Let-7d microRNA affects mesenchymal phenotypic properties of lung fibroblasts

Abstract

MicroRNAs are small noncoding RNAs that inhibit protein expression. We have previously shown that the inhibition of the microRNA let-7d in epithelial cells caused changes consistent with epithelial-to-mesenchymal transition (EMT) both in vitro and in vivo. The aim of this study was to determine whether the introduction of let-7d into fibroblasts alters their mesenchymal properties. Transfection of primary fibroblasts with let-7d caused a decrease in expression of the mesenchymal markers α-smooth muscle actin, N-cadherin, fibroblast-specific protein-1, and fibronectin, as well as an increase in the epithelial markers tight junction protein-1 and keratin 19. Phenotypic changes were also present, including a delay in wound healing, reduced motility, and proliferation of fibroblasts following transfection. In addition, we examined the effects of transfection on fibroblast responsiveness to TGF-β, an important factor in many fibrotic processes such as lung fibrosis and found that let-7d transfection significantly attenuated high-mobility group-A2 protein induction by TGF-β. Our results indicate that administration of the epithelial microRNA let-7d can significantly alter the phenotype of primary fibroblasts.

since their discovery in Caenorhabditis elegans, microRNAs have been identified in all higher organisms (29). To date, there are more than 2,233 microRNA sequences in the human genome (6). These small noncoding RNAs (19–24 bp in length) regulate protein expression by either inhibiting translation or initiating specific degradation of target mRNAs and play a fundamental role in the regulation of gene expression (29). MicroRNAs are crucial mediators in functions such as development, differentiation, proliferation, migration, and cell death (8). Expression of microRNAs has been shown to be associated with multiple diseases (15), including liver, kidney, and heart fibrosis (4, 28, 39). We and others (13, 14, 18, 21, 25, 38) have identified that microRNAs are both changed in expression and relevant mechanistically in the lungs of patients with idiopathic pulmonary fibrosis (IPF). IPF is a chronic and usually lethal fibrotic lung disease that is characterized by alveolar epithelial cell injury and progressive accumulation of extracellular matrix in the lung parenchyma when myofibroblasts play a fundamental role in the tissue remodeling (22, 26). Among the changed microRNAs were members of the Mir-154, Mir-29, Mir-17–92, and let-7 families.

The let-7 microRNA family was among the first microRNA families discovered and is highly conserved across animal species (27). Let-7 is involved in various cellular responses and is a key regulator of cell development and cancer (3, 29, 34). We have previously demonstrated that both in vitro and in vivo inhibition of let-7d resulted in upregulation of the mesenchymal markers and downregulation of epithelial markers, suggesting that changes consistent with epithelial-to-mesenchymal transition (EMT) might occur in IPF lungs (37). These changes are characterized by loss of E-cadherin and increased expression of several E-cadherin transcriptional repressors, such as Zeb-1, Zeb-2, TWIST, Snail, and Slug (17). High-mobility group-A2 protein (HMGA2) is another transcription factor that is inhibited by let-7 microRNAs and plays a fundamental role in EMT (21). Although the role of let-7 microRNAs has been studied extensively in epithelial cells, their effects on mesenchymal cells are not completely clear. In this study, we explored whether the administration of let-7d to primary fibroblasts will have an effect on their mesenchymal properties.

MATERIALS AND METHODS

Cell Culture

To ensure that we are studying a wide-range phenomena not limited to a particular cell line, we conducted our experiments on three types of primary fibroblasts: human fetal lung fibroblasts (FLF), normal human lung fibroblasts (NHLF), and human fetal foreskin fibroblasts (HFF-1). We present herein representative results, but most experiments were conducted on all three types of fibroblasts. FLF were obtained from tissues collected through the University of Pittsburgh Tissue Bank as previously described (18). CF-1 media (DMEM), which contained 10% heat-inactivated fetal bovine serum, 1 mM L-glutamine, 1% penicillin/streptomycin, and 0.1 mM nonessential amino acids (Invitrogen, Carlsbad, CA), was used for cell growth. All experiments with FLF cell line were approved by IRB protocol 0506140. NHLF were purchased from Lonza (Basel, Switzerland), and fibroblast growth media-2 was used for this cell line. HFF-1 were obtained from the ATCC and cultured according to the manufacturer's protocol. CF-1 was used for cell growth. All cell lines were cultured in a humidified chamber at 37°C with 5% CO₂.

