miR-21 may acts as an oncomir by targeting RECK, a matrix metalloproteinase regulator, in prostate cancer
- Sabrina Thalita Reis1, 2Email author,
- José Pontes-Junior1, 2, 3,
- Alberto Azoubel Antunes1, 3,
- Marcos Francisco Dall’Oglio1, 2,
- Nelson Dip1,
- Carlo Camargo Passerotti1, 3,
- Guilherme Ayres Rossini3,
- Denis Reis Morais1,
- Adriano Joao Nesrallah1, 2,
- Camila Piantino1,
- Miguel Srougi1, 2 and
- Katia R Leite1, 2
© Reis et al.; licensee BioMed Central Ltd. 2012
Received: 30 January 2012
Accepted: 4 May 2012
Published: 29 May 2012
Prognosis of prostate cancer (PCa) is based mainly in histological aspects together with PSA serum levels that not always reflect the real aggressive potential of the neoplasia. The micro RNA (miRNA) mir-21 has been shown to regulate invasiveness in cancer through translational repression of the Metaloproteinase (MMP) inhibitor RECK. Our aim is to investigate the levels of expression of RECK and miR-21 in PCa comparing with classical prognostic factors and disease outcome and also test if RECK is a target of miR-21 in in vitro study using PCa cell line.
Materials and methods
To determine if RECK is a target of miR-21 in prostate cancer we performed an in vitro assay with PCa cell line DU-145 transfected with pre-miR-21 and anti-miR-21. To determine miR-21 and RECK expression levels in PCa samples we performed quantitative real-time polymerase chain reaction (qRT-PCR).
The in vitro assays showed a decrease in expression levels of RECK after transfection with pre-miR-21, and an increase of MMP9 that is regulated by RECK compared to PCa cells treated with anti-miR-21. We defined three profiles to compare the prognostic factors. The first was characterized by miR-21 and RECK underexpression (N = 25) the second was characterized by miR-21 overexpression and RECK underexpression (N = 12), and the third was characterized by miR-21 underexpression and RECK overexpression (N = 16). From men who presented the second profile (miR-21 overexpression and RECK underexpression) 91.7% were staged pT3. For the other two groups 48.0%, and 46.7% of patients were staged pT3 (p = 0.025).
Our results demonstrate RECK as a target of miR-21. We believe that miR-21 may be important in PCa progression through its regulation of RECK, a known regulator of tumor cell invasion.
KeywordsProstate cancer Prognosis RECK Micro RNA Metaloproteinases
Prostate cancer (PCa) is the most common cancer affecting males and the second leading cause of death in men in many countries, including Brazil. For 2011 903,500 new cases and 258,400 deaths are estimated worldwide . Tumor staging, Gleason score and Prostatic specific antigen (PSA) serum levels are the most important prognostic factors; however, even taken together they cannot perfectly predict which patients are at risk for progression . There is a variety of treatment options that ranges from active surveillance to androgen deprivation. Therefore, identification of molecular markers that could contribute to prediction of PCa behavior has been an area of active research.
MicroRNAs (miRNAs) are a class of small noncoding RNA that regulate the expression of target genes by promoting translational repression or degradation of mRNAs [3, 4]. In recent years, abnormalities in miRNA expression have been identified in the progression of various cancers and consequently have been proposed as potential targets for anticancer therapies . miRNAs have been shown to function as both tumor suppressors or oncogenes in various cancers [6, 7]. Using high-throughput profiling of miRNA expression, miR-21 was identified as being strongly elevated in many tumors including breast, colorectal, and hepatocellular carcinoma [8–11]. However, our knowledge of the molecular mechanisms underlying the function of miR-21 in cancer generally, and prostate cancer specifically, is limited.
miR-21 regulates the expression of multiple mRNA targets associated with tumor invasiveness and microvascular proliferation. It has been shown that mir-21 regulates cell invasiveness by directly controlling the MMP inhibitor RECK (Reversion-inducing cysteine-rich protein with Kazal motifs), a key inhibitor of several MMPs. Indeed, RECK expression levels are predictive in determining prognoses in a number of common cancers; low levels of RECK are often associated with increased invasiveness and poor prognosis [12, 13]. Additionally, it has been shown that inhibition of miR-21 results in reduced MMP activity leading to reduction of cell motility and invasiveness, through increased expression of RECK .
