Upregulation of miR-501-5p activates the wnt/β-catenin signaling pathway and enhances stem cell-like phenotype in gastric cancer
© The Author(s). 2016
Received: 23 December 2015
Accepted: 11 April 2016
Published: 15 November 2016
miRNAs are critical post-transcriptional regulators of gene expression and key mediators of tumourigenesis. miR-501-5p is newly identified to be involved in the tumor progression, but its biological role and mechanism remain largely unknown. This study is aimed to study the role of miR-501-5p in the progression of gastric cancer.
Real-time PCR analysis was used to determine miR-501-5p expression in gastric cancer cell lines, clinical tissues and 112 clinicopathologically characterized gastric cancer specimens. The role of miR-501-5p in maintaining gastric cancer stem cell like phenotype was examined by tumor-sphere formation assay and expression of stem cell markers. Luciferase reporter assay, cellular fractionation and western blot analysis were used to determined that miR-501-5p activated the wnt/β-catenin signaling by directly targeting DKK1, NKD1 and GSK3β.
Herein, our results revealed that miR-501-5p was markedly upregulated in gastric cancer cell lines and clinical tissues. High miR-501-5p levels predicted poor overall survival in gastric cancer patients. Gain-of-function and loss-of-function studies showed that ectopic expression of miR-501-5p enhanced the cancer stem cell-like phenotype in gastric cancer cells. Notably,wnt/β-catenin signaling was hyperactivated in gastric cancer cells that overexpress miR-501-5p, and mediated miR-501-5p-induced cancer stem cell-like phenotype. Furthermore, miR-501-5p directly targeted and suppressed multiple repressors of the wnt/β-catenin signaling cascade, including DKK1, NKD1 and GSK3β. These results demonstrate that miR-501-5p maintains constitutively activated wnt/β-catenin signaling by directly targeting DKK1, NKD1 and GSK3β, which promotes gastric cancer stem cell like phenotype.
Taken together, our findings reveal a new regulatory mechanism of miR-501-5p and suggest that miR-501-5p might be a potential target in gastric cancer therapy.
Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related death worldwide. It is estimated that 951,600 new GC cases and 723,100 deaths occurred in 2012 . In most countries, survival from stomach cancer remained in the narrow range of 25–30% . Although therapeutic methods are improving in surgical combined with radiotherapy and chemotherapy, the prognosis for advanced stage patients is still very poor [3, 4]. Recent studies have revealed that gastric cancer stem cells constitute a reservoir of selfsustaining cells with the exclusive ability to self-renew and cause tumor outgrowth .. Integral to the CSC model is a subpopulation at the apex of the hierarchy (usually comprising <5% of the cancer), responsible for the formation, maintenance, and continued growth of the tumour . However, the molecular mechanisms for the maintenance of GC stem cell phenotype remains largely unknown.
The Wnt/β-catenin signaling pathway is considered as a key player in the regulation of tissue homeostasis, organ size, tumorigenesis [7–9]. Upon activation, the wnt signals will stabilize and finally lead to accumulation of β-catenin. Activated β-catenin dissociates with E-cadherin, dissembling the adherens and enters the nucleus to turn on the expression of target genes, most of which show invasion promotion functions . Notably, recent advances have shown that the wnt/β-catenin signaling pathway is involved in the maintenance of the GC stem cell population [11, 12]. It has been reported that the pathway blocked by dickkopf WNT signaling pathway inhibitor 1 (DKK-1), naked cuticle homolog 1 (NKD1) and glycogen synthase kinase 3 beta (GSK3β) caused a robust reduction in the activity of wnt/β-catenin signaling and self-renewing capacity of gastric cancer cells [12–16]. Accordingly, the wnt/β-catenin signaling pathway is considered as an important regulator in the maintenance of CSC population, and better understanding of the mechanisms that regulate wnt/β-catenin pathway may provide new clues for more effective GC therapy.
MicroRNAs (miRNAs) are a class of highly conserved, small noncoding RNAs that are approximately 22 nucleotides in length. Typically, miRNAs bind to the 3′-untranslated regions (3′-UTRs) of mRNAs, guide the formation of miRNA-mRNA-induced silencing complexes and lead to the degradation or inhibit the translation of the targeted mRNAs [17, 18]. Bioinformatic predictions indicate that miRNAs regulate more than 30 % of the protein-coding genes . The dysregulation of miRNA expression appears to be a general trait of GC . Thus, it is of particular interest to identify miRNAs that might interfere with wnt/β-catenin signaling and thereby lead to the self-renewal of GC stem cells.