Transfection

In six-well plates, 150,000 cells were plated per well at 70% confluence in CF-1 medium. After cells adhered, the medium was replaced by Opti-MEM I reduced serum medium (Invitrogen). Transfection of hsa-let-7d precursor, the negative control-scrambled and cy3-labeled scrambled (Ambion, Austin, TX) were carried out at 100 nM using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The initial calibration of transfection efficiency was done by demonstrating that, after transfection with cy3-labeled scrambled microRNA, over 90% of the cells expressed cy3. RNA was isolated 24 h posttransfection, and 6 ng/ml was used to stimulate cells with TGF-β.

RNA Isolation

RNA was isolated using the miRNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA quantity was determined using Nanodrop spectrophotometer (NanoDrop, Wilmington, DE) and its quality determined using Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).

Quantitative RT-PCR.

TaqMan MicroRNA assays (ABI, Foster City, CA) were used to determine the relative expression levels of hsa-let-7d. Results were analyzed by the ΔΔCt method using RNU43 control RNA to normalize the results. Fold change was calculated taking 0 h as the baseline. TaqMan gene expression assays (ABI) were used to determine the relative expression levels of HMGA2, SNAIL, SLUG, TWIST, LIN28, inhibitor of differentiation (ID)1, ID2, N-cadherin (N-CAD), vimentin, α-smooth muscle actin (α-SMA), fibronectin (FN1), keratin 19 (KRT19), and tight junction protein 1 (TJP-1). The results were analyzed by the ΔΔCt method, and β-glucuronidase was used for normalization. Fold change was calculated taking 0 h as the baseline. Fold change was calculated relative to scrambled. To calculate fold change in cells stimulated with TGF-β, 0 h was considered baseline.

Protein extraction.

FLF were cultured and transfected as described above. Cells were washed three times with PBS, lysed in 500 μl lysis buffer (50 mM Tris·HCl, pH, 7.5, 500 mM NaCl, NP-40, 0.25% nadeoxycholate, 20 mM NaF), aliquoted, and stored at −20°C until use.

Western Blots

Protein concentration was measured using Pierce's bicinchoninic acid protein quantitation assay kit (Pierce Chemical, Rockford, IL) and resuspended in lamellae buffer (R&D Systems, Minneapolis, MN) containing 5% β-mercaptoethanol (Sigma, St. Louis, MO). Equal concentrations of the proteins were then loaded in wells of 6% or 10% SDS-PAGE (Bio-Rad, Hercules, CA). The gels were run using Mini-Protean electrophoresis module assembly (Bio-Rad) at 150 mV at 4°C in running buffer (25 mM Tris base, 192 mM glycine, and 0.1% SDS) (Sigma) in double-distilled water, followed by wet transfer to polyvinylidene difluoride membranes (Millipore, Bedford, MA) for 90 min using the Mini Trans-Blot electrophoresis transfer cell (Bio-Rad) at 280 mA in transfer buffer (25 mM Tris, pH 7.5, 192 mM glycine, 20% methanol, and 0.025% sodium dodecyl sulfate) (Sigma). The membranes were then blocked for 45 min with 3% BSA in Tris-buffered saline/Tween 20 (Sigma) and incubated overnight with the following primary antibodies: α-SMA, N-CAD, TJP-1 (Abcam, Cambridge, MA), and β-actin (Sigma). Membranes were washed three times for 15 min each before and after they were incubated with appropriate secondary antibody. β-Actin was used as a loading control. The washed membranes were exposed to chemiluminescent substrate (PerkinElmer Life Sciences, Waltham, MA) and then visualized using autoradiography (Kodak, Pittsburgh, PA). To strip the antibodies, membranes were incubated in slow shaking stripping buffer (1 mM HCl, pH 2.2) for 45 min. The autoradiographs were scanned and processed using Adobe Photoshop CS2 software (Adobe Systems, San Jose, CA). Densitometry was performed using the shareware ImageJ (http://rsbweb.nih.gov/ij/index.html).