In this study, we will investigate if RECK is a target of miR-21 in prostate cancer cell line DU-145 in in vitro assays. Also we will evaluate the expression levels of RECK, MMP9 controlled by RECK and miR-21 in PCa tissue comparing with Gleason score, pathological stage, pre-operatory PSA serum levels and the outcome of patients with localized PCa treated with radical prostatectomy.
Patients and tissue samples
Demographic and clinical characteristics of 53 patients included in the study submitted to radical prostatectomy to treat prostate cancer
Age (years) Mean
Min - Max
50 - 79
PSA (ng/ml) Mean
Min - Max
2.0 – 37.0
<10 n (%)
≥10 n (%)
pT2 n (%)
pT3 n (%)
< 7 n (%)
≥ 7 n (%)
We analyzed miR-21, RECK and MMP9 expression levels in fresh-frozen tumor specimens randomly selected from our tumor bank from patients with clinically localized PCa who underwent radical prostatectomy by a single surgeon between 1993 and 2007 using quantitative real-time polymerase chain reaction (qRT-PCR) using TaqMan primers. The control group consisted of tissue specimens from 11 patients with benign prostatic hyperplasia (BPH) who presented lower urinary tract symptoms and underwent retropubic prostatic resection (mean age 64±6.0 years).
We found three different profiles characterized as follows:  miR-21 and RECK underexpression (n = 25)  miR-21 overexpression and RECK underexpression or  miR-21 underexpression and RECK overexpression (n = 16).
Then the three profile were correlated with Gleason score, pathological stage (TNM 2010) and pre-operative serum PSA levels (ng/mL). For this analysis, pathological stage was defined as organ-confined (pT2) or non-organ-confined (pT3). Gleason score was classified as low grade (Gleason score ≤6) or high grade (Gleason score ≥7). Preoperative PSA was divided as ≥10 ng/mL or <10 ng/mL. Additionally, we analyzed miR-21 and RECK expression levels and their relation to biochemical recurrence, which was defined as PSA levels >0.4 ng/mL, in a mean follow-up time of 66.2 months for all patients.
RNA and miRNA isolation and cDNA synthesis
All tumor samples were obtained from surgical specimens and immediately frozen at −170°C in liquid nitrogen. A section of the frozen fragment was stained with hematoxylin and eosin to verify the presence of tumor in at least 75% of the fragment in PCa patients and if there was no tumor in control BPH patients. Total RNA was isolated from tissue samples using a RNAaqueous Kit (Applied Biosystems, CA, USA) and miRNA was isolated with a mirVANA Kit (Applied Biosystems, CA, USA) according to the manufacturer’s instructions. RNA and miRNA concentration was determined by 260/280 nm absorbance using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific).
cDNA from total RNA was generated using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA). Reactions were incubated at 25°C for 10 min, followed by 37°C for 120 min and 85°C for 5 min. cDNA from miRNAs was generated using a TaqMan® miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Reactions were incubated at 16°C for 30 min, 42°C for 30 min and 85°C for 30 min. All cDNAs were stored at −20°C until further use.
Quantitative real-time PCR of miR21, RECK and MMP9
We used the ΔΔCT method to calculate the relative expression of the RECK and miR-21 using the formula ΔΔCT = (CT target gene, PC sample - CT endogenous control, PC sample) – (CT target gene, BPH sample - CT endogenous control, BPH sample). The fold change in gene expression was calculated as 2-ΔΔCT.
Human prostate cancer DU145 was purchased from ATCC (American Type Culture Collection, Manassas, USA). The cell lines were cultured in McCoy's medium supplemented with 20% (v/v) heat-inactivated fetal bovine serum (Sigma, St.Louis, MO, USA) under an atmosphere of 5% CO2 at 37°C. At the time of confluence, adherent cells were subcultured after detachment using trypsin/EDTA (0.25% trypsin- 1.0 mM ethylenediaminetetraacetate).
