Biomarkers that can facilitate disease detection, staging and prediction of outcome are highly desirable to improve survival and to help determine optimized treatment for colorectal cancer patients. microRNAs (miRNAs) are small non-coding RNAs that play a crucial role in gene regulatory networks. The deregulation of miRNA expression has been found in several types of cancer and may represent a novel class of cancer biomarkers. Our aim was to determine the miRNA signature of stage III colorectal cancer (CRC) tumors and to identify potential circulating miRNAs that may represent non-invasive biomarkers in CRC patients. Genome-wide microarray analysis of miRNA expression was performed on 12 paired tumor and non-tumor formalin-fixed paraffin-embedded tissues from stage III CRC patients. A selection of differentially overexpressed miRNAs was validated by quantitative real-time polymerase chain reaction (qRT-PCR) and determined in the serum of a set of 56 individuals (30 stage III CRC patients and 26 healthy individuals). Using 1.5-fold expression difference as a cut-off level, 43 miRNAs were identified as differentially expressed in tumor versus normal tissue. Using reverse transcription and qRT-PCR, 11 miRNAs (miR-135b, miR-141, miR-18a, miR-20a, miR-21, miR-224, miR-29a, miR-31, miR-34a, miR-92a and miR-96) were confirmed as significantly overexpressed in tumor samples when compared with normal samples. We were able to detect 9 of these 11 miRNAs in serum samples from CRC patients and healthy individuals. Serum levels of miR-18a and miR-29a were significantly higher in CRC patients when compared to levels in the controls (p<0.05). In conclusion, this study identified a substantial number of miRNAs which were differentially expressed in stage III colorectal tumors. Moreover, the findings provide relevant information concerning overexpressed tumoral miRNAs as potential circulating biomarkers and highlight serum miR-18a and miR-29a as promising biomarkers for the screening and monitoring of CRC patients.
Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and is a common cause of cancer-related deaths in developed countries (
microRNAs (miRNAs) are a newly discovered class of short 22 nucleotide non-coding RNAs that repress protein translation through binding to target mRNAs. Bioinformatics and cloning studies have estimated that miRNAs may regulate 30% of all human genes and control hundreds of gene targets (
Accumulating data indicate that miRNAs are present in body fluids including blood plasma and serum, urine, saliva and semen (
In the present study, we aimed to establish comprehensive miRNA expression patterns in stage III CRC patients. Using microarray profiling we identified differentially expressed upregulated and downregulated miRNAs between CRC tissues and paired neighboring noncancerous colorectal tissues. To investigate candidate circulating cancer-associated miRNAs we determined the expression profile of upregulated tumoral miRNAs in serum samples of stage III CRC patients and healthy individuals.
The study was approved by the institutional medical ethics boards of our hospital (Corporació Sanitària Parc Taulí, Institut Universitari Parc Taulí-UAB). Serum samples of the patients and control were prospectively obtained, and both gave written informed consent to participate in the study. The tissue samples were obtained from formalin-fixed paraffin-embedded (FFPE) blocks stored in the Pathology Department of the hospital. For these patients, informed consent was not obtained in accordance with the Spanish Law 14/2007 for Biomedical Research. According to the law, archive samples may be used for biomedical research without informed consent when obtaining informed consent is not possible or extremely difficult, provided that i) the investigation performed is of general interest, ii) there is no previous declaration of the patient against the use of the samples for research, iii) data confidentiality is granted and iv) the ethics committee of the hospital evaluates and approves the study protocol.
For the microarray analysis, 12 FFPE stage III colonic tumor tissue specimens and their surrounding non-cancerous normal mucosa were collected from 12 CRC patients who underwent surgical resection between 2000 and 2005 at the Department of Oncology at the hospital of Sabadell (Corporació Sanitària Parc Taulí, Institut Universitari Parc Taulí-UAB), Barcelona, Spain. Tumors were staged according to the Tumor-Node-Metastasis (TNM) staging system.
Peripheral blood samples (4 ml/individual) were collected from 56 individuals: 30 stage III CRC patients and 26 healthy volunteers (13 female and 13 male with a median age of 64.1±7.4 years) with no prior history of solid or hematological malignancy. No neoadjuvant therapy was used at the time when the blood samples were obtained.