Immunofluorescence

Immunofluorescence was performed as previously described (21). Briefly, human FLF cells were plated on coverslips. Coverslips were removed, washed with PBS three times for 5 min each, and then fixed in 1% paraformaldehyde (Sigma) for 45 min. Permeabilization of cells was carried out using 0.1% Triton X-100 in PBS for 15 min, and then they were air dried and dehydrated with three washes in PBS each for 5 min followed by three washes each for 5 min with 0.3% BSA and 5% goat serum in PBS. Coverslips were blocked with 2% BSA in PBS for 60 min, incubated with anti-cytokeratin, N-CAD, α-SMA, and TJP-1 (Abcam) in 0.5% BSA in PBS for 60 min, washed three times with 0.5% BSA in PBS (5 min each), and incubated with rabbit anti-mouse conjugated to Alexia 488 and mouse anti-rabbit conjugated to Alexia 586 (Invitrogen) for 1 h. After the staining, coverslips were washed with 0.1% Triton X-100 in PBS twice for 5 min each followed by three washes with PBS for 5 min each. Coverslips were inverted onto slides and mounted in Vectashield anti-fade medium that contained DAPI for nuclei staining (Vector Laboratories, Burlingame, CA) to prevent photobleaching. Slides were examined using a Leica TCS-SP2 laser-scanning confocal microscope equipped with appropriate lasers for simultaneous imaging of up to four fluorophores.

Fibroblast Phenotyping

Scratch assay.

Cells were cultured and transfected as described above in six-well plates. When culture was confluent (24 h posttransfection), a p-200 pipette tip was used to score two vertical lines and one horizontal line (average width: 700–900 μm) simulating a “wound” by scratching the culture. Photographs were taken every 3 h with a camera attached to a light microscope.

Senescence.

Senescence was determined 24 h after transfection by the senescence detection kit (Abcam) according to the manufacturer's protocol. Cells were seeded and transfected as described above. For stable transfected cells, senescence was determined 24 h after the cells were seeded.

Migration.

Migration was determined by counting the number of cells that migrated through Matrigel inserts with 8-μm pores (Becton Dickinson, Woburn, MA) according to the manufacturer instructions and as previously described (18).

Proliferation.

Proliferation was performed as previously described (18). In the [3H]-thymidine incorporation assay, cells were briefly pulsed with [3H]-thymidine (1 μCi/well, PerkinElmer) and were incubated for 48 h.

Stable Transfection

Approximately 60 μl of concentrated let-7d or a control (scrambled sequence) lentiviral vector (Openbiosystems, Waltham, MA) were transduced into fibroblasts (HFF-1, FLF, or NHLF) that were ∼85% confluent in a T-25 flask (∼5 ml/flask). Cells were incubated overnight (37°C, 5% CO₂) with concentrated virus and 6 mg/ml polybrene. Cells were selected using 2 μg/ml puromycin solution (Sigma) in growth media. Cells were grown in puromycin selection media until most cells were dead, and all living cells appeared red under the fluorescent microscope because the vector carried a red fluorescent protein (RFP) for selection. At this point, cells were treated as described above.

Luciferase Reporter Assay

Cells were plated and transfected as mentioned above. The HMGA2 3′-UTR miRNA target sequence expression clone in pEZX-MT01 vector with fLuc was commercially obtained (Genecopoeia, Rockville, MD). According to the manufacturer's instructions, the effects of let-7d on HMGA2 were estimated by the ratios of Renilla luciferase readings to Firefly luciferase readings.

