MicroRNA-7 functions as a tumor-suppressor gene by regulating ILF2 in pancreatic carcinoma.

Interleukin enhancer binding factor 2 (ILF2) has been found to be markedly upregulated in pancreatic carcinoma and is involved in the pathogenesis of pancreatic carcinoma. Thus, ILF2 may be a potential target for therapy. Yet, the regulatory mechanisms of ILF2 in pancreatic carcinoma remain largely elusive. In the present study, we demonstrated that ILF2 functioned as an oncogene and regulated epithelial-mesenchymal transition (EMT)-associated genes in pancreatic carcinoma PANC-1 cells. MicroRNA-7 (miR-7) suppressed ILF2 mRNA expression and the protein level in PANC-1 cells. Contrary to ILF2, miRNA-7 functioned as a tumor-suppressor gene and negatively regulated EMT-associated genes in the PANC-1 cells. Curcumin, a polyphenol natural product isolated from the rhizome of the plant Curcuma longa, has emerged as a promising anticancer therapeutic agent. We found that treatment with curcumin increased miR-7 expression and suppressed ILF2 protein in the PANC-1 cells. Thus, we identified ILF2 as a new downstream target gene of curcumin. The results revealed that ILF2 is regulated by miR-7 and suggest that downregulation of miR-7 may be an important factor for the ILF2 overexpression in pancreatic carcinoma.

Introduction

Pancreatic carcinoma is the fourth most common cause of cancer-related mortality in Western countries and the 5-year survival rate of these patients is only 5% (1). Thus, improving the long-term survival of pacreatic carcinoma patients is urgently needed. Elucidating the molecular mechanisms of the pathogenesis and progression of pancreatic carcinoma not only will aid in the further understanding of the disease, but may also provide novel targets for effective therapy.

Interleukin enhancer binding factor 2 (ILF2), also known as NF45, is a nuclear factor of activated T cells (NFAT), to regulate interleukin-2 transcription with its binding partner NF90/NF110 isoforms (2,3). ILF2 is ubiquitously expressed in human tissues, especially the brain, thymus, pancreas, kidney and testis indicating that it is involved in the physiology of various cell types (4). ILF2 has been reported to promote the progression of multiple cancer types including cervical cancer, esophageal squamous cell carcinoma, glioma, pancreatic carcinoma and non-small cell lung cancer (5–8). Yet, the regulatory mechanisms of ILF2 remain largely elusive.

MicroRNAs (miRNAs or miRs) are small, non-coding RNAs that can post-transcriptionally regulate gene expression (9) as well as play significant roles in maintaining normal cellular functions (10). Deregulation of miR expression leads to progression of cancers (11) as exemplified by their differential expression in carcinomas (12), sarcomas (13,14) and hematologic tumors (15). They have been demonstrated to regulate the expression levels of major cancer-related genes and hence may be useful in the treatment of cancer (16,17).

In the present study, we demonstrated that ILF2 functioned as an oncogene and regulated epithelial-mesenchymal transition (EMT)-associated genes in pancreatic carcinoma PANC-1 cells. miR-7 was found to suppress ILF2 mRNA expression and the protein level in the PANC-1 cells. Contrary to ILF2, miR-7 functioned as a tumor-suppressor gene and negatively regulated EMT-associated genes in the PANC-1 cells. Curcumin, a polyphenol natural product isolated from the rhizome of the plant Curcuma longa, has emerged as a promising anticancer therapeutic agent. We found that treatment with curcumin increased miR-7 expression and suppressed ILF2 protein in the PANC-1 cells. Thus, we identified ILF2 as a novel downstream target gene of curcumin. The results revealed that ILF2 is regulated by miR-7 and suggest that downregulation of miR-7 may be an important factor for ILF2 overexpression in pancreatic carcinoma.

Materials and methods

Pancreatic carcinoma tissues, cell line and curcumin/DMSO

A total of 73 patients diagnosed with pancreatic carcinoma were recruited at the Department of Gastroenterology, No. 307 Hospital of PLA, Academy of Military Medical Science, Beijing, China. The use of human tissue samples followed internationally recognized guidelines as well as local and national regulations. Informed consent was obtained from each individual. The Medical Ethics Committee of No. 307 Hospital of PLA, Academy of Military Medical Science, approved the experiments undertaken. The pancreatic carcinoma cell line PANC-1 was obtained from the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Briefly, the cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2. Curcumin and DMSO were purchased from Sigma (St. Louis, MO, USA).

