Up-regulation of miR-224 promotes cancer cell proliferation and invasion and predicts relapse of colorectal cancer
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 10 July 2013
Accepted: 20 October 2013
Published: 23 October 2013
MicroRNAs (miRNAs) are small, non-coding RNAs that can function as oncogenes or tumor suppressors in human cancer. Abnormally expressed miR-224 was found to play a fundamental role in several types of cancer. The aim of this study was to investigate the prognostic and biological values of miR-224 in colorectal cancer (CRC).
Quantitative RT-PCR (qRT-PCR) was used to evaluate expression levels of miR-224. The postoperative survival rate was analyzed with Kaplan–Meier method. The roles of miR-224 in cell proliferation, migration and invasion were analyzed with pre-miR-224 transfected cells. In addition, the regulation of SMAD4 by miR-224 was evaluated by qRT-PCR, Western blotting and luciferase reporter assays.
In the present study, we demonstrated that miR-224 was significantly up-regulated in CRC tissue samples and associated with disease relapse and a relative poorer disease-free survival rate. Moreover, ectopic expression of miR-224 potently promoted tumor cell proliferation, migration and invasion in vitro. Furthermore, the over-expression of miR-224 in CRC cell lines decreased SMAD4 expression at the translational level and decreased SMAD4-driven luciferase-reporter activity.
Our data suggest that miR-224 could play an oncogenic role in the cellular processes of CRC and represent a novel biomarker for tumor relapse of CRC patients.
Colorectal carcinoma (CRC) is one of the most common cancers, and is a significant contributor to cancer death. Although surgery currently offers the possibility of prolonged survival for CRC patients, a significant number of patients with CRC who undergo curative surgery develop local recurrence or distant metastasis, leading to shorter survival. A better understanding of the molecular mechanisms underlying tumor recurrence or metastasis is essential to facilitate the prevention and treatment of advanced CRC.
MicroRNAs (miRNAs) are endogenous non-coding RNAs that negatively regulate target gene expressions by binding to 3′-untranslated region (UTR)[3, 4]. MiRNAs participate in gene regulation, apoptosis, hematopoietic development, the maintenance of cell differentiation, and tumor genesis[5, 6]. The dysregulation of miRNAs is common in various carcinomas and plays an important role in tumorigenesis, tumor progression, metastasis and relapse in cancers[7–9].
Recently, miR-224 has been shown to be up-regulated in cervical cancer and pancreatic ductal adenocarcinomas[10, 11], and the involvement of miR-224 in the tumorigenesis and development of breast cancer and hepatocellular carcinoma has also been reported[12, 13]. Previous reports revealed that miR-224 was upregulated in CRC by miRNA microarray analysis[14, 15]. Moreover, miR-224 is one of the most highly differentially expressed miRNAs in methotrexate-resistant cells, and its over-expression induces the resistant phenotype in HT29 colon cancer cells. Taken together, these studies suggest that miR-224 functions as an oncogenic miRNA. However, the association between miR-224 and relapse of colorectal cancer has not been evaluated yet, and the biological roles of miR-224 in CRC remain poorly understood.
Thus, we investigated the relationship between expression level of miR-224 and prognosis in CRC, and further studied the possible function of miR-224 in the CRC cell line. Our study results showed the high expression level of miR-244 in CRC was significantly associated with a relative poorer disease-free survival rate. Moreover, we also demonstrated miR-224 promoted proliferation, migration and invasion of SW480 cells, at least partially through suppression of SMAD4 expression.
Materials and methods
Patients and tissue samples
A total of 108 stage I-II (UICC, 6th ed., 2002) colorectal patients received radical surgery at the First Department of General Surgery, the Affiliated Hospital of North Sichuan Medical College, from January 2004 to January 2009, were collected. All clinicopathological characteristics of patients with disease relapse (n=40) or without disease relapse (n=68) within 3 years after surgery were available for all patients. Disease relapse was defined as local recurrence or distant metastasis of colorectal cancer. All tissue specimens were derived from 108 patients who did not received neoadjuvant therapy before surgery. The patients who received postoperative adjuvant therapy were also excluded. To test whether miR-224 was differentially expressed between paired tumor and adjacent normal tissue in the same subject, we recruited a second cohort comprising 20 CRC patients. All tissue samples were immediately frozen in liquid nitrogen and stored at -80°C for subsequent analysis. The median follow-up time was 48.3 months (12.6–62.5 months) until June, 2012. Disease-free survival (DFS) was calculated from radical surgery to the first disease relapse. Informed written consent was obtained from each patient, and research protocols were approved by the Medical Ethics Committee of North Sichuan Medical College.