Microarray

Microarrays were performed as previously described (5). Briefly, transfected cells were scraped with Trizol (Invitrogen), and total RNA was extracted and used as a template for double-stranded cDNA synthesis. RNA quantity was determined by NanoDrop at 260 nm and RNA integrity by Bioanalyzer (Agilent Technologies). Labeling was performed using the Agilent Low RNA Input Linear Amplification Kit PLUS, one color (5184-3523, Agilent Technologies). Briefly, first-strand cDNA synthesis was performed using an oligo(dT) 24 primer containing a T7 RNA polymerase promoter site. The cDNA was used as a template to generate Cy3-labeled cRNA that was used for hybridization. After purification and fragmentation, aliquots of each sample were hybridized to Agilent Whole Human Genome 4 × 44K arrays (G4112F, Agilent Technologies). After hybridization, each array was sequentially washed and scanned by Agilent microarray scanner. Arrays were individually visually inspected for hybridization defects, and quality control procedures were applied as recommended by the manufacturer of the arrays. For array readout, Agilent Feature Extraction 9.5.3 Software was used, and microarray data were normalized using cyclic Lowess by in-house software built in the R programming environment. Differentially expressed genes were identified using Significant Analysis of Microarrays (http://www-stat.stanford.edu/∼tibs/SAM). Genes were considered to be differentially expressed if a P value <0.05 was observed. A heat map was generated using score-gene software (1). The data are deposited on the Gene Expression Omnibus and are publically available (GEO accession number of GSE38530).

RESULTS

Overexpression of Let-7d in Fibroblasts Causes Reduction in Its Targets as Well as Mesenchymal Markers

Transfection of let-7d in FLF resulted in a significant increase of its expression compared with endogenous levels of this microRNA (Fig. 1A). HMGA2 is a known target of let-7d (16). We examined the effect of let-7d transfection on HMGA2 mRNA levels. As expected, let-7d transfection caused a decrease in HMGA2 mRNA in both FLF and HFF-1 (Fig. 1B). To confirm that HMGA2 is a direct target of let-7d in fibroblast, we have performed an HMGA2 3′ UTR luciferase assay. We observed a significant decrease in the Firefly/Renilla luminescence, which suggests that let-7d directly affects HMGA2 (Fig. 1C). Furthermore, we have found in our microarray gene expression data a significant decrease in both Myc (fold change = −0.85, P < 0.05) and cyclin d2 (fold change = −0.3, P < 0.05), which are known let-7d targets. To confirm our transfection efficiency, we overexpressed FLF with cy-3-scrambled sequence and found all cells to appear red under the microscope (data not shown). Using qPCR, we examined the gene expression of both mesenchymal markers and epithelial markers following transfection of let-7d. We found significant decreases in the mRNA levels of mesenchymal markers α-SMA, N-CAD, fibroblast-specific protein-1, FN1 but not vimentin (Fig. 2A). At the protein level, we observed a significant decrease in α-SMA and N-CAD (Fig. 2, B and C) as well as a significant increase in epithelial marker TJP-1 (Fig. 2D). This was consistent with mRNA level trends, which was also found in epithelial marker KRT19 (Fig. 2A).

Fig. 1.

let-7d transfection efficiency. A: fetal lung fibroblasts (FLF) were transfected with let-7d or a scrambled sequence as a control, as described in materials and methods. 24 h posttransfection let-7d levels were evaluated using qPCR (n = 3). B: fibroblasts [FLF and human fetal foreskin fibroblasts (HFF)] were transfected with let-7d and a scrambled control (n = 3). High-mobility group-A2 protein (HMGA2) gene expression levels were examined. C: HMGA2 3′ UTR luciferase assay was performed (n = 9) as described in materials and methods to confirm direct effect of let-7d on HMGA2 expression. 24 h posttransfection with let-7d and a scrambled ratio of Renilla luciferase readings to Firefly luciferase readings were determined. *P < 0.05.

Fig. 2.