Plasmids, pre-miR-7/control miR and transfection

ILF2-expressing plasmids/empty vectors (pcDNA3.1) were purchased from Tiangene (Tianjin, China). The amount of the ILF2-expressing plasmids or empty vector (pcDNA3.1) used for each transfection was 10 µg, except for the dose-dependent experiments. Pre-miR-9 and control miR were purchased from Ambion Inc. (Ambion, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Transfection was performed using Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the instructions provided by the manufacturer.

Western blot analysis

Western blot analysis was performed as previously described (18). In brief, after incubation with the primary antibodies, anti-ILF2 (Cat no. ab154791; 1:250), anti-E-cadherin (Cat no. ab40772; 1:250), anti-N-cadherin (Cat no. ab18203; 1:250), anti-vimentin (Cat no. ab45939; 1:250), anti-fibronectin (Cat no. ab2413; 1:250) and anti-β-actin (Cat no. ab8227; 1:500) (all from Abcam, Cambridge, MA, USA) overnight at 4°C, goat anti-rabbit IgG H&L (HRP) secondary antibody (Cat no. ab7090; Abcam) was used for 30 min at room temperature. The specific proteins were visualized using the Odyssey™ infrared imaging system (Li-COR, Biosciences).

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay

Cell viability was examined by MTT assay was performed as previously described (19). Briefly, the transfected cells were seeded into 96-well plates and 10 µl MTT (concentration, 5 mg/ml; Sigma) was added into 100 µl medium after 48 h of transfection. The cells were incubated with MTT for ~4 h at 37°C, followed by the removal of MTT and the addition of 150 µl DMSO. Following incubation with DMSO for 10 min in the dark, the absorbance was measured at 570 nm (A570 nm) using a microplate reader (Biorad-168-1000XC; Bio-Rad Laboratories, Hercules, CA, USA).

Immunofluorescence analyses

Immunofluorescence analyses were performed as previously described (19). For immunofluorescence analyses, the cells were plated on glass coverslips in 6-well plates and transfected with 30 nM pre-miR-7 or control miR. At 36 h after transfection, coverslips were stained with the above-mentioned anti-ILF2 antibody. Goat anti-rabbit IgG was used as a secondary antibody (same as above). Coverslips were counterstained with DAPI (ab104139, 1:500; Abcam) for visualization of the nuclei. Microscopic analysis was performed with a confocal laser-scanning microscope (Leica Microsystems, Bensheim, Germany). Fluorescence intensities were measured in a few viewing areas for 200–300 cells per coverslip and analyzed using ImageJ 1.37v software (http://rsb.info.nih.gov/ij/index.html).

Colony formation

For the colony formation assay, cells were transfected for 24 h, and then seeded in a 6-well plate. A total of 0.5 ml FBS was added per well on day 5. After a 10-day incubation, the plates were washed with PBS and stained with 0.1% crystal violet. Colonies consisting of >50 cells were manually counted. Plating efficiency was calculated by dividing the number of colonies formed in the treated group by the number of colonies formed in the control.

Migration and invasion assays

The migration and invasion assays were performed as previously described (20). For Transwell migration assays, 2.5×104 cells were plated in the top chamber with the non-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences, San Jose, CA, USA). For invasion assays, 1.25×105 cells were plated in the top chamber with Matrigel-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences). In both assays, cells were plated in medium without serum or growth factors, and medium supplemented with serum was used as a chemoattractant in the lower chamber. The cells were incubated for 24 h and cells that did not migrate or invade through the pores were removed by a cotton swab. Cells on the lower surface of the membrane were stained with the Diff-Quick Staining Set (Dade) and counted.

Bioinformatic methods

Analysis of potential microRNA target sites was performed using the commonly used prediction algorithm, miRanda (http://www.microrna.org/microrna/home.do).

Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR for ILF2

RT-PCR and real-time PCR were performed as previously described (19). The PCR primer sequences were: GAPDH F, 5′-ATTCAACGGCACAGTCAAGG-3′ and R, 5′-GCAGAAGGGGCGGAGATGA-3′; ILF2 F, 5′-GAATCAGGACCTGGCTCCCA-3′ and R, 5′-GGTCAAGGATCCAGGGTGTG-3′. PCR was conducted according to manufacturer's instructions and the PCR products were analyzed by agarose gel electrophoresis. Gels were photographed and densities of the bands were determined using a computerized image analysis system (Alpha Innotech, San Leandro, CA, USA). The area of each band was calculated as the integrated density value (IDV). Real-time PCR for ILF2 was carried out using Power SYBR-Green PCR Master mix (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's instructions.