The human CRC cell line SW480 was purchase from American Type Culture Collection. The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 u/ml penicillin and 100 mg/ml streptomycin, at 37°C in a humidified atmosphere of 5% CO2.
RNA extraction and real-time RT–PCR
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The PCR primers for miR-224 and U6 were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA, USA). The PCR primers for SMAD4 were 5′-TGGCCCAGGATC AGTAGGT-3′ and 5′-CATCAACACCAATTCCAGCA-3′.The primers for β-actin: 5′-CCAAGGCCAACCGCGAGAAGATGAC-3′ and 5′-AGGGTACAT GGTGGTGCCGCCAGAC-3′. The first-strand cDNA was synthesized using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Real-time PCR was performed using SYBR Premix Ex Taq (TaKaRa) and measured in a LightCycler 480 system (Roche, Basel, Switzerland). U6 or β-actin was used as internal control. Relative gene expression was calculated using 2-ΔCT method, and fold change of gene was calculated using the equation 2-ΔΔCT.
Transfection of miRNA
Ectopic expression of miR-244 in cells was achieved by transfection with Pre-miR-224 precursor (pre-miR-224) (Ambion, Foster City, CA, USA) using Lipofectamine 2000 (Invitrogen). 2 ×105 cells were seeded into each well of a 6-well plate and transfected for 24 h or 48 h. Transfected cells were used in further assays or RNA/protein extraction.
2×104 SW480 cells were plated onto 96-well plates for 24 h. The cells were then transfected with 50 nM pre-miR-224 or pre-miR-nc. At different time points (24 h, 48 h and 72 h), the culture medium was removed and replaced with culture medium containing 10μl of sterile MTT dye (5 mg/ml). After incubation at 37°C for 4 h, the MTT solution was removed, and 150μl dimethyl sulfoxide (DMSO) was added to each well followed by measuring the absorbance at 570 nm on an enzyme immunoassay analyzer (Bio-Rad).
Migration and invasion assays
For migration assays, 5×104 cells transfected with either pre-miR-224 or pre-miR-nc were placed into Boyden chambers (Corning, Cambridge, MA, USA) with an 8.0mm pore membrane. For invasion assays, 5×104 cells were placed into chambers coated with 150μg of Matrigel (BD Biosciences, Bedford, MD, USA). Medium containing 10% fetal bovine serum in the lower chamber served as the chemoattractant. After the cells were incubated for 48 h at 37°C in a humidified incubator with 5% CO2, the cells remaining on the upper surface of the membranes were removed, whereas the cells adhering to the lower surface were fixed, stained with hematoxylin and counted under a microscope at a magnification of 400× to calculate their relative numbers.
Western blot analysis
Immunoblotting was performed to detect the expression of SMAD4 in CRC cell lines. Transfected cells were lysed in RIPA lysis buffer (ProMab Biotechnology). Protein was loaded onto a SDS-PAGE minigel and transferred onto PVDF membrane. After probed with 1:500 diluted mouse polyclonal SMAD4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, the blots were subsequently incubated with HRP-conjugated secondary antibody (1:5000). Signals were visualized using ECL Substrates (Millipore, MA, USA). GAPDH was used as an endogenous protein for normalization.
PCR primers and oligonucleotide sequences of constructs in luciferase reporter assay
Oligonucleotide sequence (5′-3′)
(site 1, site 2)
All data presented in this study have been repeated at least three times from three independent experiments. Continuous variables were expressed as the mean ± standard deviation. Measurement data were analyzed using Student's t-test, while categorical data were studied using chi-square test. Receiver operating characteristic (ROC) curve was used to determine the cut off value of miR-224 expression. The postoperative survival rate was analyzed with Kaplan–Meier method, and differences in survival rates were assessed with log-rank test. All statistical analyses were performed using SPSS 16.0 software (SPSS, Chicago, IL, USA). Two-sided P-values were calculated, and differences were considered significant at P-values of <0.05.