Gene expression and protein expression in fibroblasts are altered by transfection of let-7d. A: FLF were transfected with let-7d (n = 3), and gene expression was evaluated 24 h later by qPCR for both mesenchymal markers [α-smooth muscle actin (α-SMA), N-cadherin (N-CAD), vimentin (VIM), fibroblast-specific protein 1 (FSP-1), and fibronectin (FN1)] and epithelial markers [tight junction protein 1 (TJP-1) and keratin 19 (KRT19)]. B–D: Western blot analysis (n = 3) confirmed gene expression results. α-SMA (B), N-CAD (C), TJP-1 (D) are shown. Left: Western blot. Right: quantification using ImageJ. *P < 0.05.

Overexpression of Let-7d Induces Changes In Fibroblast Function

We next examined whether differences in gene and protein expression seen after microRNA transfection led to changes in fibroblast functional properties. We determined whether let-7d transfection affected the ability of fibroblasts to repopulate a scratched area in a confluent culture. As depicted in Fig. 3A, let-7d-transfected cells were slower to regain full confluence over a wounded area in the scratch assay. Because this phenomenon is explained by an inhibitory effect of let-7 on fibroblast proliferation, migration, or both, we looked at the effects of overexpression of let-7 on fibroblast proliferation and migration separately. Impressively, let-7d-transfected fibroblasts exhibited a significantly slower proliferation rate (Fig. 3B) and a slower migration rate (Fig. 3C) compared with scrambled-control-transfected fibroblasts.

Fig. 3.

Fibroblast cellular properties are altered by let-7d transfection. Let-7d-transfected cells (n = 3) were subjected to 3 methods for evaluating their cellular properties: scratch assay (A), proliferation assay (B), and migration assay (C). For details see materials and methods. All show that transfected cells are less mobile and less proliferative. *P < 0.05. CPM, counts per minute.

Let-7d Inhibits TGF-β-Induced Expression of HMGA2 and α-SMA

Because HMGA2 is a TGF-β target, we tested let-7d ability to modify the response to TGF-β without directly effecting TGF-β levels. We examined plasminogen activator inhibitor-1 (PAI-1), a known direct TGF-β1 pathway target that is not a target of let-7d. As expected, let-7d transfection did not affect TGF-β-induced expression of PAI-1 (Fig. 4A). Consistent with previous reports (21, 36), TGF-β caused an increase in HMGA2 (Fig. 4B) and α-SMA (Fig. 4C). Both increases were significantly modified by let-7d transfection, suggesting that let-7 overexpression can be used to prevent the effects of TGF-β on primary lung fibroblasts.

Fig. 4.

Effects of TGF-β are inhibited by cotransfection with let-7d. To examine the reciprocal effects of let-7d and TGF-β, FLF fibroblasts were transfected with let-7d (n = 3) and 24 h later treated with TGF-β for 6 h. Plasminogen activator inhibitor-1 (PAI-1), a known target of TGF-β (A), HMGA2 (B), and α-SMA (C) levels were all evaluated using qPCR. *P < 0.05.

Stable Transfection of Let-7d Causes Global Phenotypic Changes in Fibroblasts

Because transient transfection of let-7d caused distinct phenotypic changes in transfected fibroblast, we set out to establish primary fibroblast lines that constitutively overexpressed let-7d. For this, we utilized a lentivirus system, which carried Neo selection and RFP for selection markers. These transfected cells are resistant to puromycin and also appear red under the florescence microscope. As seen in Fig. 5A, all cells appear red following establishment of stably transfected lines. Using qPCR, we confirmed high let-7d expression levels following transfection and puromycin selection (Fig. 5B).

Fig. 5.

Stable transfection of let-7d alters mesenchymal properties of fibroblasts. Normal human lung fibroblasts cells were stably transfected with let-7d or a scrambled sequence as control. A: let-7d-expressing cells were larger and scarcer as seen under the florescent microscope. Red cell appearance is due to the expression of red fluorescence protein in the expression construct. B: let-7d expression levels were examined using qPCR (n = 3). C: gene expression of stably transfected cells (n = 3) was evaluated for mesenchymal markers (α-SMA, N-CAD) and the epithelial marker TJP-1 and confirmed using immunofluorescence and specific antibodies (D), mesenchymal markers (α-SMA and N-CAD), and epithelial markers (TJP-1 and cytokeratin). *P < 0.05.