The transfection of Pre-miR-21 and its antagomir anti-miR-21 via NeoFX was performed by following the manufacturer’s manual. Briefly, 2.5 μl de Pre e anti – miR-21 was mixed with 50 μl OptiMEM, and 1.5 μl of NeoFX was mixed with 50 μl OptiMEM. This dilutions were mixture and then, were incubated at room temperature for 10 min. Untreated cells and cells transfected by scrambled microRNA precursor with no target were negative controls in our work.
Quantification of miR-21 and RECK, MMP9 mRNA expression in DU145 cells by quantitative real time PCR
Twenty-four hours post transfection, the cells were washed with 1xPBS and then RNA and miRNA were extracted with mirVANA Kit (ambion), cDNA from total RNA was generated using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA) and from miRNAs was generated using a TaqMan® miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). The qRT-PCR amplification of cDNA was performed using TaqMan MicroRNA assay for miR-21, and TaqMan gene expression assay for RECK and MMP-9.
Qualitative variables were expressed as numbers and percentages. To compare the clinical characteristics of patients with PCa, we used the Mann–Whitney or T Student, Chi-squared and Fisher exact tests. Statistical analysis was performed using SPSS 15.0 for Windows using a significance of p ≤0.05.
miR-21 and RECK expression data in the malignant prostatic tissue according to Gleason score, Pathological stage, PSA value and Biochemical Recurrence
< 7 (n = 22)
≥ 7 (n = 31)
pT2 (n = 22)
pT3 (n = 30)
<10 (n = 27)
≥10 (n = 26)
W/n (n = 27)
With (n = 20)
Relative expression of miR-21 and RECK in the malignant prostatic tissue in three profiles
Additionally, there was a marginal statistical difference between the three profiles when we examined biochemical recurrence. From patients presenting miR-21 overexpression and RECK underexpression, 63.6% had biochemical recurrence compared with 50.0% and 25.0% of patients in the others profiles (p = 0.087) (Table4).There were no differences in the three groups considering Gleason score or PSA pre-operatory serum levels.
The expression level of MMP9 having as a control BPH tissue was 5.32 times when miR21 was overexpressed and RECK underexpressed, and 5.48 times when both miR21 and RECK were underexpressed. When miR21 was underexpressed and RECK overexpressed MMP9 was 3.1 times.
The in vitro assays after 24 h of DU145 exposition to pre-miR21 and anti-miR-21, showed a 20 times increase in miR21 expression, confirming the success of transfection (Figure 1.B). The supposed target gene RECK showed two times reduction in expression when exposed to pre-miR-21. Also, MMP9, regulated by RECK had doubled its expression levels after exposition to pre-miR21 (Figure 1.C).
To understand the role of any miRNA in disease such as cancer, the mRNA targets of that miRNA must be identified. Each miRNA has hundreds of targets and each mRNA is target for different miRNAs. miR-21 plays a role in the regulation of cell proliferation, apoptosis and tumor invasiveness by targeting PTEN, PDCD4, and RECK , and has been shown to be involved in gliomas carcinogenesis process. This is the first study to explore the role of miR-21 controlling RECK in PCa cell lines and PCa specimens. In PCa cell line DU-145 miR-21 showed that directly inhibits RECK, a tumor suppressor gene that is involved in the control of MMP9. Studying 53 PCa patient samples, 22.6% exhibited miR-21 overexpression and RECK underexpression associated with non-organ confined tumors (pT3). We therefore believe that miR-21 may be playing a role in a subset of prostate cancers through regulation of RECK expression levels.
The same phenomenon has been observed in other types of human cancers. Liu et al. showed that inhibition of miR-21 lead to a decrease in invasiveness in hepatocellular carcinoma . Inhibition of miR-21 leads to increasing levels of RECK, a membrane-anchored glycoprotein that inhibits tumor cell invasion by regulating MMP-2, MMP-9 and MT1-MMP . In tumors in which RECK is absent or diminished, MMPs will be highly active, facilitating tumor promotion and progression. In our study, we showed in PCa cell line transfected with pre-miR-21, a decreased in the levels of RECK mRNA and consequently an increased in the level of MMP9.