Clinicopathological characteristics of colorectal cancer patients
Correlations between miR-224 expressions and disease relapse
MiR-224 promotes CRC cell proliferation
MiR-224 regulates CRC cell invasion and migration in vitro
MiR-224 binds to the 3′UTR of SMAD4
We performed a luciferase reporter assay to verify that miR-224 directly targets SMAD4.Sequences of the 3′UTR of the SMAD4 mRNA surrounding the two close miR-224 potential binding sites (site 1 and site 2) containing the wild-type (WT1).we cloned the regions of 3′UTR each containing one putative miR-224 binding site into the psicheck-2 vector and named as WT1 (site1, site2) and WT2 (site3) (Figure 4A). The reporter constructs harboring mutation of the miR-224 target sites were generated similarly (MUT1, MUT2 and MUT3) (Figure 4A). The luciferase reporter constructs were transfected into HEK 293T cells, along with pre-miR-224 or pre-miR-nc. Luciferase activites were then measured. The luciferase activity of WT1 (site1, site2) reporter transfected with pre-miR-224 was significantly decreased compared with control (P<0.01, Figure 4B), while the luciferase activity of the WT2 (site3) reporter was not interfered with after transfection with pre-miR-224 compared with control (P>0.05, Figure 4B). These data indicate that miR-224 may target SMAD4 gene through the seeding region of wild type 3′UTR (site1, site2). However, the luciferase reporter activity was not inhibited by miR-224 when the seeding sites were mutated.
MiR-224 inhibits SMAD4 protein expression but not mRNA level
It was reported that disease relapse was an important factor leading to the poor survival of colorectal cancer patients. At present, poor clinicopathological characteristics and high carcinoembryonic antigen (CEA) level were known as high risk factors for relapse but with varying reliability reported. Therefore, effective biomarkers were wanted to distinguish between patients with and without high relapse risk followed by appropriate therapy in CRC.
Differential miRNA expression in tumor samples compared to normal samples or between groups of tumor samples with a favourable and poor clinical outcome have been used to generate miRNA signatures with potential prognostic and/or predictive value[19, 20]. In the current study, we confirmed that miR-224 expression in CRC tumor tissues was significantly higher than that in normal tissues. Furthermore, miR-224 expression levels were significantly up-regulated in the tissues of CRC patients with disease relapse compared with those without disease relapse, and the CRC patients with up-regulated miR-224 in tumor tissues had a high risk of relapse. Thus, miR-224 could function as a potential predictive marker for relapse following radical surgery of colorectal cancer.
In this study, we investigated the biological role of miR-224 in regulating CRC cancer progression. Our results revealed that miR-224 promoted CRC cells growth, migration and invasion in vitro. To address the molecular mechanisms involved in miR-224-mediated changes of biological properties, SMAD4 was selected for further study because it was predicted to be a target of miR-224 by bioinformatics analysis. SMAD4 belongs to the evolutionarily conserved family of SMAD proteins which are transmitters of signals from the transforming growth factor-β (TGF-β) superfamily of cytokines. It is suggested that SMAD4 can function as a tumor suppressor gene in gastrointestinal carcinoma[22, 23]. Previous study showed that patients with tumors expressing low SMAD4 levels had significantly worse overall and disease-free survival than patients with high levels in colorectal cancer. Moreover, Loss of SMAD4 expression was found to be associated with liver metastasis, and reduced SMAD4 expression enhances tumorigenicity in CRC. A recent study also reported that loss of SMAD4 promoted migration and invasion, and mediated epithelial– mesenchymal transition (EMT) in CRC cell line SW480[26, 27]. Hence, it is an attractive target for anti-cancer therapy in colorectal cancer.
