MicroRNA-26b Represses Colon Cancer Cell Proliferation by Inhibiting Lymphoid Enhancer Factor 1 Expression Free

microRNAs (miR) are a group of endogenous noncoding RNAs that posttranscriptionally regulate expression of protein-coding genes by recognizing specific mRNAs with complementary sequence (1, 2). miRs imperfectly match the 3′ untranslated region (UTR) of target mRNAs and inhibit translation and/or promote degradation. miRs can also target regions other than 3′UTR to trigger degradation of target mRNAs or repress protein synthesis. miRs are estimated to represent about 1% of all genes in human genome and the importance of their biologic functions are being intensively investigated (3–5). miRs are associated with cancer as either oncogenes or tumor-suppressor genes and in cancer cells they are either up- or downregulated (6–8). Many miRs upregulated in colon adenomas or adenocarcinomas have been reported (6). Recent reports have shown that miR26b is underexpressed in human breast cancer, parathyroid tumor, oral lichen planus disease, glioma cells, hepatocellular carcinomas, nasopharyngeal carcinomas, primary squamous cell lung carcinomas, and squamous cell carcinoma of the tongue (9–16). Recently, two new target genes of miR26b have been reported in glioma cells (EphA2) and human breast cancer cells (SLC7A11; refs. 9, 12). EphA2 is an erythropoietin-producing hepatocellular A receptor and activation of this receptor tyrosine kinase is associated with cancer cell growth (12). SLC7A11 is a solute carrier family seven member 11 that may play a role in providing resistance to apoptosis in cells (9). Finally, human embryonic stem cells and metastatic colorectal cancer cells also express miR26b, which may regulate TAF12, PTP4A1, CHFR, and ALS2CR2 gene expression (17).

Wnt signaling is one of the most important and best characterized signaling pathways involved in embryonic development, cell proliferation, and cancer progression. The canonical Wnt signaling pathway is activated when Wnt ligand binds to cell surface receptor Frizzled and lipoprotein receptor-related protein (LRP), which activate Dishevelled and releases β-catenin from its destroying complex formed by Axin, adenomatous polyposis coli (APC), and GSK3β. The released β-catenin enters nucleus and binds to Lef/Tcf transcription factors to activate downstream gene expression (18). Among those components, many of them are potential targets of miR regulation.

In the case of colorectal cancer, mutation of Wnt signal components is estimated to cause approximately 90% of the cancer (19). Most are associated with truncated APC and point mutations of phosphorylation sites in β-catenin involved in its degradation. Targeting the Wnt/β-catenin pathway, especially the downstream transcriptional regulation, would be effective in therapeutic treatment. We previously demonstrated that miR26b was a potent inhibitor of LEF1 expression (20). Given the critical role of LEF1 in canonical Wnt signaling, colon cancer progression (21), and human sebaceous tumors (22), we asked whether miR26b repression of LEF1 would inhibit colon cancer cell proliferation.

hsa-miR26b precursor (addition of six thymine deoxyriboside at 3′) was cloned from the genomic DNA of HEK 293 cell and was linked to the U6 promoter in pLL3.7 backbone. Human LEF1 cDNA was cloned from total mRNA of HEK 293 cell and was inserted downstream of the EF1α promoter in pWPI backbone. The 7xTopFlash reporter plasmid was constructed into luciferase vector by inserting seven T-cell factor/lymphoid enhancer factor (LEF/TCF)–binding sites upstream of the minimal thymidine kinase (TK) promoter (20). The 7xFopFlash-negative control contains mutations within each LEF/TCF–binding site. All constructs were confirmed by DNA sequencing.

A second-generation Lentiviral vector system was used. The packaging vectors are psPAX2 and pMDG. The gene expression vectors were transfected with packaging vectors into HEK 293 FT cells to generate lentiviral vectors as previously described (23). The virus containing cell medium was harvested 48 hours after transfection. The viral vectors were concentrated by ultracentrifuge at 26,000 rpm for 2 hours at 4°C and added to the SW480 cell medium with 8 μg/mL polybrene. Infected cells were subject to the cell proliferation assay. Cells (150,000) were seeded in 60-mm plates on day 0 and then trypsinized and counted after 24, 48, 72, and 96 hours by a Coulter Z1 cell counter as previously described (24). Experiments were run in triplicate.