Clinical and pathologic data were extracted from the medical records and reviewed for the purpose of this study. Detailed patient information is documented in
miRNA-enriched total RNA from tissue specimens was extracted using RecoverAll™ Total Nucleic Acid isolation kit for FFPE samples (Ambion, Austin, TX, USA) following the manufacturer’s protocol. Purity and concentration of the RNA were determined from OD260/280 readings using a spectrophotometer. RNA integrity was determined using the Bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA, USA).
A blood sample from each participant was collected in a serum clot activator tube (Vacuette ref. 454067) before surgical resection and centrifuged at 3,500 × g for 10 min at 4°C to spin down the blood cells. Serum was then aliquoted and stored at −80°C until use. RNA was isolated from 200 μl serum using TRIzol LS (Invitrogen Life Technologies, Paisley, UK) as described by the manufacturer and finally eluted into a 50-μl volume of RNA-free water.
Microarray analysis was carried out by an external core facility (
Total RNA (100 ng) was labeled using Agilent miRNA Complete Labeling and Hyb kit (Agilent, cat. no. 5190-0456) following the manufacturer’s instructions. Briefly, total RNA from each sample was dephosphorylated using calf intestinal alkaline phospatase (CIP), and cyanine 3-pCp was then ligated using the T4 RNA ligase. Labeled samples were dried and resuspended in 18 μl of nuclease-free water and co-hybridized with
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate miRNAs upregulated in the microarray experiment data to determine the level of the same miRNAs in serum samples of CRC patients and healthy controls.
cDNA synthesis was performed using miRCURY LNA™ Universal RT microRNA PCR kit (Exiqon A/S, Vedbaek, Denmark) according to the manufacturer’s instructions. Briefly, 20 ng of total RNA from tissue or 4 μl of total RNA from 200 μl of serum was polyadenylated, and cDNA was synthesized using a poly(T) primer with a 3′ degenerate anchor and a 5′ universal tag. Then, cDNA served as a template for miRNA qPCR using FastStart Universal SYBR-Green Master (Rox) (Roche Applied Science, Indianapolis, IN, USA; cat no. 04913850001) and Exiqon-validated specific primer sets. The amplification profile was denatured in 96-well optical plates at 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 60 sec. At the end of the PCR cycles, melting curve analyses were performed. For each sample, both the cDNA synthesis and real-time PCR assays were performed in duplicate in a final volume of 20 μl using an Applied Biosystems 7500 Sequence Detection system (Applied Biosystems, Foster City, CA, USA).
For statistical analysis of microarray data, extracted intensities were background corrected using the normexp method with an offset of 50 (
According to Exiqon’s guidelines for microRNA qPCR in serum/plasma, we chose miR-16, miR-103 and miR-let-7a as reference miRNAs for normalization of expression levels. Using DataAssist™ Software v3.0 (Applied Biosystems) the fold-change in the miRNA expression was calculated by the equation 2−ΔΔCT(
Comparison of the expression profiles of 887 human miRNAs between normal and tumor tissue revealed a total of 43 differentially expressed miRNAs between tumors and corresponding non-tumor samples with a fold change (FC) >1.5 and adjusted p-value below the threshold level of 0.05. This miRNA signature was able to separate the tumors from the normal group into two main unsupervised hierarchial clusters shown in
Thirteen overexpressed miRNAs in CRC tumors were selected for validation using qRT-PCR in the same cohort of 12 paired samples investigated initially by the microarray assay. Ten highly upregulated miRNAs (FC >1.9, p<0.05): miR-135b, miR-142-3p, miR-20a, miR-21, miR-223, miR-224, miR-29a, miR-34a, miR-92a, miR-96, along with 3 miRNAs which were selected on the basis of previous studies supporting their involvement in CRC prognosis: miR-141 (FC 1.76, p=0.04), miR-18a (FC 1.36, p=0.0035) and miR-31 (FC 1.57, p=0.0035).
Statistically significant increases in 11 miRNAs (miR-135b, miR-141, miR-18a, miR-20a, miR-21, miR-224, miR-29a, miR-31, miR-34a, miR-92a, miR-96) were validated by qRT-PCR in the 12 CRC tissue samples compared with non-cancerous adjacent colorectal mucosa. No significant differences were observed for miR-142-3p and miR-223 (data not shown).