Our study suggested that SMAD4 was a possible target of miR-224. Firstly, the luciferase reporter assay demonstrated its down-regulation was mediated by the direct binding of miR-224 to the SMAD4 3′-UTR, because the alteration of this region abolished this effect. Secondly, over-expression of miR-224 suppressed SMAD4 protein levels without any change in SMAD4 mRNA expression. Therefore, we proposed that the main mechanism of miR-224-induced SMAD4 suppression was post-transcriptional. In addition, SMAD4 has been confirmed as a target gene of miR-224 in Granulosa Cells. In our study, restoration of miR-224 promoted CRC cell proliferation, migration and invasion, this could possibly be due to miR-224-mediated down-regulation of SMAD4 expression.
Cancer stem cells (CSCs) are predicted to be critical drivers of tumor progression due to CSC characteristics including self-renewal and pluripotency, drug resistance, limitless proliferative potential and metastatic capability, suggesting that targeting CSC characteristics would likely eliminate CSCs which are the “seeds” of tumor recurrence and metastasis. Specific miRNAs have been shown to be involved in CSC regulation in CRC, such as miR-328 and miR-449b[29, 30]. Recently, Fellenberg et al. showed that the miR-224 functions as an important regulator of stem cells induction by targeting the apoptosis inhibitor, API5. The generation of CSCs involves a process of mesenchymal-to-epithelial transition (MET), therefore factors inducing MET or blocking the EMT by inhibiting TGF-β signaling play an essential role in cell reprogramming. It is also known that TGF-β/Smad4 signaling plays a crucial role in the regulation of EMT as well as cell stemness in CRC[27, 33]. We have discovered a novel target of miR-224 (Smad4), which has key function in TGF-β signaling, providing the possibility that miR-224 may mediate CSC by suppressing TGF-β/Smad4 activity. Thus, our studies might provide a potential molecular mechanism and crosstalk of CSC regulation and tumor metastasis.
In summary, the association between increased levels of miR-224 and disease relapse in CRC patients indicated that miR-224 was a potential biomarker for identifying high-risk CRC patients after radical resection. The present data showed that miR-224 had oncogenic effects, including the promotion of CRC cell proliferation, migration and invasion, at least in part by targeting the anti-oncogene SMAD4, highlighting the function of miR-224 in the process of tumor progression.
This study was supported by Scientific Research Fund of Sichuan Provincial Education Department of China (CBY12-A-ZD16).
- Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ: Cancer statistics, 2008. CA Cancer J Clin. 2008, 58 (2): 71-96. 10.3322/CA.2007.0010.View ArticlePubMedGoogle Scholar
- Kobayashi H, Mochizuki H, Sugihara K, Morita T, Kotake K, Teramoto T, Kameoka S, Saito Y, Takahashi K, Hase K: Characteristics of recurrence and surveillance tools after curative resection for colorectal cancer: a multicenter study. Surgery. 2007, 141 (1): 67-75. 10.1016/j.surg.2006.07.020.View ArticlePubMedGoogle Scholar
- Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004, 116 (2): 281-297. 10.1016/S0092-8674(04)00045-5.View ArticlePubMedGoogle Scholar
- Bartel DP: MicroRNAs: target recognition and regulatory functions. Cell. 2009, 136 (2): 215-233. 10.1016/j.cell.2009.01.002.PubMed CentralView ArticlePubMedGoogle Scholar
- Munker R, Calin GA: MicroRNA profiling in cancer. Clin Sci (Lond). 2011, 121 (4): 141-158. 10.1042/CS20110005.View ArticleGoogle Scholar