In the present study, we sought to elucidate the the effect of miR-501-5p on wnt/β-catenin signaling in GC, as well as the related molecular mechanism by which miR-501-5p affected wnt/β-catenin signaling. Our data showed that miR-501-5p was upregulated in GC tissues and correlated with a more aggressive phenotype of GC in patients. miR-501-5p activates the wnt/β-catenin pathway by directly targeting DKK1, NKD1 and GSK3β, and consequently enhances stem cell-like phenotype of GC.
Tissues and cells
Fresh GC tissue samples from GC patients, and their matched adjacent non-tumor gastric mucosal tissues (>5 cm laterally from the edge of tumor region) were obtained from the First Affiliated Hospital of Guangzhou University of Chinese Medicine. The samples had been clinically and histopathologically diagnosed according to the World Health Organization criteria. Tumor and non-cancerous tissues were confirmed histologically by hematoxylin and eosin staining. All samples were collected from consenting individuals according to the protocols approved by the Ethics Review Board at Guangzhou University of Chinese Medicine. GC cell lines SGC-7901, HGC-27, MGC-803, MKN-28 and BGC-823 were routinely maintained in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT).
Plasmids and oligonucleotides
Western blot analysis
Cells were harvested in cell lysis buffer (Cell Signaling Technology; Cat#: 9803) and heated for 5 min at 100 °C. Equal quantities of denatured protein samples were resolved on 10% SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (Roche). After blocking with 5% non-fat dry milk in Tris-buffered saline/0.05% Tween 20 (TBST), the membrane was incubated with a specific primary antibody followed by a horseradish peroxidase-conjugated secondary antibody. Proteins were visualized using ECL reagents (Pierce). The antibodies used were as follows: anti-β-catenin, anti-DKK1, anti-NKD1 and anti-GSK3β (Abcam, Cambridge, MA, USA), and p-GSK3β (Ser9) (Cell Signaling Technology). The membranes were stripped and reprobed with an anti–α-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA) as the loading control.
MiRNA extraction and real-time quantitative PCR
Total miRNA from cultured cells and fresh surgical gastric cancer tissues was extracted using a mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. We synthesised cDNA from 10 ng total RNA using a TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA), and quantified the expression levels of miR-501-5p using a miRNA-specific TaqMan MiRNA Assay Kit (Applied Biosystems). miRNA expression was defined based on the threshold cycle (Ct), and relative expression levels were calculated as 2-[(Ct of miR-501-5p) – (Ct of U6)] after normalization with reference to expression of U6 small nuclear RNA.
Sphere formation assays
1 × 103 cells were seeded in 6-well ultra low cluster plates (Corning, NY) and about 10 cells were seeded in 24-well ultra low cluster plates (Corning, NY) for 15 days. Spheres were cultured in DMEM/F12 serum-free medium (Invitrogen, Grand Island, NY) supplemented with 2% B27 (Invitrogen, Grand Island, NY), 20 ng/ml of EGF, and 20 ng/ml of bFGF (PeproTech, Offenbach, Germany), 0.4% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA), and 5 μg/ml insulin.
Cells (4 × 104) were seeded in triplicate in 24-well plates and cultured for 24 h. Cells were transfected with 100 ng TOP/FOP reporter luciferase plasmid, or pGL3-DKK1-3′UTR, pGL3-NKD1-3′UTRor pGL3-GSK3B-3′UTR luciferase plasmids, plus 5 ng pRL-TK Renilla plasmid (Promega) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommendation. Luciferase and Renilla signals were measured 36 h after transfection using a Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s protocol.
All statistical analyses were carried out using SPSS statistical software (SPSS Inc., Chicago, IL, USA). The 2-tailed Student’s t-test was used to evaluate the significance of the differences between two groups of data in all pertinent experiments; a P value <0.05 was considered significant.
miR-501-5p is upregulated in human gastric cancer tissues and cell lines
High miR-501-5p predicts poor prognosis
miR-501-5p enhances the stem cell-like phenotype in gastric cancer cells
miR-501-5p activates wnt/β-catenin signaling pathway
We further investigated the functional significance of wnt/β-catenin signaling activation in miR-501-5p–mediated self-renewal of gastric cancer cells by silencing β-catenin or expressing TCF4-dn in miR-501-5p–overexpressing MGC-803 and SGC-7901 cells. As expected, the stimulatory effect of miR-501-5p on TOP/FOP luciferase reporter activity was impaired by silencing β-catenin or expressing TCF4-dn (Fig. 4d). Moreover, sphere formation assays indicated that silencing β-catenin or expressing TCF4-dn abrogated the promotive effects of miR-501-5p on self-renewal of gastric cancer cells (Fig. 4e). Thus, these results reveal that activation of wnt/β-catenin signaling is essential for miR-501-5p–promoted stem cell like phenotype in gastric cancer.