The CCD 841 CoN, SW480, DLD-1, Colo 320, and HEK 293 cells were obtained from the ATCC (cultured and frozen immediately for storage and used within 3 months; cell cultures used in revision experiments were subsequently thawed and cultured and genotypes were confirmed to previous cell cultures) and cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. SW480 cells were seeded in 12-well plates and transfected by Fugene HD (Roche) with 500 ng of reporter plasmid, 500 ng of expression plasmid, and 50 ng SV-40–driven β-galactosidase plasmid. Transfected cells were incubated for 48 hours and then lysed for reporter activities and protein content by Bradford assay (Bio-Rad) as previously described (25). Luciferase activity was measured with reagents from Promega and β-galactosidase activity was assessed by Galacto-Light Plus reagents (Tropix). Experiments were run in triplicate and all luciferase activities were normalized to β-galactosidase activity.

Cells were cultured in T-75 flasks and two flasks of 80% confluent cells were harvested by scraping and miR isolation. Total RNA, including miR from cells, was prepared using the miRNeasy Mini Kit (Qiagen). Mulitple isolations were performed and separate qPCR assays were run on each sample (n = 3). The amount and integrity of the RNA samples were assessed by measurement at 260 and 280 nm and gel analyses. Quantitative real-time PCR for miR26b mature expression was done with TaqMan MicroRNA assay probes (Applied Biosystems), including U6B as a reference gene. Total RNA was reverse transcribed into cDNA by the iScript Select cDNA Synthesis Kit (Bio-Rad). Real-time PCR was carried out in a total reaction of 25 μL containing 12.5 μL iQ SYBR Green Supermix, 0.1 μmol/L forward primer, 0.1 μmol/L reverse primer, 0.25 μL cDNA template in the MyiQ Single color Real-Time Detection System and analyzed by the MyiQ Optical System Software 2.0 (Bio-Rad). β-Actin served as a reference gene for normalization of LEF1, c-MYC, and cyclin D1 mRNA levels and ΔΔC_t values are reported. The thermal cycling profile consisted of 95°C for 4 minutes followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds and elongation at 72°C for 18 seconds. Samples were run in triplicate. No-template control was run in each experiment. Melting curve analyses were performed to confirm amplification specificity of the PCR products. PCR primers are LEF1 F 5′-TTCCTTGGTGAACGAGTCTG-3′, R 5′-CTCTGGCCTTGTCGTGGTAG-3′; cyclin D1 F 5′-ACACGCGCAGACCTTCGTTG-3′, R 5′-GTAGGACAGGAAGTTGTTGG-3′; c-MYC F 5′-GATTCTCTGCTCTCCTCGAC-3′, R 5′- GTGATCCAGACTCTGACCTT-3′. β-Actin primers were described previously (26).

Endogenous LEF1 isoforms were identified in colon cells and HEK 293 cells using the LEF1 antibody (Cell Signaling Technology; C12A5). c-MYC and cyclin D1 expressions in colon cells were detected using c-MYC antibody (Cell Signaling Technology; D84C12) and cyclin D1 antibody (Cell Signaling Technology; 92G2). GAPDH antibody was from Santa Cruz Biotechnology (sc-32233). Approximately 20 to 30 μg of cell lysates was analyzed in Western blots. Following SDS gel electrophoresis, the protein were transferred to PVDF filters (Millipore), immunoblotted, and detected by specific antibodies and ECL plus reagents from GE Healthcare.

Male nude mice (Foxn1^nu; ages 6–7weeks) were purchased from Harlan and were maintained in laminar flow cabinets under pathogen-free conditions. SW480 cells (1 × 10⁷ cells) stably expressing scramble shRNA (control) or miR26b in serum-free DMEM were injected into either side of flank area of nude mice. The mice were weighed, and tumor sizes were measured every third day with calipers for calculation of tumor volumes, V = LW²/2, where L and W were length and width, respectively. After 33 days, mice were sacrificed and the tumors were collected for weighting and analysis of the expression levels of miR26b and other related genes. Tumors were flash-frozen in LN2, tissue was crushed and lysed, and RNA extracted using the Qiagen miRNeasy Extraction Kit (Qiagen).