We next examined the expression of epithelial and mesenchymal markers of let-7d stably transfected fibroblasts. Consistent with previous results in transiently transfected cells (Fig. 2), a significant decrease in the mRNA of mesenchymal markers N-CAD and α-SMA was observed in stably transfected cells. A nonsignificant decrease was found in TJP-1 mRNA levels (Fig. 5C). To determine whether changes in gene expression correlated to changes in protein levels, immunofluorescence and specific antibodies were used. As can be seen in Fig. 5D, a decrease in staining of α-SMA and a concomitant increase in staining intensity of the epithelial markers TJP-1 and cytokeratin are seen. Interestingly, the let-7d-expressing cells were larger and more cuboidal, and there were consistently fewer cells on the plate although the same numbers of cells were seeded on coverslips for staining, potentially reflecting slower proliferation rates (Fig. 5D).

Stable-Transfection Let-7d Changes Fibroblast Functionality

We next examined the functional characteristics of the stably transfected let-7d fibroblasts (NHLF). Similar to the transiently transfected cells, stably transfected fibroblasts were slower to close a gap in the scratch assay (Fig. 6A). We looked at the cell proliferation and migration and determined that cells stably transfected with let-7d had slower proliferation and migration rates (Fig. 6, B and C). We next examined the cell cycle properties of HFF-1 and FLF stably transfected cells using florescence-activated cell sorting. We found a decrease in HFF-1 S phase and an increase in G2/M phase in both cell types. These observations were less evident in FLF cells compared with HFF-1 cells, indicating that cell cycle results could be cell line specific (Fig. 6, D and E). To have a better understanding of whether the phenomena we observed were related to cell senescence, we performed β-galactosidase staining on FLF cells. We found that a few let-7d-expressing cells expressed senescence markers (Fig. 6F) compared with no staining in the control scrambled, but this finding was rare and did not explain the significant changes in fibroblast phenotype.

Fig. 6.

Let-7d stably transfected fibroblasts undergo phenotypical changes. Cells (n = 3) were less mobile as seen by the scratch assay (A) and migration assay-measured Boyden chamber (B). C: cells (n = 3) were less proliferative as measured by thymidine incorporation. For details, see materials and methods. D and E: cell cycle was evaluated using florescence-activated cell sorting (FACS) and propidium iodide. A typical analysis is depicted in D; cells in S-phase are shown in striped area. E: quantification of FACS experiments using 2 stably transfected cell lines (n = 3). Let-7d-transfected cells are in black and scrambled transfected-controls in white. Percentage of cells in each phase of the cell cycle are demonstrated. F: senescence was evaluated by x-gal staining and visualization under the light microscope. No major differences were found between scrambled and control (n = 3). *P < 0.05.

Let-7d Induces Global Decreases in Mesenchymal Markers in Primary Lung Fibroblasts

To determine whether let-7 transfection affected the global mesenchymal phenotype of primary fibroblasts, we examined the expression of 67 genes known from the literature (30) to be mesenchyme markers in our gene expression microarray dataset. We found that 35 of 67 genes were significantly changed (Fig. 7A), while 26 were also differentially expressed in IPF (unpublished data). Because let-7 microRNAs are decreased in IPF lungs, we looked at genes that were increased in IPF lungs and decreased in fibroblasts in response to transfection with let-7. We identified 13 such genes, suggesting that they indeed represent effects of downregulation of let-7 microRNAs in the IPF lung. qPCR of transcription factors associated with regulation of cellular mesenchymal phenotype such as SLUG, a downstream target of HMGA2, ID1, ID2, SNAIL1, and TWIST revealed that SLUG, ID1, and ID2 were significantly decreased but not SNAIL and TWIST (Fig. 7B). Taken together, these results suggest that let-7 introduction to fibroblasts modulates their mesenchymal phenotype, potentially through HMGA2, SLUG, ID1, and ID2. Furthermore, upregulation of epithelial markers was observed. KRT19 was also found to be significantly upregulated in the array data, along with KRT14, and TJP-1 is significantly upregulated at the protein level. These changes in expression of epithelial markers emphasize the significance of let-7d in affecting cellular phenotype.

