Introduction
In developed countries, endometrial cancer (EC) is the most common malignancy among women, accounting for ~25% of all deaths related to cancer of the female genital tract (1). Unopposed estrogen therapy, obesity, nulliparity, diabetes mellitus and arterial hypertension have been linked to an increased risk of ECs (2). ECs are clinicohistologically classified into two subgroups: type I and type II. Type I tumors, which account for ~80% of all cases, are estrogen-dependent, low-grade tumors, while type II tumors are more aggressive and exhibit invasion into the myometrium (3,4). Currently, there is a lack of effective treatments for patients with advanced stage and recurrent EC (5); thus, more effective treatment strategies based on genomic data are needed.
In the post-genome sequencing era, the discovery of non-coding RNA (ncRNA) has been a conceptual breakthrough in cancer research fields (6). For example, microRNAs (miRNAs) are small ncRNA molecules (19–22 bases in length) that function to regulate the expression of multiple protein-coding genes by repressing translation or cleaving RNA transcripts in a sequence-specific manner (7,8). Bioinformatic predictions indicate that miRNAs regulate 30–60% (or more) of the protein-coding genes in the human genome. Numerous studies have reported that various miRNAs are aberrantly expressed in many types of human cancers, affecting the development and metastasis of cancers through oncogenic or tumor-suppressive functions (9,10).
Elucidation of cancer-related miRNA networks has provided important new information about human cancers. In our previous studies, we used our miRNA expression signatures to investigate several tumor-suppressive miRNAs and their regulated cancer pathways. We recently showed that miR-1/133a clustered miRNAs are significantly down-regulated in several cancer tissues (11–14). From our miRNA signatures, we have sequentially reported functional roles of the miR-1/133a cluster and the molecular targets/pathways regulated by these miRNAs. However, the contributions of these miRNAs in EC cells have not been fully elucidated.
The aim of the present study was to investigate the functional significance of the miR-1/133a cluster and to identify the molecular targets regulated by these miRNAs in EC cells. We found that restoration of mature miR-1 or miR-133a in EC cells significantly inhibited cell migration and invasion. Gene expression data and in silico analysis demonstrated that phosphodiesterase 7A (PDE7A), an enzyme that hydrolyzes intracellular cAMP, was a potential target of the miR-1/133a cluster. Elucidation of the cancer-related signaling pathways and targets regulated by the tumor-suppressive miR-1/133a cluster will provide new insights into the potential mechanisms of EC oncogenesis and metastasis.
Materials and methods
Clinical specimens
A total of 27 primary EC specimens were collected from patients who had undergone surgical treatment at Chiba University Hospital. Eight non-cancer endometrial specimens were obtained from patients who underwent total hysterectomy because of other gynecologic diseases (Table I). The samples were processed and stored in liquid nitrogen until RNA extraction. Our study was approved by the Bioethics Committee of Chiba University; prior written informed consent and approval was given by each patient.
Cell lines and cell culture
Hec1B cells (derived from endometrioid adenocarcinoma G1) and Hec265 cells (derived from endometrioid adenocarcinoma G2) were used in this analysis. Hec1B cells were grown in E-MEM medium supplemented with 10% fetal bovine serum. Hec265 cells were grown in E-MEM medium supplemented with 15% fetal bovine serum. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.
RNA isolation
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA concentrations were determined spectrophotometrically. RNA quality was confirmed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
First-strand cDNA was synthesized from 1 μg of total RNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Gene-specific PCR products were assayed continuously using a 7300-HT Real-Time PCR system according to the manufacturer’s protocol. The initial PCR step consisted of a 10- min hold at 95°C, followed by 40 cycles consisting of denaturation for 15 sec at 95°C and annealing/extension for 1 min at 60°C.
The expression levels of miR-1 (assay ID: 002222) and miR-133a (assay ID: 0002246) were analyzed by TaqMan qPCR (TaqMan MicroRNA assay; Applied Biosystems) and normalized to RNU48 (assay ID: 001006). All reactions were performed in duplicate. TaqMan probes and primers for PDE7A (P/N: Hs00300285_m1), DDX3X (P/N: Hs00606179_m1), CORO1C (P/N: Hs00170938_m1), SPTBN1 (P/N: Hs00162271_m1) and GUSB (P/N: Hs00939627_m1; used as an internal control) were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products). All reactions were performed in triplicate and included negative control reactions that lacked cDNA. The ΔΔCt method was adopted and applied to calculate the relative quantities of target genes.