SW480 cells stably expressing scramble shRNA or miR26b were grown to 75% confluence on glass slides and incubated for 2 hours with or without 0.5 mmol/L H₂O₂. The TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling) assay was performed with the DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer's instruction.

Statistics of growth curves were performed by the repeated measures of ANOVA. Other statistics were performed by the two-sample t test. P values less than 0.05 were considered to be significant.

The nontumor colon epithelial cell line (CCD 841 CoN; from the ATCC) was set as a control to compare the relative expression of LEF1 and miR26b in noncancer colon versus colon cancer cells. miR26b was significantly downregulated in colon cancer cell lines compared with the normal cells. miR26b expression in normal cells was approximately 7.4, 12.3, and 37 times higher than observed in DLD-1, SW480, Colo 320 colon cancer cells, respectively (Fig. 1A). In contrast, LEF1 isoform expression was upregulated in the cancer cells at both the mRNA level and protein level (Fig. 1B and C). A gradient of LEF1 expression was detected in which normal cells <DLD-1 <SW480 <Colo 320 (Fig. 1B and C) and the data indicated an inverse relationship between miR26b and LEF1 isoform expression in these cell lines. c-Myc, a LEF1 target gene, was also correlated with increased LEF1 expression (Fig. 1B). This inverse correlation suggests an inhibitory role of miR26b on LEF1 expression and indicated that miR26b may have a potent regulatory role during cancer progression. miR26b targets all LEF1 isoforms as they contain identical 3′UTRs and miR26b–binding sites. Furthermore, we analyzed miR26b expression in 54 human colon cancer specimens compared with 20 normal colon specimens from the NCBI Gene Expression Omnibus public database (GSE30454; ref. 27). These data show decreased miR26b expression (P < 0.005) in human colon cancer tissue (Fig. 1D). Because SW480 cells exhibited moderate expression of both LEF1 and miR26b, we used these cells for overexpression and knockdown treatments of both genes in cell proliferation assays.

To test whether miR26b effected colon cancer proliferation, we established a variety of overexpression, knockdown, and rescue cell lines and analyzed their proliferation (Fig. 2). Human miR26b and control scramble shRNA were inserted downstream of the U6 promoter in pLL3.7 plasmid. Human LEF1 FL mRNA (full-length isoform) was inserted downstream of the EF1α promoter in pWPI plasmid (Fig. 2A). The gene expression vectors were transfected with packaging vectors into HEK 293 FT cells to generate lentiviral vectors. The virus containing cell medium was harvested 48 hours after transfection. The viral vectors were concentrated and added to the SW480 cell medium with 8 μg/mL polybrene. Approximately 100% infection efficiency was achieved, as indicated by expression of selection marker EGFP in virus-treated cells (Fig. 2B). Three stable cell lines were established: the control scramble cell line, the miR26b overexpression cell line, and the LEF1 FL overexpression cell line. These cell lines, together with SW480 cells transfected with anti-miR miRNA inhibitor (anti-miR26b; Ambion) and empty vector (mock) cells, were subjected to cell proliferation assays and the growth rates over 96 hours are summarized in Fig. 2C. LEF1 FL overexpression in SW480 cells significantly increased growth compared with the control cells. In contrast, miR26b overexpression decreased growth. The anti-miR26b–treated cells also exhibited increased growth albeit not as high as cells in which LEF1 FL was overexpressed. These data suggest that miR26b is a potent inhibitor of colon cancer cell growth through regulation of LEF1 expression.

To demonstrate that LEF1 isoform expression was targeted by miR26b in the cells used for cell proliferation assays, LEF1 expression was analyzed by real-time PCR and Western blots. LEF1 mRNA levels were significantly decreased in the miR26b-overexpressing cells compared with control cells, whereas LEF1 mRNA was significantly increased in cells transfected with anti-miR26b or overexpressing LEF1 FL (Fig. 3A). Western blots showed that LEF1 protein and mRNA levels were similar in these cell lines (Fig. 3B). A Topflash reporter assay was used to examine the overall Wnt/β-catenin signal activity. SW480 cells were transfected with 7xTopflash reporter plasmid and indicated expression plasmids, together with SV-40 β-galactosidase as a control. Transfection with miR26b-decreased Topflash activity compared with control cells, whereas anti-miR26b and LEF1 transfection increased reporter activity. The Fopflash plasmid with a mutation in each LEF/TCF–binding site was used as a negative control (Fig. 3C). These data indicated that miR26b had a potent inhibitory effect on LEF1 expression as well as Wnt/β-catenin signal activity in colon cancer cells.