- Farazi TA, Spitzer JI, Morozov P, Tuschl T: miRNAs in human cancer. J Pathol. 2011, 223 (2): 102-115. 10.1002/path.2806.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee DY, Deng Z, Wang CH, Yang BB: MicroRNA-378 promotes cell survival, tumor growth, and angiogenesis by targeting SuFu and Fus-1 expression. Proc Natl Acad Sci USA. 2007, 104 (51): 20350-20355. 10.1073/pnas.0706901104.PubMed CentralView ArticlePubMedGoogle Scholar
- Paterson EL, Kazenwadel J, Bert AG, Khew-Goodall Y, Ruszkiewicz A, Goodall GJ: Down-regulation of the miRNA-200 family at the invasive front of colorectal cancers with degraded basement membrane indicates EMT is involved in cancer progression. Neoplasia. 2013, 15 (2): 180-191.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang GJ, Xiao HX, Tian HP, Liu ZL, Xia SS, Zhou T: Upregulation of microRNA-155 promotes the migration and invasion of colorectal cancer cells through the regulation of claudin-1 expression. Int J Mol Med. 2013, 31 (6): 1375-1380.PubMedGoogle Scholar
- Shen SN, Wang LF, Jia YF, Hao YQ, Zhang L, Wang H: Upregulation of microRNA-224 is associated with aggressive progression and poor prognosis in human cervical cancer. Diagn Pathol. 2013, 8: 69-10.1186/1746-1596-8-69.PubMed CentralView ArticlePubMedGoogle Scholar
- Mees ST, Mardin WA, Sielker S, Willscher E, Senninger N, Schleicher C, Colombo-Benkmann M, Haier J: Involvement of CD40 targeting miR-224 and miR-486 on the progression of pancreatic ductal adenocarcinomas. Ann Surg Oncol. 2009, 16 (8): 2339-2350. 10.1245/s10434-009-0531-4.View ArticlePubMedGoogle Scholar
- Huang L, Dai T, Lin X, Zhao X, Chen X, Wang C, Li X, Shen H, Wang X: MicroRNA-224 targets RKIP to control cell invasion and expression of metastasis genes in human breast cancer cells. Biochem Biophys Res Commun. 2012, 425 (2): 127-133. 10.1016/j.bbrc.2012.07.025.View ArticlePubMedGoogle Scholar
- Zhang Y, Takahashi S, Tasaka A, Yoshima T, Ochi H, Chayama K: Involvement of microRNA-224 in cell proliferation, migration, invasion, and anti-apoptosis in hepatocellular carcinoma. J Gastroenterol Hepatol. 2013, 28 (3): 565-575. 10.1111/j.1440-1746.2012.07271.x.View ArticlePubMedGoogle Scholar
- Motoyama K, Inoue H, Takatsuno Y, Tanaka F, Mimori K, Uetake H, Sugihara K, Mori M: Over- and under-expressed microRNAs in human colorectal cancer. Int J Oncol. 2009, 34 (4): 1069-1075.PubMedGoogle Scholar
- Arndt GM, Dossey L, Cullen LM, Lai A, Druker R, Eisbacher M, Zhang C, Tran N, Fan H, Retzlaff K: Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer. BMC cancer. 2009, 9: 374-10.1186/1471-2407-9-374.PubMed CentralView ArticlePubMedGoogle Scholar
- Mencia N, Selga E, Noe V, Ciudad CJ: Underexpression of miR-224 in methotrexate resistant human colon cancer cells. Biochem Pharmacol. 2011, 82 (11): 1572-1582. 10.1016/j.bcp.2011.08.009.View ArticlePubMedGoogle Scholar
- Li S, Gao J, Gu J, Yuan J, Hua D, Shen L: MicroRNA-215 inhibits relapse of colorectal cancer patients following radical surgery. Medical oncology. 2013, 30 (2): 549-View ArticlePubMedGoogle Scholar
- Tsai HL, Chu KS, Huang YH, Su YC, Wu JY, Kuo CH, Chen CW, Wang JY: Predictive factors of early relapse in UICC stage I-III colorectal cancer patients after curative resection. J Surg Oncol. 2009, 100 (8): 736-743. 10.1002/jso.21404.View ArticlePubMedGoogle Scholar
- Garzon R, Marcucci G: Potential of microRNAs for cancer diagnostics, prognostication and therapy. Curr Opin Oncol. 2012, 24 (6): 655-659. 10.1097/CCO.0b013e328358522c.View ArticlePubMedGoogle Scholar