miR-501-5p directly targets multiple repressors of wnt/β-catenin signaling pathway
MicroRNAs have been demonstrated to negatively regulate target mRNAs in a sequence-specific manner, and are key regulators in a wide variety of oncogenic processes, such as cell proliferation, differentiation, invasion and metastasis, functioning as either tumor suppressors or oncogenes [21–23]. Therefore, elucidating the underlying mechanism of miRNAs in tumor development may provide valuable diagnostic and therapeutic strategies for malignancy. Previous evidence has demonstrated that miR-501-5p is overexpressed in human hepatocellular carcinoma, and upregulation of miR-501-5p contributes to cancer cell proliferation, migration, invasion and drug resistance via different mechanisms . Herein, combining with the TCGA gastric cancer microRNA data set analysis, our results revealed that miR-501-5p was markedly upregulated in GC tissues compared with paired adjacent normal tissues, and was significantly correlated with a more aggressive phenotype of GC in patients.
Gastric cancer continues to be a highly lethal malignancy, despite the use of multimodal treatment approaches. The CSC model has been proposed to explain the high rate of relapse and subsequent resistance of cancer to current systemic treatments . CSCs have been identified in many solid malignancies, including gastric cancer, and have significant clinical implications, as targeting the CSC population may be essential in preventing the recurrence and spread of a tumour [26, 27]. Herein, we found that miR-501-5p acted as a potent CSC-promoting factor in GC. Overexpression of miR-501-5p promoted the self-renewal of GC cells and upregulated the pluripotency associated markers, including CD133, Bmi1, Nanog, MYC and Sox2. More importantly, inhibition of miR-501-5p by antagomiR-501-5p potentially reduced self-renewal of GC cells. Thus, our results suggest that upregulation of miR-501-5p in GC is involved in the malignant progression of GC, and propose that miR-501-5p might be a potential therapeutic target for human gastric cancer.
Accumulating evidence revealed that cancer stem cells have higher metastatic potency by inducing Epithelial–mesenchymal transition (EMT) . Notably, Yoshida et al. recently found that oxidative stress-induced canonical Wnt activation play an important role in the heterogeneous cancer stem cell population at the invasive area by regulating CD44 and c-Myc . Herein, we found that miR-501-5p increased the stemness in gastric cancer by activating the Wnt/β-catenin signaling and increasing the expression of CD44 and c-Myc. Thus, miR-501-5p might contribute to gastric cancer metastasis, and this hypothesis remains to be examined. For example, does miR-501-5p induce EMT; is miR-501-5p increased at the invasive edge of gastric cancer tissues; does miR-501-5p regulate the heterogeneous cancer stem cell population; these questions remain to be answer. Moreover, it was reported that Wnt activation could be induced by micro-environmental stress . Therefore, it would be interesting to investigate whether micro-environmental stress is responsible for miR-501-5p upregulation in gastric cancer.
Several signaling pathways, including the wnt/β-catenin, Notch, and PI3K/Akt pathways, have been found to be aberrantly activated and play vital roles in the development and progression of gastric cancer [11, 30, 31] Among these pathways, the functions of wnt/β-catenin signaling in gastric cancer development and progression have been well documented. TCF4 and coactivator β-catenin are two key downstream effectors of the wnt/β-catenin signaling pathway, and considered as potent oncogenes in tumor progression . Notably, unlike in other cancer types, the mutations in β-catenin are rare, but the expression and/or nuclear localization of β-catenin is often abnormal in gastric cancer, indicating a constitutive activation of Wnt/β-catenin signaling [7, 11, 12]. However, how cancer cells evade the negative regulation by wnt/β-catenin signaling cascade, leading to constitutively activated β-catenin/TCF remains unclear. Herein, we demonstrated that miR-501-5p was substantially overexpressed in gastric cancer and induced hyper-activation of β-catenin/TCF via directly targeting DKK1, NKD1 and GSK3β. Therefore, our findings not only suggest activation of Wnt/β-catenin pathway contributes to the malignant behavior of gastric cancer, but also reveal a novel mechanism for activation of wnt/β-catenin pathway involving miR-501-5p in gastric cancer.
It was reported that GSK-3β exhibited both oncogenic and tumor-suppressive roles in the context of different cancer types. For example, Tang et al. found that loss of GSK-3β expression activated β-Catenin to upregulate the expression of miR-183-96-182 cluster, leading to proliferation and migration of gastric cancer . In contrast, inhibition of GSK3β compromises the survival, proliferation and invasion of glioblastoma cells . Herein, we found that miR-501-5p repressed GSK3β to activate Wnt/β-Catenin pathway and enhance gastric cancer stem cell phenotype, further suggesting a tumor-repressive role of GSK3β in gastric cancer. Moreover, the role of miR-501-5p in glioblastoma remains to be further examined in future. Appropriate reference and discussion have been incorporated into revised manuscript.