Transfections with mature miRNA and small-interfering RNA (siRNA)
Cells were transfected with 10 nM mature miRNA or siRNA molecules using Lipofectamine RNAiMAX transfection reagent (Invitrogen) and Opti-MEM (Invitrogen). The following RNA species were used in this study: mature miRNA, Pre-miR™ miRNA Precursors (hsa-miR-1; P/N: PM10617, hsa-miR-133a; P/N: PM10413; Applied Biosystems), negative control miRNA (P/N: AM17111; Applied Biosystems), siRNA (Stealth siRNAs, si-PDE7A, P/N: HSS107737 and HSS107739; Invitrogen) and negative control siRNA (Stealth RNAi Negative Control Med GC, P/N: 12935-300; Invitrogen).
Cell proliferation, migration and invasion assays
For cell proliferation assays, cells were transfected with 10 nM miRNA or siRNA by reverse transfection and plated in 96-well plates at 3×103 cells per well. After 72 h, cell proliferation was determined with XTT assays using a Cell Proliferation Kit II (Roche Applied Science, Tokyo, Japan).
Cell migration assay
Modified Boyden Chambers (Trans-wells, no. 3422; Corning, NY, USA) were used. Cells were transfected with 10 nM miRNA or siRNA by reverse transfection and plated in 10-cm dishes at 8×105 cells/dish. After 48 h, 1×105 cells were added to the upper chamber of each migration well and were allowed to migrate for 48 h. After gentle removal of the non-migratory cells from the filter surface of the upper chamber, the cells that migrated to the lower side were fixed and stained with Diff-Quick (no. 16920; Sysmex Corp., Japan). The number of cells migrating to the lower surface was determined microscopically by counting four areas of constant size per well. Cell invasion assays were carried out using modified Boyden chambers in 24-well tissue culture plates at 1×105 cells per well (Matrigel invasion chamber, no. 354480; BD Biocoat, USA). All experiments were performed in duplicate.
Search for miR-1 and miR-133a target genes
To identify putative miR-1- and miR-133a-regulated genes, we searched the TargetScan database (http://www.targetscan.org) for genes having conserved sites for both miR-1 and miR-133a. Then, we analyzed gene expression using the GEO database. Gene expression data for clinical EC specimens were entered into the GEO database (accession no. GSE17025). The procedure used for the selection of miR-1 and miR-133a genes is shown in Fig. 3.
Western blot analysis
Cells were harvested and lysed 72 h after transfection. Cell lysates (50 μg of protein each) were separated using Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA), followed by subsequent transfer to PVDF membranes. Immunoblotting was performed with polyclonal anti-PDE7A antibodies (ab154857; Abcam, Cambridge, UK). Anti-GAPDH antibodies (ab8245; Abcam) were used as an internal control.
Plasmid construction and dual-luciferase reporter assays
Partial sequences of the PDE7A 3′ untranslated region (3′UTR) containing the miR-1 and miR-133a target sites were inserted between the XhoI and PmeI restriction sites in the 3′UTR of the hRluc gene in the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). Hec265 cells were then transfected with 5 ng vector or 10 nM mature miRNA.
Statistical analysis
The relationships between 2 variables and numerical values were analyzed using the Mann-Whitney U test, and the relationships between 3 variables and numerical values were analyzed using the Bonferroni-adjusted Mann-Whitney U test. Expert StatView analysis software (ver. 4; SAS Institute Inc., Cary, NC, USA) was used in both analyses. In the comparison of 3 variables, an unadjusted statistical level of significance of P<0.05 corresponded to the Bonferroni-adjusted level of P<0.0083.
Results
Expression levels of miR-1 and miR-133a in EC specimens and cell lines
To validate our previous miRNA expression signatures, we evaluated the expression levels of miR-1 and miR-133a in 27 EC specimens and 8 non-cancer endometrial specimens. The backgrounds and clinicopathological characteristics of patients are summarized in Table I. Quantitative stem-loop RT-PCR demonstrated that miR-1 and miR-133a expression levels were significantly lower in cancer specimens compared with non-cancer specimens (P<0.0001; Fig. 1A and B, respectively). The expression levels of miR-1 and miR-133a were also reduced in 2 EC cell lines (Hec1B and Hec265). Spearman’s rank test showed a positive correlation between the expression of miR-1 and that of miR-133a (r=0.887, P<0.0001; Fig. 1C).
Effects of transfection with miR-1 and miR-133a on cell proliferation, migration and invasion in EC cell lines
To examine the functional roles of miR-1 and miR-133a, we performed gain-of-function assays by transfecting mature miRNAs into Hec1B and Hec265 cells. XTT assays showed that cell proliferation was inhibited by transfection with miR-1 and miR-133a in both Hec1B and Hec265 cells compared with mock and miRNA-control transfections (P<0.0083, Fig. 2A).
Cell migration assays demonstrated that cell migration was significantly inhibited in miRNA-transfected cells compared with mock- or miRNA-control-transfected cells (P<0.0083, Fig. 2B).