Recently, Zhang et al. have described the similar results in gastric cancer . Cancers which overexpressed miR-21 and had Helicobacter pylori infection had a higher capacity for invasion and migration of tumor cells. Here, we have demonstrated a similar association in PCa where 91.7% of tumors presenting overexpression of miR-21 were staged pT3.
The miR-21 is one of the most commonly implicated miRNAs in cancer. Its expression is highly upregulated in a variety of solid tumors including gliomas, breast, lung and pancreas carcinomas [18, 19]. Elevated miR-21 expression has been causally linked to increase in cell proliferation, reduction in apoptosis, and migration of several cancer cell lines . However, the molecular mechanisms mediating miR-21 function in cancer generally, and PCa specifically, are poorly understood. MiR-21 regulates RECK, which we have observed can function as a tumor suppressor gene in PCa (unpublished data). RECK decreases the amount of active MMP-2 and MMP-9 in conditioned medium and inhibits metastatic activity in vitro  and in vivo  through modulation of these MMPs, which are known to be involved in cancer progression .
Our data show a tendency in association between miR-21 overexpression and biochemical recurrence. When miR-21 was overexpressed and RECK was underexpressed 63.4% of patients suffered recurrence with a mean follow up of 60 months (p = 0.087). We may hypothesize that this specific profile is unfavorable for prostate adenocarcinoma, and a higher number of patients should be studied to confirm this preliminary finding.
In this first study we identify a possible role of miR-21 in the behavior of prostate cancer promoting a decay in the levels of RECK mRNA allowing the overexpression of MMP9. Interesting would be to prove also the decay in protein levels using westerbloting, and this is a theme of our next study.
In conclusion, we believe that miR-21 may be important in PCa progression through its regulation of RECK, a known regulator of tumor cell invasion. Experimental studies must be performed in order to show the precise role of miR-21 and MMP and its regulators, specially RECK in prostate carcinogenesis. Consequently, suppression of miR-21 would be considered as a potential therapeutic tool in the treatment of the neoplasia.
Benign prostatic hyperplasia
Complementary deoxyribonucleic acid
Quantitative real-time polymerase chain reaction
Reversion-inducing cysteine-rich protein with Kazal motif
Tissue inhibitor of metalloproteinases 1.
This study was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de Sao Paulo) under protocol number 2009/50368-9, we acquired all necessary reagent with this support. This article was reviewed by American Journal Expert.
- Jemal A, Siegel R, Ward E, Brawley O: Cancer statistics, 2011. CA Cancer J Clin. 2011, 61: 212-236. 10.3322/caac.20121.View ArticlePubMedGoogle Scholar
- Berger AP, Spranger R, Kofler K, Steiner H, Bartsch G, Horninger W: Early detection of prostate cancer with low PSA cut-off values leads to significant stage migration in radical prostatectomy specimens. Prostate. 2003, 57: 93-98. 10.1002/pros.10278.View ArticlePubMedGoogle Scholar
- Peek RM, Blaser MJ: Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat Rev Cancer. 2002, 2: 28-37. 10.1038/nrc703.View ArticlePubMedGoogle Scholar
- Pillai RS: MicroRNA function: multiple mechanisms for a tiny RNA?. RNA. 2005, 11: 1753-1776. 10.1261/rna.2248605.View ArticlePubMedPubMed CentralGoogle Scholar
- Croce CM: Oncogenes and cancer. N Engl J Med. 2008, 358: 502-511. 10.1056/NEJMra072367.View ArticlePubMedGoogle Scholar
- He L, He X, Lowe SW, Hannon GJ: microRNAs join the p53 network-another piece in the tumour-supression puzzle. Nat Rev Cancer. 2007, 7: 819-822. 10.1038/nrc2232.View ArticlePubMedPubMed CentralGoogle Scholar