In conclusion, our study has revealed that miR-501-5p upregulation plays an important role in gastric cancer progression and miR-501-5p is a critical activator of Wnt/ β-catenin signaling by targeting DKK1, NKD1 and GSK3β. Understanding the precise role of miR-501-5p in gastric cancer pathogenesis and in the wnt/β-catenin signaling pathway promises to increase our knowledge of the biological basis of cancer development and may also facilitate the development of new therapeutic strategies against gastric cancer.
DF and BR carried out most of the experimental work. DF and XY examined miR-501-5p expression in cell lines and clinical samples. BR and JL conducted luciferase reporter assay and western blot analysis. BR and ZZ conducted Real-time PCR analysis and data analysis. DF supervised the project and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108.View ArticlePubMedGoogle Scholar
- Allemani C, Weir HK, Carreira H, Harewood R, Spika D, Wang XS, Bannon F, Ahn JV, Johnson CJ, Bonaventure A, et al. Global surveillance of cancer survival 1995–2009: analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries (CONCORD-2). Lancet. 2015;385(9972):977–1010.View ArticlePubMedGoogle Scholar
- Cui J, Yin Y, Ma Q, Wang G, Olman V, Zhang Y, Chou WC, Hong CS, Zhang C, Cao S, et al. Comprehensive characterization of the genomic alterations in human gastric cancer. Int J Cancer. 2015;137(1):86–95.View ArticlePubMedGoogle Scholar
- Oliveira C, Pinheiro H, Figueiredo J, Seruca R, Carneiro F. Familial gastric cancer: genetic susceptibility, pathology, and implications for management. Lancet Oncol. 2015;16(2):e60–70.View ArticlePubMedGoogle Scholar
- Brungs D, Aghmesheh M, Vine KL, Becker TM, Carolan MG, Ranson M. Gastric cancer stem cells: evidence, potential markers, and clinical implications. J Gastroenterol. 2016;51(4):313-26.Google Scholar
- Jang BG, Lee BL, Kim WH. Prognostic significance of leucine-rich-repeat-containing G-protein-coupled receptor 5, an intestinal stem cell marker, in gastric carcinomas. Gastric Cancer. 2016;19(3):767-77.Google Scholar
- Chiurillo MA. Role of the Wnt/beta-catenin pathway in gastric cancer: an in-depth literature review. World J Exp Med. 2015;5(2):84–102.View ArticlePubMedPubMed CentralGoogle Scholar
- Clevers H, Loh KM, Nusse R. Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science. 2014;346(6205):1248012.View ArticlePubMedGoogle Scholar
- Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell. 2012;149(6):1192–205.View ArticlePubMedGoogle Scholar
- Tian X, Liu Z, Niu B, Zhang J, Tan TK, Lee SR, Zhao Y, Harris DC, Zheng G. E-cadherin/beta-catenin complex and the epithelial barrier. J Biomed Biotechnol. 2011;2011:567305.PubMedPubMed CentralGoogle Scholar
- Mao J, Fan S, Ma W, Fan P, Wang B, Zhang J, Wang H, Tang B, Zhang Q, Yu X, et al. Roles of Wnt/beta-catenin signaling in the gastric cancer stem cells proliferation and salinomycin treatment. Cell Death Dis. 2014;5:e1039.View ArticlePubMedPubMed CentralGoogle Scholar
- Cai C, Zhu X. The Wnt/beta-catenin pathway regulates self-renewal of cancer stem-like cells in human gastric cancer. Mol Med Rep. 2012;5(5):1191–6.PubMedGoogle Scholar
- Katoh M. Molecular cloning, gene structure, and expression analyses of NKD1 and NKD2. Int J Oncol. 2001;19(5):963–9.PubMedGoogle Scholar
- Guo J, Cagatay T, Zhou G, Chan CC, Blythe S, Suyama K, Zheng L, Pan K, Qian C, Hamelin R, et al. Mutations in the human naked cuticle homolog NKD1 found in colorectal cancer alter Wnt/Dvl/beta-catenin signaling. PLoS One. 2009;4(11):e7982.View ArticlePubMedPubMed CentralGoogle Scholar