Wnt/β-Catenin signaling regulates cell proliferation through several different mechanisms (18, 19). One of the most important mechanisms is through regulation of cyclin D1 and c-Myc, which are essential regulators of cell proliferation and direct targets of the LEF1/β-catenin transcription complex (28–30). Therefore, we wanted to determine whether expression of these two genes was altered in our lentiviral-infected cells, which exhibit different rates of cell proliferation (Fig. 2C). Real-time PCR showed that cyclin D1 and c-Myc mRNA levels were decreased in miR26b-overexpressing cells compared with control cells, whereas they were increased in anti–miR26b-transfected and LEF1 FL–overexpressing cells (Fig. 4A and B). Western blots showed that there were parallel changes in c-Myc and cyclin D1 protein and mRNA levels (Fig. 4C), suggesting that miR26b repressed c-Myc and cyclin D1 expression through inhibition of LEF1 in colon cancer cells. As a control miR26b-expressing cells were transfected with the LEF1 FL cDNA, which is not targeted by miR26b to demonstrate Lef1 FL expression by the Western blot analysis (Fig. 4C).

To test the effect of miR26b inhibition on growth of colon cancer cells in vivo, we injected male nude mice (Foxn1^nu; ages 6–7weeks) with SW480 cells (1 × 10⁷ cells) stably expressing scramble shRNA (control) or miR26b in serum-free DMEM at either side of the flank area. Tumor sizes were measured every third day with calipers to calculate tumor volumes (V = LW²/2; L, length; W, width; Fig. 5A). After 33 days, mice were sacrificed and the tumors were collected for weighing and expression of miR26b and other related genes. The average weight of miR26b tumors was about 50% of the control tumors (Fig. 5B and C) and real-time PCR analyses showed that miR26b expression was significantly increased in miR26b tumors, whereas LEF1, c-Myc, and cyclin D1 were significantly decreased in tumors derived from miR26b-overexpressing SW480 cells (Fig. 5D and E). These data were consistent with the in vitro cell proliferation assay and real-time PCR results.

To test whether miR26b inhibited colon cancer cells through apoptosis, we performed TUNEL assays. SW480 cells stably expressing scramble miR (control) or miR26b were stained with Promega TUNEL Assay Kit and almost no apoptosis was detected in either control or miR26b cell lines (Fig. 6A and B). The same cell lines were treated with 0.5 mmol/L H₂O₂ for 2 hours as positive controls. After H₂O₂ treatment, apoptosis was induced in both cell lines (Fig. 6A and B). These data suggest that miR26b expression does not affect apoptosis of colon cancer cells in the presence or absence of oxidative stress, indicating that miR26b primarily regulates colon cancer cell/tumor proliferation and not survival.

We previously demonstrated that miR26b not only targets the β-catenin–responsive isoform of LEF1 FL but also the dominant negative isoform (LEF1ΔN) and intermediate isoform because they share the same 3′UTR. Thus, miR26b also reduces expression of the dominant negative isoform. However, many colon cancer cells lack expression of the dominant negative isoform of LEF1 (31). The full-length LEF1 isoform is responsive to Wnt/β-catenin signaling in colon cancer cells (19). LEF1 and TCF belong to a family of DNA-binding transcription factors that interact with β-catenin to activate genes involved in cell proliferation, morphogenesis, epithelial–mesenchymal transition, and stemness, which can lead to cancer progression (32–36). The LEF1 full-length and intermediate isoforms are produced in colon cancers through Wnt signaling and the LEF1ΔN isoform is produced by an alternative promoter, which is inactive in some cancers (31, 37). Furthermore, β-catenin also activates cyclin D1 expression in colon carcinoma cells through TCF/LEF1–binding elements in the cyclin D1 promoter (29, 30). The c-Myc promoter contains multiple Tcf-4–binding sites and is activated by β-catenin and presumably by LEF1 (28). These data demonstrate the role for LEF1/TCF and β-catenin in activating cyclin D1 and c-Myc expression in cancer cells and our studies demonstrate that miR26b targets LEF1 expression, which in turn regulates cyclin D1 and c-Myc expression.