Moreover, in Matrigel invasion assays, transfection with miR-1 and miR-133a significantly inhibited cell invasion as compared with mock or miRNA-control transfection (P<0.0083, Fig. 2C). These results suggested that the miR-1/133a cluster could represent a putative tumor suppressor in EC cells.
Identification of common targets of miR-1 and miR-133a by in silico analysis and gene expression data
To identify putative genes regulated by the miR-1/133a cluster (i.e., both miR-1 and miR-133a), we searched the TargetScan database (Release 6.2, http://www.targetscan.org/) and analyzed expression data of EC clinical specimens using the Gene Expression Omnibus (GEO accession no. GSE 17025). Our strategy for identification of target genes of the miR-1/133a cluster is shown in Fig. 3. We found that 23 genes were upregulated in EC specimens and had putative target sites for miR-1 and miR-133a in their 3′UTRs. Therefore, these genes were annotated as putative targets of the miR-1/133a cluster (Table II). Among 23 genes, we evaluated the expression of 4 genes (PDE7A, DDX3X, CORO1C and SPTBN1) in clinical specimens. As a result, the expression of PDE7A mRNA was significantly higher in clinical EC specimens.
PDE7A was a direct target of the miR-1/133a cluster in EC cells
Next, we performed qRT-PCR and western blotting to confirm downregulation of PDE7A mRNA and protein following restoration of miR-1 or miR-133a in Hec1B and Hec265 EC cells. The mRNA and protein expression levels of PDE7A were significantly repressed in miR-1 and miR-133a transfectants in comparison with mock or miR-control transfectants (P<0.0083, Fig. 4A and B).
We then performed luciferase reporter assays in EC cells to determine whether PDE7A was directly regulated by miR-1 and miR-133a. The TargetScan database predicted that there was one binding site for miR-1 in the 3′UTR of PDE7A (positions 1333–1340; Fig. 4C) and one binding site for miR-133a in the 3′UTR of PDE7A (positions 1192–1198; Fig. 4C). We then used vectors encoding the partial wild-type sequence of the 3′UTR of PDE7A mRNA, including the predicted miR-1 or miR-133a target sites. We found that the luminescence intensity was significantly reduced by cotransfection with miR-1 or miR-133a and the vector carrying the wild-type 3′UTR of PDE7A. In contrast, transfection with the mutant vector, in which the sequence within positions 1333–1340 or 1192–1198 had been changed, blocked the decrease in luminescence (P<0.0001, Fig. 4C). These data suggested that miR-1 and miR-133a bound directly to specific sites in the 3′UTR of PDE7A mRNA.
Expression levels of PDE7A in EC clinical specimens
Twenty-seven EC and 8 normal endometrium specimens were subjected to PDE7A mRNA expression analysis in this study. qRT-PCR analysis showed that the expression of PDE7A mRNA was significantly higher in clinical EC (differentiation G3) specimens than in normal specimens (P=0.0022, Fig. 5).
Downregulation of PDE7A expression in EC cells affected cell proliferation, migration and invasion activities
To investigate the functional role of PDE7A, we performed loss-of-function studies using si-PDE7A transfectants. First, we evaluated the knockdown efficiency of si-PDE7A transfection in Hec1B and Hec265 EC cells. Western blotting and qRT-PCR indicated that si-PDE7A effectively downregulated PDE7A expression in EC cells (P<0.0083, Fig. 6A and B).
Next, we analyzed the functional effects of PDE7A knockdown in EC cells. XTT assays demonstrated that cell proliferation was significantly inhibited in si-PDE7A transfectants in comparison with mock or si-control transfectants (P<0.005, Fig. 6C). Moreover, cell migration assays revealed significant inhibition of cell migration in si-PDE7A transfectants in comparison with mock or si-control transfectants (P<0.0001, Fig. 6D). Similarly, Matrigel invasion assays revealed that the number of invading cells was significantly decreased when EC cells were transfected with si-PDE7A (P<0.0001, Fig. 6E). These findings suggested that PDE7A acted as an oncogene in EC cells.