- Niehrs C. Head in the WNT: the molecular nature of Spemann’s head organizer. Trends Genet. 1999;15(8):314–9.View ArticlePubMedGoogle Scholar
- Dajani R, Fraser E, Roe SM, Yeo M, Good VM, Thompson V, Dale TC, Pearl LH. Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex. EMBO J. 2003;22(3):494–501.View ArticlePubMedPubMed CentralGoogle Scholar
- Leung AK. The whereabouts of microRNA actions: cytoplasm and beyond. Trends Cell Biol. 2015;25(10):601–10.View ArticlePubMedPubMed CentralGoogle Scholar
- Su Z, Yang Z, Xu Y, Chen Y, Yu Q. MicroRNAs in apoptosis, autophagy and necroptosis. Oncotarget. 2015;6(11):8474–90.View ArticlePubMedPubMed CentralGoogle Scholar
- Oulas A, Karathanasis N, Louloupi A, Pavlopoulos GA, Poirazi P, Kalantidis K, Iliopoulos I. Prediction of miRNA targets. Methods Mol Biol. 2015;1269:207–29.View ArticlePubMedGoogle Scholar
- Guo LL, Song CH, Wang P, Dai LP, Zhang JY, Wang KJ. Competing endogenous RNA networks and gastric cancer. World J Gastroenterol. 2015;21(41):11680–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281–97.View ArticlePubMedGoogle Scholar
- Hata A, Kashima R. Dysregulation of microRNA biogenesis machinery in cancer. Crit Rev Biochem Mol Biol. 2016;51(3):121–34.Google Scholar
- Esquela-Kerscher A, Slack FJ. Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer. 2006;6(4):259–69.View ArticlePubMedGoogle Scholar
- Huang DH, Wang GY, Zhang JW, Li Y, Zeng XC, Jiang N. MiR-501-5p regulates CYLD expression and promotes cell proliferation in human hepatocellular carcinoma. Jpn J Clin Oncol. 2015;45(8):738–44.View ArticlePubMedGoogle Scholar
- Vermeulen L, de Sousa e Melo F, Richel DJ, Medema JP. The developing cancer stem-cell model: clinical challenges and opportunities. Lancet Oncol. 2012;13(2):e83–9.View ArticlePubMedGoogle Scholar
- Dewi DL, Ishii H, Kano Y, Nishikawa S, Haraguchi N, Sakai D, Satoh T, Doki Y, Mori M. Cancer stem cell theory in gastrointestinal malignancies: recent progress and upcoming challenges. J Gastroenterol. 2011;46(10):1145–57.View ArticlePubMedGoogle Scholar
- Yoshida GJ, Saya H. Therapeutic strategies targeting cancer stem cells. Cancer Sci. 2016;107(1):5–11.View ArticlePubMedGoogle Scholar
- Scheel C, Weinberg RA. Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links. Semin Cancer Biol. 2012;22(5-6):396–403.View ArticlePubMedGoogle Scholar
- Yoshida GJ, Saya H. Inversed relationship between CD44 variant and c-Myc due to oxidative stress-induced canonical Wnt activation. Biochem Biophys Res Commun. 2014;443(2):622–7.View ArticlePubMedGoogle Scholar
- Demitrack ES, Gifford GB, Keeley TM, Carulli AJ, VanDussen KL, Thomas D, Giordano TJ, Liu Z, Kopan R, Samuelson LC. Notch signaling regulates gastric antral LGR5 stem cell function. EMBO J. 2015;34(20):2522–36.View ArticlePubMedPubMed CentralGoogle Scholar
- Singh SS, Yap WN, Arfuso F, Kar S, Wang C, Cai W, Dharmarajan AM, Sethi G, Kumar AP. Targeting the PI3K/Akt signaling pathway in gastric carcinoma: a reality for personalized medicine? World J Gastroenterol. 2015;21(43):12261–73.View ArticlePubMedPubMed CentralGoogle Scholar
- Tang X, Zheng D, Hu P, Zeng Z, Li M, Tucker L, Monahan R, Resnick MB, Liu M, Ramratnam B. Glycogen synthase kinase 3 beta inhibits microRNA-183-96-182 cluster via the beta-Catenin/TCF/LEF-1 pathway in gastric cancer cells. Nucleic Acids Res. 2014;42(5):2988–98.View ArticlePubMedGoogle Scholar
- Chikano Y, Domoto T, Furuta T, Sabit H, Kitano-Tamura A, Pyko IV, Takino T, Sai Y, Hayashi Y, Sato H, et al. Glycogen synthase kinase 3beta sustains invasion of glioblastoma via the focal adhesion kinase, Rac1, and c-Jun N-terminal kinase-mediated pathway. Mol Cancer Ther. 2015;14(2):564–74.View ArticlePubMedGoogle Scholar