- Albulescu R, Neagu M, Albulescu L, Tanase C: Tissular and soluble miRNAs for diagnostic and therapy improvement in digestive tract cancers. Expert Rev Mol Diagn. 2011, 11 (1): 101-120. 10.1586/erm.10.106.View ArticlePubMedGoogle Scholar
- Massague J, Chen YG: Controlling TGF-beta signaling. Genes Dev. 2000, 14 (6): 627-644.PubMedGoogle Scholar
- Wang LH, Kim SH, Lee JH, Choi YL, Kim YC, Park TS, Hong YC, Wu CF, Shin YK: Inactivation of SMAD4 tumor suppressor gene during gastric carcinoma progression. Clin Cancer Res. 2007, 13 (1): 102-110. 10.1158/1078-0432.CCR-06-1467.View ArticlePubMedGoogle Scholar
- Maitra A, Molberg K, Albores-Saavedra J, Lindberg G: Loss of Dpc4 expression in colonic adenocarcinomas correlates with the presence of metastatic disease. Am J Pathol. 2000, 157 (4): 1105-1111. 10.1016/S0002-9440(10)64625-1.PubMed CentralView ArticlePubMedGoogle Scholar
- Alazzouzi H, Alhopuro P, Salovaara R, Sammalkorpi H, Jarvinen H, Mecklin JP, Hemminki A, Schwartz S, Aaltonen LA, Arango D: SMAD4 as a prognostic marker in colorectal cancer. Clin Cancer Res. 2005, 11 (7): 2606-2611. 10.1158/1078-0432.CCR-04-1458.View ArticlePubMedGoogle Scholar
- Zhang B, Halder SK, Kashikar ND, Cho YJ, Datta A, Gorden DL, Datta PK: Antimetastatic role of Smad4 signaling in colorectal cancer. Gastroenterology. 2010, 138 (3): 969-980. 10.1053/j.gastro.2009.11.004. e961-963PubMed CentralView ArticlePubMedGoogle Scholar
- Muller N, Reinacher-Schick A, Baldus S, van Hengel J, Berx G, Baar A, van Roy F, Schmiegel W, Schwarte-Waldhoff I: Smad4 induces the tumor suppressor E-cadherin and P-cadherin in colon carcinoma cells. Oncogene. 2002, 21 (39): 6049-6058. 10.1038/sj.onc.1205766.View ArticlePubMedGoogle Scholar
- Pohl M, Radacz Y, Pawlik N, Schoeneck A, Baldus SE, Munding J, Schmiegel W, Schwarte-Waldhoff I, Reinacher-Schick A: SMAD4 mediates mesenchymal- epithelial reversion in SW480 colon carcinoma cells. Anticancer Res. 2010, 30 (7): 2603-2613.PubMedGoogle Scholar
- Yao G, Yin M, Lian J, Tian H, Liu L, Li X, Sun F: MicroRNA-224 is involved in transforming growth factor-beta-mediated mouse granulosa cell proliferation and granulosa cell function by targeting Smad4. Mol Endocrinol. 2010, 24 (3): 540-551. 10.1210/me.2009-0432.View ArticlePubMedGoogle Scholar
- Xu XT, Xu Q, Tong JL, Zhu MM, Nie F, Chen X, Xiao SD, Ran ZH: MicroRNA expression profiling identifies miR-328 regulates cancer stem cell-like SP cells in colorectal cancer. Br J Cancer. 2012, 106 (7): 1320-1330. 10.1038/bjc.2012.88.PubMed CentralView ArticlePubMedGoogle Scholar
- Fang Y, Gu X, Li Z, Xiang J, Chen Z: miR-449b inhibits the proliferation of SW1116 colon cancer stem cells through downregulation of CCND1 andE2F3 expression. Oncol Rep. 2013, 30 (1): 399-406.PubMedGoogle Scholar
- Fellenberg J, Saehr H, Lehner B, Depeweg D: A microRNA signature differentiates between giant cell tumor derived neoplastic stromal cells and mesenchymal stem cells. Cancer Lett. 2012, 321 (2): 162-168. 10.1016/j.canlet.2012.01.043.View ArticlePubMedGoogle Scholar
- Li R, Liang J, Ni S, Zhou T, Qing X, Li H, He W, Chen J, Li F, Zhuang Q: A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell. 2010, 7 (1): 51-63. 10.1016/j.stem.2010.04.014.View ArticlePubMedGoogle Scholar
- Roy S, Majumdar AP: Signaling in colon cancer stem cells. J Mol Signal. 2012, 7 (1): 11-10.1186/1750-2187-7-11.PubMed CentralView ArticlePubMedGoogle Scholar
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