Fig. 7.

Gene expression of known epithelial-to-mesenchymal transition markers after let-7d transfection. FLF were transfected with let-7d and scrambled (n = 5), and gene expression was evaluated 24 h posttransfection using microarray and demonstrated using a heatmap (A). 67 genes were examined. Genes highlighted in red are significant by Student's t-test (P < 0.05). Genes that are marked with * have been confirmed using qPCR. B: inhibitor of differentiation (ID)1, ID2, Slug, Snail, and TWIST were evaluated using qPCR in FLF-transfected cells with let-7d and scrambled (n = 3). *P < 0.05, #genes that are changed in the opposite direction in the idiopathic pulmonary fibrosis lung.

DISCUSSION

In this study, we described the transfection of let-7d into fibroblasts and examined its ability to cause changes in their mesenchymal and phenotypic properties, perhaps reducing their mesenchymal properties. We found transfecting let-7d into three different types of fibroblasts caused a decrease in mesenchymal gene and protein expression levels, which accompanied changes in cellular properties that included a decrease in proliferation and migration both in transient and stable transfection. Furthermore, we have demonstrated how let-7d attenuated the TGF-β effect on downstream targets such as HMGA2 and α-SMA. Gene expression microarray revealed decreases in multiple mesenchymal markers and transcription factors but increases in some epithelial markers, suggesting that the introduction of let-7d has profound effects on the phenotype of primary lung fibroblasts.

Overexpression and inhibition of microRNAs in the lung have been demonstrated to be vital for cell properties and phenotypes (18, 21, 22). In this work, we were able to change fibroblast phenotype and properties that caused fibroblasts to be less mesenchymal and more similar to epithelial cells with a single microRNA molecule, let-7d. Microarray data revealed global changes in gene expression that were confirmed by qPCR and Western blot analysis. Interestingly, some of the genes showed expression and regulation in the opposite direction compared with IPF, which is known to have downregulation of let-7d. Another interesting finding was the increase in TJP-1, which is a tight junction protein known to be highly expressed in epithelial cell lines. Interestingly, this result is consistent with previous reports showing increases in TJP-1 during wound healing in corneal fibroblasts (32). Taken together, these findings highlight the influence of microRNA, specifically let-7d, on cellular phenotype and properties. Let-7d overexpression dramatically affects the ability of primary fibroblasts to migrate and proliferate, their responsiveness to TGF-β, and their expression of myofibroblast markers, suggesting that let-7d mimicry will potentially have a beneficial effect on epithelial cells and fibroblasts.

To have a better understanding of the phenomena caused by let-7d, we investigated relevant transcription factors known for their involvement in changes consistent with EMT (33). The ID protein family has two main functions: to maintain proliferation and to inhibit differentiation (2, 20). Therefore, changes in ID expression levels can cause changes in cell behavior. In our results, we observed decreases in both ID2 and (to a lesser extent) in ID1 after let-7d transfection, suggesting that this decrease can halt the proliferation and migration of fibroblasts (Figs. 3, 6, and 7). These findings are compatible with previous research, showing that an increase of ID1 during changes consistent with EMT (12) and the absence of ID2 slows proliferation and inhibits differentiation (35). It has also been shown that, when ID1 and ID2 are downregulated, fibroblasts were prevented from entering the cell cycle, specifically S phase followed by an increase in G2 to M phase (7). Our findings in the current study show that, after let-7d transfection, fibroblasts cannot fully progress through the cell cycle (Fig. 6), which confirms previous observations (11) and supports a potential therapeutic role for let-7d supplementation in preventing fibroblast accumulation typical of multiple fibrotic disorders.