Real-time PCR for miRNAs

Total RNA from cultured cells, with efficient recovery of small RNAs, was isolated using the mirVana miR Isolation kit (Ambion, Thermo Fisher Scientific, Inc.). Detection of the mature form of miRNAs was performed using the mirVana qRT-PCR miR detection kit and qRT-PCR primer sets, according to the manufacturer's instructions (Ambion, Thermo Fisher Scientific, Inc.). The U6 small nuclear RNA was used as an internal control.

Statistical analysis

Data are presented as the means ± SEM Student's t-test (two-tailed) was used to compare two groups (p<0.05 was considered significant), unless otherwise indicated (x2 test).

Results

ILF2 functions as an oncogene and regulates EMT-associated genes in pancreatic carcinoma cells

To investigate whether ILF2 affects the proliferation of pancreatic carcinoma cells, firstly using western blot analysis, we tested whether ILF2-expressing plasmids could stably express ILF2 protein in PANC-1 cells. The results showed that ILF2 protein was significantly increased by the ILF2-expressing plasmids in the PANC-1 cells (Fig. 1A). In addition, we performed an MTT assay to detect the proliferation of PANC-1 cells following transfection with the ILF2-expressing plasmids. The results showed that ILF2 promoted the proliferation of the PANC-1 cells after 48 h of transfection and the increase was dose-dependent (Fig. 1B). In order to identify the effect of ILF2 on colony formation, we performed a colony formation assay. The results showed that the overexpression of ILF2 significantly increased the colony formation rate of the PANC-1 cells after transfection (Fig. 1C).

Figure 1

ILF2 functions as an oncogene and regulates EMT-associated genes in pancreatic carcinoma PANC-1 cells. (A) Western blot analysis of ILF2 in PANC-1 cells transfected with ILF2-expressing plasmids or an empty vector (mock). β-actin was used as a loading control (n=3). (B) MTT assay of PANC-1 cells transfected with ILF2-expressing plasmids as indicated (n=3). (C) Colony formation assay of PANC-1 cells transfected with ILF2-expressing plasmids or an empty vector (mock). Colonies with >50 cells were counted. Representative micrographs (left) and quantification of colonies (right) after transfection with ILF2-expressing plasmids or an empty vector (mock) (n=3). (D) Matrigel migration and Transwell invasion assays in PANC-1 cells. Mock groups were transfected with empty vectors. Representative micrographs (left) and quantification of colonies (right) after transfection of the cells with ILF2-expressing plasmids or an empty vector (mock). Magnification, ×200 (n=3). (E) Western blot analysis of E-cadherin, N-cadherin, vinmentin, and fibronectin in PANC-1 cells transfected with ILF2-expressing plasmids or an empty vector (mock). β-actin was used as a loading control (n=3). ILF2, interleukin enhancer binding factor 2; EMT, epithelial-mesenchymal transition; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

In an attempt to identify the role of ILF2 in regulating the migration and invasion of PANC-1 cells, we performed migration and invasion assays to detect the migration and invasion abilities of the PANC-1 cells following transfection with the ILF2-expressing plasmids and empty vectors. Ectopic expression of ILF2 promoted the migration and invasion capacities by ~4–6 fold in the PANC-1 cells (Fig. 1D). Since certain genes that promote migration and invasion can regulate epithelia-mesenchymal transition (EMT) (21–23), next we aimed to ascertain whether ILF2 expression is associated with EMT. Thus, we performed western blot analysis to detect levels of N-cadherin, vimentin and fibronectin (mesenchymal markers) and E-cadherin (epithelial marker). Our results demonstrated that E-cadherin was suppressed and N-cadherin, vimentin and fibronectin were upregulated in the PANC-1 cells following transfection with IFL2 (Fig. 1E).

miR-7 suppresses ILF2 in pancreatic carcinoma cells

It has been reported that ILF2 is abundantly expressed in pancreatic carcinoma (8) and it functions as an oncogene. Thus, we aimed to elucidate the mechanisms underlying the increased ILF2 expression in pancreatic carcinoma. miRs are a class of small noncoding RNAs (~22 nucleotides) and negatively regulate protein-coding gene expression by targeting mRNA degradation or translational inhibition (24–26). Downregulation of specific miRs can contribute to the upregulation of oncogenes (27). Thus, we hypothesized that ILF2 was upregulated by specific miRs in pancreatic carcinoma.