Discussion
The 5-year survival rate of patients with stage I EC is >90%; however, that in patients with stages III or IV EC is much lower, ranging from 40 to 80% (3,4). Previous studies have demonstrated that mutation of K-ras or PTEN is common in low-grade EC, while high-grade EC is associated with P53 mutation (15). These data suggest that differences in expression of cancer-related genes have substantial effects on disease progression. However, EC is a complex disease and cannot be explained only by mutations in these few genes; thus, elucidation of the involvement of other unknown genetic abnormalities and signaling pathways, including ncRNAs, is critical.
miRNAs are unique in their ability to regulate multiple protein-coding genes. Recent bioinformatic predictions have shown that miRNAs regulate >30–60% of the protein-coding genes in the human genome (9,10). Accumulating evidence has suggested that aberrantly expressed miRNAs disrupt tightly regulated RNA networks in cancer cells. These events are believed to initiate cancer cell development and metastasis. Therefore, identification of key miRNAs and the networks regulated by these miRNAs will provide new insights into the potential mechanisms of cancer initiation, development and metastasis. Recent studies have reported the differential expression of miRNAs in EC cells; for examples, miR-205, miR-210, miR-429 and miR-449 are upregulated in EC tissues, whereas let-7e, miR-30c, miR-204 and miR-221 are downregulated in EC tissues (16). Upregulation of miR-205 is significantly correlated with disease survival in EC, and thus, miR-205 is considered a potential prognostic marker in EC. Interestingly, miR-205 directly regulates the expression of PTEN and inhibits apoptosis in EC cells (17).
To identify novel miRNA-mediated RNA networks in cancer cells, we have constructed miRNA expression signatures in several types of cancers and investigated the roles of miRNAs in oncogenesis and metastasis using differentially expressed miRNAs (12,18,19). These miRNA signatures have revealed that the miR-1/133a cluster is frequently down-regulated in several types of cancers, including head and neck squamous cell carcinoma, prostate cancer, bladder cancer and lung cancer (11–14). Our present study demonstrated that miR-1 and miR-133a were significantly downregulated in EC specimens and cell lines. Moreover, restoration of these miRNAs significantly inhibited cancer cell migration and invasion, suggesting that this miRNA cluster may function as a tumor suppressor in EC cells, similar to its function in other cancers (20–22). A full understanding of the targets in EC cells that are regulated by the miR-1/133a cluster may contribute to our knowledge on EC oncogenesis and metastasis. Recently, we established a strategy for identification of pathways and genes regulated by tumor-suppressive miRNAs (14,22). In the present study, we used this strategy and found 23 putative candidate genes potentially regulated by the miR-1/133a cluster in EC cells. This is the first report demonstrating that PDE7A is directly regulated by the miR-1/133a cluster in EC cells.
PDEs are enzymes that regulate the cellular levels of the secondary messengers cAMP and cGMP by controlling their rates of degradation. PDEs can be categorized into 11 families (PDE1-11), which are structurally related but functionally distinct (23). PDE7, including isoforms PDE7A and PDE7B, is a high-affinity cAMP-specific PDE (24–26). Three variant forms of PDE7A have been annotated: PDE7A1 and PDE7A2 are N-terminal variants, and PDE7A3 is a C-terminal variant (25,27). The expression of PDE7A is elevated in pro-inflammatory and immune cells, supporting the role of PDE7A as a therapeutic target for inflammation disorders (28). A recent study indicated that the PDE7A-specific inhibitor ASB16165 suppresses keratinocyte proliferation on TPA-induced skin inflammation (29).
Many tumor cells exhibit significantly decreased cAMP levels as a consequence of overexpression of PDEs in chronic lymphocytic leukemia (CLL) and malignant carcinoma cells (30–32). PDE7A is overexpressed in CLL, and stimulation of the cAMP signaling pathway has been shown to induce apoptosis and augment the effects of glucocorticoids in inducing apoptosis in CLL cells (33). Increasing intracellular concentrations of cAMP may arrest growth, induce apoptosis and attenuate cancer cell migration in various cancers (34–37). The effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) (38). The cAMP/PKA signaling pathway may have an important role in tumor migration. Indeed, activation of the cAMP/PKA pathway inhibits cancer cell migration in various cancers by targeting matrix metalloproteinase (MMP)2, actin, integrin, MMP9 and MMP4 (39–42). Interestingly, PDE7A contains a PKA pseudosubstrate site within 2 repeated sequences at the N-terminal region of PDE7A. The PDE7A1 N-terminal repeat region inhibits the C subunit of PKA (C) activity and suppresses C-dependent, cAMP-independent, physiological responses. These observations demonstrate that PDE7A1 can inhibit cAMP signaling via direct binding to C (43).
In conclusion, downregulation of the miR-1/133a cluster was a frequent event in EC. Moreover, the tumor-suppressive miR-1/133a cluster directly regulated PDE7A, a high-affinity cAMP-specific enzyme. Restoration of miR-1/miR-133a or silencing of PDE7A inhibited cancer cell migration and invasion, suggesting that the miR-1/miR-133a-PDE7A pathway contributes to the metastasis of EC. Identification of molecular targets regulated by tumor-suppressive miRNAs will provide insights into the potential mechanisms of EC oncogenesis and metastasis, facilitating the development of novel therapeutic strategies for the treatment of this disease.