Another transcription factor that is involved in cell movement, cell invasion, cell cycle regulation, and metastasis is Slug (31). Slug is a member of the Snail family of zinc finger transcription factors that is activated through the TGF-β-SMAD-signaling pathway (31). As we show in our results, let-7d overexpression causes a significant decrease in Slug mRNA levels, as well as a prevention of TGF-β1-induced increases in HMGA2. We suggest that the inhibition of Slug through HMGA2 contributes to the phenotypic changes observed in our experiments (Fig. 7). Interestingly, Snail (an E-CAD repressor) and TWIST, which are also known to play a fundamental role in regulating changes that are consistent with EMT (9, 10, 23), did not change after let-7d transfection, suggesting that the effects of let-7d in fibroblast are mediated through suppression of the HMGA2 and SLUG axis, without effects on TWIST and SNAIL, which may be more important in epithelial cells.

It is important that our results are taken in context. Although overexpression of a single microRNA was enough to change fibroblast properties and phenotype, it did not transform them into epithelial cells, even after stable transfection. The lack of full cell differentiation could happen for many different reasons, the most probable of which is that the use of a single microRNA is not enough to cause such a dramatic effect on cell differentiation. Perhaps the use of several microRNAs simultaneously is required to achieve full cell differentiation. During expression of mesenchymal markers in epithelial cells and cell differentiation, different pathways are involved; therefore changes in expression of just a few transcription factors probably is not sufficient to cause this undesired change. Notably, when we inhibited let-7d levels in FLF cells, we did not observe any changes in mesenchymal/epithelial markers or cell migration (data not shown), potentially reflecting the low endogenous levels of let-7d in fibroblasts. One conclusion we can draw safely from our experiments is that, if let-7d supplementation was used to treat lung fibrosis, its effects on fibroblasts would be mainly beneficial by slowing down proliferation and migration, and prevention of TGF-β mediated fibroblast-to-myofibroblast differentiations through let-7d-suppressive effects on HMGA2, SLUG, and the prevention of α-SMA expression. However, these changes will not permanently induce the unexpected consequence of generating “rogue” epithelial cells out of lung fibroblasts and probably would be dependent on therapy, thus reversible when stopped.

In summary, our study shows that transfection of primary fibroblasts with let-7d causes them to lose some of their mesenchymal properties and responsiveness to TGF-β, a potential desirable effect in treatment of IPF or other fibrotic diseases. Whereas let-7d supplementation in IPF may exert its potential benefits through improving the function of epithelial cells in IPF, it may also have an important effect on the other cellular populations important in IPF and lung myofibroblasts (19, 24). The let-7d effects on fibroblasts that include slower proliferation rate, reduced migration capacity, and suppression of myofibroblast markers suggest that let-7 supplementation may have a beneficial effect on both epithelial cells and fibroblasts. Thus we believe that let-7d supplementation should be further studied as a therapeutic strategy in human lung fibrosis.

GRANTS

This work was funded by Grants R01LM009657, R01HL095397, and U01HL108642.

DISCLOSURES

N. Kaminski is an inventor on use of microRNAs for the diagnosis and treatment of lung fibrosis.

AUTHOR CONTRIBUTIONS

Author contributions: L.H., A.B.-Y., K.P., H.Y., E.M., G.S., and N.K. conception and design of research; L.H., A.B.-Y., J.M., G.Y., K.S., M.L., T.A.C., C.J.R., and L.C. performed experiments; L.H., A.B.-Y., J.M., G.Y., K.S., M.L., and N.K. analyzed data; L.H., A.B.-Y., K.P., M.L., E.M., and N.K. interpreted results of experiments; L.H., A.B.-Y., G.Y., and N.K. prepared figures; L.H., A.B.-Y., and N.K. drafted manuscript; L.H., A.B.-Y., E.M., and N.K. edited and revised manuscript; L.H. and N.K. approved final version of manuscript.