To further confirm this, on the one hand, we utilized the commonly used prediction algorithm miRanda (http://www.microrna.org/microrna/home.do) to analyze the 3′ untranslated region (UTR) of ILF2. The algorithm predicted that dozens of miRs could target the 3′UTR of ILF2. miR-7 attracted our interest as it has been reported that targeting miR-7 by curcumin could be a novel strategy for the treatment of pancreatic cancer (28). Target sites on the 3′UTR of ILF2 are shown in Fig. 2A. We reasoned that miR-7 could downregulate ILF2 expression by targeting its 3′UTR in pancreatic cancer and that ILF2 was upregulated in pancreatic cancer cells due to a lack of miR-7. In an attempt to identify the role of miR-7 in regulating ILF2 expression in pancreatic cancer PANC-1 cells, the cells were transfected with pre-miR-7 and control miR. After transfection, miR-7 expression was detected by real-time PCR. The results showed that miR-7 was significantly increased by pre-miR-7 in the PANC-1 cells (Fig. 2B). We next performed western blot analysis to detect ILF2 protein expression in PANC-1 cells following transfection with pre-miR-7 or control miR. We found that ILF2 protein was downregulated by miR-7 (Fig. 2C). Next, we performed immunofluorescent analysis to detect ILF2 protein expression in the PANC-1 cells transfected with pre-miR-7 or control miR. The results showed that ILF2 protein (Fig. 2D) was significantly downregulated in the cells transfected with pre-miR-7. To detect whether ILF2 mRNA was affected by miR-7, we performed RT-PCR in the PANC-1 cells transfected with pre-miR-7 or control miR. The results showed that ILF2 mRNA was evidently suppressed in the cells transfected with pre-miR-7 (Fig. 2E). Consistent with the results of RT-PCR, real-time PCR demonstrated that ILF2 mRNA was reduced in the PANC-1 cells transfected with pre-miR-7, compared with that observed in the control miR-transfected group (Fig. 2F).

Figure 2

ILF2 is a target of miR-7 in pancreatic carcinoma PANC-1 cells. (A) Diagram showing that ILF2 is a target gene of miR-7, predicted by miRanda and schematic for miR-7 and 3′ UTR of ILF2. (B) Detection of miR-7 by real-time PCR in PANC-1 cells transfected with pre-miR-7 or control miR (mock) (n=3). (C) Western blot analysis of ILF2 in PANC-1 cells transfected with pre-miR-7 or control-miR (mock). β-actin was used as a loading control (n=3). (D) Immunofluorescent analyses of ILF2 in PANC-1 cells transfected with pre-miR-7 or control miR (mock) (n=3). (E) RT-PCR of ILF2 in PANC-1 cells transfected with pre-miR-7 or control-miR (mock). GAPDH was used as a loading control (n=3). (F) Real-time PCR of ILF2 in PANC-1 cells transfected with pre-miR-7 or control-miR (mock). GAPDH was used as a loading control (n=3). ILF2, interleukin enhancer binding factor 2; miR, microRNA; UTR, untranslated region.

miR-7 functions as a tumor-suppressor gene and regulates EMT-associated genes in pancreatic carcimoma cells

To investigate whether miR-7 affects the proliferation of pancreatic carcinoma cells, we performed an MTT assay to detect the proliferation of PANC-1 cells transfected with pre-miR-7. The results showed that miR-7 inhibited the proliferation of the PANC-1 cells after 48 h of transfection (Fig. 3A). In order to identify the effect of miR-7 on colony formation, we performed a colony formation assay. The results showed that overexpression of miR-7 significantly inhibited the colony formation rate of the PANC-1 cells after transfection (Fig. 3B).

Figure 3

miR-7 functions as a tumor-suppressor gene and regulates EMT-associated genes in pancreatic carcinoma PANC-1 cells. (A) MTT assay of PANC-1 cells transfected with pre-miR-7 or control miR (mock) (n=3). (B) Colony formation assay of PANC-1 cells transfected with pre-miR-7 or control miR (mock). Colonies with >50 cells were counted. Representative micrographs (left) and quantification of colonies (right) after transfection as indicated (mock) (n=3). (C) Matrigel migration and Transwell invasion assays of PANC-1 cells transfected with pre-miR-7 or control miR (mock). Representative micrographs (left) and quantification (right) after transfection as indicated. Magnification, ×200 (n=3). (D) Western blot analysis of E-cadherin, N-cadherin, vinmentin, and fibronectin in PANC-1 cells transfected with pre-miR-7 or control miR (mock). β-actin was used as a loading control (n=3). (E) Real-time PCR of miR-7 in 73 pairs of pancreatic carcinoma and adjacent normal tissues. U6 was used as a loading control. miR, microRNA; EMT, epithelial-mesenchymal transition; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

In an attempt to identify the role of miR-7 in regulating the migration and invasion of PANC-1 cells, we performed migration and invasion assays to detect the migration and invasion abilities of the PANC-1 cells following transfection with pre-miR-7 and control miR. Ectopic miR-7 inhibited the migration and invasion capacities of the cells (Fig. 3C).