- Kent OA, Mendell JT: A small piece in the cancer puzzle: microRNAs as tumor suppressors and oncogenes. Onocogene. 2006, 25: 6188-6196. 10.1038/sj.onc.1209913.View ArticleGoogle Scholar
- Asangani IA, Rasheed SA, Nikolova DA, Leupold JH, Colburn NH, Post S, Allgayer H: MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene. 2008, 27: 2128-2136. 10.1038/sj.onc.1210856.View ArticlePubMedGoogle Scholar
- Meng F, Henson R, Wehbe-Janek H, Gholshal K, Jacob ST, Patel T: MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology. 2007, 133: 647-658. 10.1053/j.gastro.2007.05.022.View ArticlePubMedPubMed CentralGoogle Scholar
- Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY: miR-21-mediated tumor growth. Oncogene. 2007, 26: 2799-2803. 10.1038/sj.onc.1210083.View ArticlePubMedGoogle Scholar
- Zhu S, Si ML, Wu H, Mo YY: MicroRNA-21 targets the tumor suppressor genes tropomyosin 1 (TPM1). J Biol Chem. 2007, 282: 14328-14336. 10.1074/jbc.M611393200.View ArticlePubMedGoogle Scholar
- Kotzsch M, Farthmann J, Meye A, Fuessel S, Baretton G, Tjan-Heijnen VC, Schmitt M, Luther T, Sweep FC, Magdolen V, Span PN: Prognostic relevance of uPAR-del4/5and TIMP-3 mRNA expression levels in breast cancer. Eur J Cancer. 2005, 41: 2760-2768. 10.1016/j.ejca.2005.09.002.View ArticlePubMedGoogle Scholar
- Takenaka K, Ishikawa S, Kawano Y, Yanagihara K, Miyahara R, Otake Y, Morioka Y, Takahashi C, Noda M, Wada H, Tanaka F: Expression of a novel matrix metalloproteinase regulator, RECK, and its clinical significance in resected non-small cell lung cancer. Eur J Cancer. 2004, 10: 1617-1623.View ArticleGoogle Scholar
- Gabriely G, Wurdinger T, Kesari S, Esau CC, Burchard J, Linsley PS, Krichevsky AM: MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol. 2008, 28: 5369-5380. 10.1128/MCB.00479-08.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu C, Yu J, Yu S, Lavker RM, Cai L, Liu W, Yang K, He X, Chen S: MicroRNA-21 acts as an oncomir through multiple targets in human hepatocellular carcinoma. J Hepatol. 2010, 53: 98-107. 10.1016/j.jhep.2010.02.021.View ArticlePubMedGoogle Scholar
- Takahashi C, Sheng Z, Horan TP, Kitayama H, Maki M, Hitomi K, Kitaura Y, Takai S, Sasahara RM, Horimoto A, Ikawa Y, Ratzkin BJ, Arakawa T, Noda M: Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK. Proc Natl Acad Sci U S A. 1998, 95: 13221-13226. 10.1073/pnas.95.22.13221.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang Z, Li Z, Gao C: miR-21 plays a pivotal role in gastric cancer pathogenesis and progression. Lab Invest. 2008, 88: 1358-1366. 10.1038/labinvest.2008.94.View ArticlePubMedGoogle Scholar
- Chan JA, Krichevsky AM, Kosik KS: MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005, 65: 6029-6033. 10.1158/0008-5472.CAN-05-0137.View ArticlePubMedGoogle Scholar
- Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM: A microRNA expression signature of human solid tumors defines cancer gene targets. Procl. Natl. Acad. Sci. USA. 2006, 103: 2257-2261. 10.1073/pnas.0510565103.View ArticleGoogle Scholar
- Zhu S, Wu H, Wu G, Nie D, Sheng S, Mo YY: MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res. 2008, 18: 350-359. 10.1038/cr.2008.24.View ArticlePubMedGoogle Scholar
- Oh J, Takahashi R, Kondo S, Kawashima S, Kitayama H, Takahashi C, Noda M: The membrane anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell. 2001, 107: 789-800. 10.1016/S0092-8674(01)00597-9.View ArticlePubMedGoogle Scholar
- Chang CK, Hung WC, Chang HC: The Kazal motifs of RECK protein inhibit MMP-9 secretion and activity and reduce metastasis of lung cancer cells in vitro and in vivo. J Cell Mol Med. 2008, 12: 2781-2789. 10.1111/j.1582-4934.2008.00215.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Oh J, Takahashi R, Kondo S: The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell. 2001, 107: 789-800. 10.1016/S0092-8674(01)00597-9.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2490/12/14/prepub