growth surface area). This indicated that, under space-limiting conditions, overexpression of miR-92a promoted proliferation of HCT116 and A549 cells, at least in part through downregulation of NF2 mRNA/protein expression.

Merlin also exerts its tumor suppressor effects by promoting apoptosis via the Hippo/SWH (Sav/Wts/Hpo) signaling pathway (14). Assessment of apoptosis induction in HCT116 cells transfected with miR-92a and maintained under low-serum conditions (2%) revealed significantly lower levels of activated caspase-3/7 compared with empty vector control in both the absence or presence of the apoptosis inducer sodium butyrate (Fig. 3C). Similarly, overexpression of miR-92a in A549 cells incubated under serum-deprived conditions (0.5%) was associated with significantly lower levels of active caspase-3/7 compared with empty vector control (Fig. 3D). Overall, these results demonstrated that miR-92a inhibited caspase-3/7 activation and promoted resistance to apoptosis in HCT116 and A549 cells, possibly, at least in part, by downregulation of NF2 mRNA/protein expression.

To assess whether overexpression of miR-92a inhibits the negative regulatory effect of Merlin on cell motility, the wound healing capacity of transfected HCT116 and A549 cells was assessed. Cells overexpressing miR-92a migrated over the introduced wound gap at a significantly faster rate compared with cells transfected with empty vector control in both HCT116 (Fig. 4A) and A549 (Fig. 4B) cell lines under serum-depleted conditions (2%). This indicated that overexpression of miR-92a enhanced the motility of colorectal and lung cancer cells, possibly in part by inhibition of NF2 mRNA/protein expression.

To explore a possible molecular mechanism underlying the observed increase in the migration capacity of cells overexpressing miR-92a, the expression of EMT markers was assessed. At the transcriptional level, normalized band intensities from semi-RT-qPCR consistently showed downregulation of NF2 mRNA expression in cells transfected with miR-92a vs. empty vector control (Fig. 4C). Consequently, a decrease in the transcript expression of the epithelial marker E-cadherin was observed in miR-92a-overexpressing cells, with a corresponding increase in mRNA expression of the mesenchymal marker N-cadherin (Fig. 4C). Contrary to what was expected, a decrease in the mRNA expression of vimentin was observed in miR-92a-overexpressing cells (Fig. 4C). At the protein level, normalized band intensities from western blot analyses revealed downregulation of NF2 protein expression in cells transfected with miR-92a compared with empty vector control (Fig. 4D). No significant change in E-cadherin protein expression was observed in cells overexpressing miR-92a, although a marked increase in protein expression of both N-cadherin and vimentin was detected (Fig. 4D). Overall, these results demonstrated that downregulation of NF2 by overexpression of miR-92a led to an apparent shift in EMT marker expression from an epithelial to a mesenchymal-like phenotype. These results were consistent with the observed increase in the migration capacity of cells overexpressing miR-92a, demonstrating that negative regulation of NF2 by miR-92a in HCT116 cells promoted cell migration by inducing partial EMT.

Merlin is known to act as an organizer of the actin cytoskeleton primarily through the F1 and F2 subdomains within its N-terminal 4.1 ezrin-radixin-moesin (FERM) domain. Although not required per se, the domains also facilitate the function of Merlin in contact-inhibition of growth (15). To determine whether miR-92a overexpression exerts deleterious effects on the cytoskeletal dynamics of epithelial carcinoma cells, the filamentous actin network of transfected A549 cells was visualized through fluorescence staining.

Untransfected A549 cells displayed a typical resting epithelial cell morphology, with an overall flat, spread-out cell shape, sparse transverse stress fibers and high intercellular cohesion, as also observed in control cells transfected with empty pmR-ZsGreen1 vector and negative control target protector oligonucleotide (Fig. 5). By contrast, A549 cells transfected with miR-92a alone or co-transfected with negative control target protector displayed gross morphological changes in cytoskeletal organization, as evidenced by cytoplasmic shrinkage and a spindle-like morphology, which are characteristic of highly motile cells (Fig. 5). Co-transfection of miR-92a with the target protector for its conserved MRE within NF2−3′UTR appeared to abolish the cytoskeletal changes induced by miR-92a overexpression (Fig. 5). Collectively, these results indicated that miR-92a induced a highly dynamic actin network typical of cells with a more motile phenotype, possibly in part through downregulation of NF2 mRNA/protein expression.

MicroRNAs target multiple genes within the cell via partial seed sequence complementarity with their respective 3′UTRs. Thus, phenotypic readouts observed upon overexpression of a single miRNA species is typically the overall effect of the miRNA on its endogenous targets expressed in the cell system tested. To confirm the contribution of NF2 knockdown in the observed phenotypes consequent to miR-92a overexpression, RNA interference via small interfering RNA (siRNA) was used as a gene-specific alternative to knockdown by a miRNA. An NF2-specific pre-designed siRNA oligonucleotide was used to induce short-term silencing of the NF2 gene. Knockdown of endogenous NF2 was confirmed by RT-qPCR (Fig. 6A) and western blot analysis (Fig. 6B).

Similar to cells transiently overexpressing miR-92a, cell proliferation experiments performed on HCT116 (Fig. 6C) and A549 (Fig. 6D) cells revealed a marked increase in the number of viable cells in setups transfected with NF2 siRNA upon reaching semi-confluence (>90%) at 24 h post-transfection. Abolition of the pro-apoptotic function of Merlin was demonstrated in A549 cells by the significantly lower activity of caspase-3/7 measured in cells transfected with NF2 siRNA compared with control cells (Fig. 6F). On the other hand, NF2 siRNA-transfected HCT116 cells did not display any significant difference in caspase-3/7 activity compared with controls (Fig. 6E). This finding suggests that the function of Merlin in promoting apoptosis is cell context-dependent.

Knockdown of Merlin by transfection of NF2 siRNA also resulted in a significant increase in motility compared with controls for both HCT116 (Fig. 6G) and A549 (Fig. 6H) cells. Furthermore, HCT116 cells transfected with NF2 siRNA consequently exhibited a decrease in the expression of E-cadherin with a concurrent increase in the mRNA expression of N-cadherin and vimentin at both the mRNA (Fig. 6I) and protein levels (Fig. 6J). Additionally, A549 cells transfected with NF2 siRNA displayed gross morphological changes brought about by cytoskeletal organization, as evidenced by cytoplasmic shrinkage and a more spindle-like morphology, which are characteristic of highly motile cells (Fig. 6K).

Overall, these results demonstrated that NF2 knockdown by RNA interference phenocopies the oncogenic effects of miR-92a overexpression. Given that miR-92a has also been shown to exert its oncogenic effects in colorectal and lung cancer by potentially targeting phosphatase and tensin homolog (PTEN) (16,17) and Krüppel-like factor 4 (KLF4) (18), the results of the present study confirm the role of miR-92a in simultaneously targeting the tumor suppressors NF2, PTEN and KLF4, and possibly other targets, in order to bring about a congruent phenotypic readout.