Myc is a well-known oncogenic transcription factor that controls many cellular processes, including cell growth, differentiation, cell adhesion, and motility. Aberrant Myc activity is associated with many human cancers and Myc-mediated transactivation of target genes results in deregulated gene expression. Interestingly, some of these Myc target genes are miRs. Myc directly activates transcription of the polycistronic miR17–92 cluster and miR9 (38–40). The miR17–92 cluster is classified as an oncogene as its expression is associated with several solid tumors (for reviews see refs. 41, 42). Conversely, Myc represses several miRs that are tumor suppressors, including let-7 family members, miR34a, miR29 members, miR26a, and miR15a/16-1 (43). Myc-regulated miRs control cell-cycle programs, apoptosis, angiogenesis, metabolism and, therefore, tumorigenesis through a variety of molecular mechanisms and gene targets (41, 42). miR26a overexpression has been used in an MYC-driven mouse hepatocellular carcinoma model to reverse disease progression (44). The molecular mechanism of miR26a involved decreased expression of cyclin D1 and D2 and induced cell-cycle arrest (44). Our data suggest that miR26b overexpression could potentially be used in patients as a therapeutic, targeting hepatocellular carcinoma in these patients or a combination of miR26a and b. Similar to miR26a, which is expressed at low levels in human liver cancer samples, miR26b is also expressed at low levels in colon cancer cells (Fig. 1A; ref. 17). Interestingly, there is a direct correlation with the severity of colon cancer with a decrease in miR26b expression and a concomitant increase in LEF1 expression, suggesting that miR26b could be an effective therapeutic agent in treating this disease. miR26b was also decreased in LoVo colon cancer cells and miR26b overexpression in these cells suppressed cell growth and tumor growth in vivo (17). However, LEF1 was not included as a miR26b target in the LoVo cells and tumors.

In summary, miR26b is decreased and LEF1 expression is increased in colon cancer cells. We show an inverse correlation between miR26b and LEF1 expression in multiple colon cell lines (Fig. 1A and B) and that miR26b has a potent inhibitory effect on colon cancer cell proliferation (Fig. 2). Furthermore, in patient colon cancer samples miR26b expression is decreased (Fig. 1D). Endogenous LEF1 expression is significantly decreased by miR26b overexpression and the Wnt/β-catenin target genes cyclin D1 and c-Myc are also repressed, and this represents the underlying mechanism of miR26b-dependent inhibition of colon cancer cell proliferation. Thus, miR26b is a tumor repressor that is a potent inhibitor of colon cancer cell proliferation that blocks Wnt/β-catenin–dependent activation of LEF1 and a model for tumor suppressor–like activity of miR26b is illustrated in Fig. 6C. These findings also have important therapeutic implications.

Given the prevalence of increased Wnt/β-catenin signaling in colon cancer patients, miR26b can be a potential candidate for gene therapy of multiple cancer types that express LEF1. The efficient delivery of miRs and other nucleic acids are being extensively investigated with some clinical trials already in progress (4, 5).

No potential conflicts of interest were disclosed.

Conception and design: Z. Zhang, K.H. Kim, X. Li, T. Sharp, B.A. Amendt

Development of methodology: Z. Zhang, X. Li, T. Sharp, B.A. Amendt

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Z. Zhang, K.H. Kim, X. Li, M. Moreno, T. Sharp, M.J. Goodheart, S. Safe, B.A. Amendt

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Z. Zhang, K.H. Kim, X. Li, T. Sharp, S. Safe, A.J. Dupuy, B.A. Amendt

Writing, review, and/or revision of the manuscript: Z. Zhang, K.H. Kim, X. Li, M. Moreno, T. Sharp, M.J. Goodheart, S. Safe, A.J. Dupuy, B.A. Amendt

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Li, T. Sharp, B.A. Amendt

Study supervision: S. Safe, B.A. Amendt

This work was supported by the NIH grant DE13941 to B.A. Amendt.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.