Next, in order to identify whether miR-7 expression is associated with EMT, we performed western blot analysis to detect N-cadherin, vimentin and fibronectin (mesenchymal markers) and E-cadherin (epithelial marker). Our results demonstrated that E-cadherin protein was upregulated and N-cadherin vimentin and fibronectin were suppressed in the PANC-1 cells transfected with miR-7 (Fig. 3D). In order to identify whether miR-7 is deregulated in pancreatic carcinoma, we detected its expression in pancreatic carcinoma and adjacent normal tissues. Our results showed that miR-7 expression was decreased in the pancreatic carcinoma tissues (Fig. 3E).

Curcumin increases miR-7 expression and suppresses ILF2 protein in pancreatic carcinoma cells

Curcumin was previously demonstrated to inhibit cell growth and invasion through upregulation of miR-7 in pancreatic cancer cells and to subsequently decrease expression of SET8, one of the miR-7 targets (28). Consistent with this report, we found that curcumin upregulated the miR-7 level in the PANC-1 cells (Fig. 4A). Moreover, we showed that curcumin inhibited ILF2 mRNA (Fig. 4B) and protein (Fig. 4C) levels. Thus, we identified ILF2 as a new downstream target gene of curcumin.

Figure 4

Curcumin promotes miR-7 expression and suppresses ILF2 expression in pancreatic carcinoma PANC-1 cells. Real time-PCR for miR-7 in PANC-1 cells. (A) PANC-1 cells were treated with curcumin or DMSO (mock). U6 was used as a loading control (n=3). (B) RT-PCR of ILF2 in PANC-1 cells treated with curcumin or DMSO (mock). GAPDH was used as a loading control (n=3). (C) Western blot analysis of ILF2 in PANC-1 cells treated with curcumin or DMSO (mock). β-actin was used as a loading control (n=3). miR, microRNA; ILF2, interleukin enhancer binding factor 2.

Discussion

Pancreatic carcinoma, with a median patient survival rate of less than 6 months from the time of diagnosis, is one of the most devastating types of cancer (29). Although significant improvement has been achieved in the understanding of the molecular mechanism underlying pancreatic carcinoma initiation and progression, the use of individual targeted agents has currently failed to provide meaningful improvement in the outcome of patients with the disease (30,31). Thus, further understanding of the pathogenesis of the disease and the development of novel targets for effective therapeutic approaches are urgently required.

ILF2 is markedly upregulated in pancreatic carcinoma and is involved in the pathogenesis of pancreatic carcinoma. Thus, ILF2 may be a potential therapeutic target (8). Yet, the regulatory mechanisms of ILF2 remain largely elusive. Consistent with the report that ILF2 is abundantly expressed in pancreatic cancer tissues, and the expression of ILF2 is correlated with tumor size, histological differentiation, and TNM stage, we found in the present study that ILF2 overexpression inhibited proliferation, colony formation, migration and invasion as well as regulated EMT-associated gene expression. Yet, the regulatory mechanisms of ILF2 in pancreatic carcinoma remain largely elusive.

miRNAs, small non-coding RNA molecules that suppress gene expression by interacting with the 3′ untranslated regions (3′UTRs) of target mRNAs, have also been linked to EMT and cancer (32–35). We found that miR-7 suppressed ILF2 protein expression and inhibited the proliferation, colony formation, migration and invasion as well as negatively regulated EMT-associated gene expression in PANC-1 cells. It has been reported that ILF2 is markedly upregulated in pancreatic carcinoma (8). Our results further demonstrated that miR-7 was significantly downregulated in pancreatic tissues, implying that ILF2 upregulation may be due to the lack of miR-7.

Curcumin has been extensively studied in several types of malignancies and has emerged as a promising anticancer therapeutic agent (28,36). Curcumin inhibited cell growth and invasion through upregulation of miR-7 in pancreatic cancer cells and subsequently decreased expression of SET8, one of the miR-7 targets (28) (Fig. 5). These findings suggest that targeting miR-7 by curcumin could be a novel strategy for the treatment of pancreatic cancer (28). Our results identified ILF2 as a novel target of miR-7 and further confirmed the restoration of miR-7 as a promising direction for the development of novel targets for effective therapeutic approaches.

Figure 5

Mechanism illustrating that deregulation of miR-7 is involved in pancreatic carcinoma. Curcumin may be a novel strategy for the treatment of pancreatic cancer as it upregulates miR-7 and decreases expression of SET8, an miR-7 target in pancreatic cancer cells consequently inhibiting cell growth, invasion and migration and inducing apoptosis. miR, microRNA.