Employing qRT-PCR assay we then compared the expression levels of the 11 validated miRNAs in serum samples from 56 individuals: 30 stage III CRC patients and 26 healthy donors. Our data reflected that the relative levels of miR-135b, miR-141 and miR-224 were not consistently measurable in serum. miR-31 showed two peaks in the derivative melting curve and was therefore not considered for the analysis. Eventually, 7 miRNAs (miR-18a, miR-20a, miR-21, miR-29a, miR-34a, miR-92a and miR-96) were efficiently extracted and amplified from the serum of stage III CRC patients and healthy individuals. The levels of expression of these miRNAs are shown in
The identification of molecular markers that can predict prognosis is an important aim in cancer research. Accumulating evidence suggess that miRNAs play an active role in controlling the development, differentiation and cancer progression. miRNAs are well preserved in tissue samples even after formalin-fixation and paraffin-embedding and can be efficiently extracted and evaluated in tumors and also in body fluids such as serum or plasma. Therefore, currently, there is a growing interest in the study of miRNAs as molecular biomarkers with potential clinical applicability to tumor classification, cancer diagnosis and disease follow-up.
In the present study, we used a commercial platform including 887 validated human miRNAs to investigate miRNA expression profiling in stage III colon adenocarcinomas and adjacent non-tumor tissues. In agreement with previous reports, our data confirmed that numerous miRNAs are aberrantly expressed in CRC in relation to normal tissue. Heat map and cluster analyses showed that miRNA signatures were able to separate cancer tissue and non-tumor tissue in most cases. We found concordant expression of previously reported miRNAs altered in CRC (i.e. downregulation of miR-143, miR.145 or miR-195 and upregulation of miR-21, miR-17-92 cluster or miR-135b). Moreover, we identified several other miRNAs which have not previously been reported in CRC, probably since they have been more recently included in array platforms (i.e. downregulation of miR-1274a, miR-1274b, miR-1260 and upregulation of miR-1246, miR-199b-5p, miR-29b). The current results support other published studies and contribute to the better characterization of differentially expressed miRNAs in stage III CRC tumors.
The discovery of the presence of miRNAs in blood (serum or plasma) has generated great scientific and clinical interest. The expression pattern of miRNAs in the circulation could be relevant to identify the high risk of disease progression in CRC. While many studies have shown the overexpression or downregulation of specific miRNAs in CRC tissue or cell lines, there is less data concerning circulating tumor-associated miRNAs.
Therefore, we investigated the circulating levels of 11 miRNAs overexpressed in tumors and identified in serum samples of stage III CRC patients and healthy individuals. Our results identified at least 2 highly overexpressed miRNAs (miR-135b and miR-224) in tumor tissue that have not been previously detected in the serum of patients nor in healthy individuals. Future studies determining the cellular localization of these 2 miRNAs as well as an increasing understanding of miRNA transportation outside cells are warranted to address the absence of circulating miR-135b and miR-224.
The present study identified 7 miRNAs that were stable and detectable in all serum samples analyzed. To our knowledge, this is the first study to investigate circulating levels of miR-18a, miR-142-3p, miR-20a and miR-223 in serum samples of CRC patients. Our results identified miR-18a and miR-29a, whose expression was found to differ significantly between CRC patients and normal donor sera. miR-18a, located at region 13q31.3, is one of the 6 miRNAs encoded by the miR-17-92 cluster. This cluster, also named oncomir-1, is known as a body of oncogenic miRNAs and typically exhibits increased expression in tumors, including CRC. Previous studies have reported significantly elevated levels of both miR-17-3p and miR-92 in the blood of patients with CRC (
Consistent with another published report, we found that serum miR-29a was significantly higher in CRC cancer patients than in healthy individuals (
In summary, the data presented in this study confirm that miRNAs are differentially expressed between colonic tumor tissues and adjacent tissues. Our results provide a list of 43 miRNAs that are differentially expressed between stage III CRC tissue and corresponding normal tissue, which could contribute to further explain the role of miRNAs in colonic tumors with lymph node metastasis. Our data showed that not all overexpressed tumor miRNAs are detectable in serum fluids. Further investigation of miR-18a and miR-29a, preferably in large prospective studies, to validate their potential use as circulating biomarkers in CRC patients is warranted.
We are grateful to all the patients and healthy individuals who kindly allowed samples to be collected and used in this study. The authors would also like to acknowledge the nursing staff Francisca Cuadras-Lacasa and Marta Fernández-Ros of Corporació Sanitària Parc Taulí, Institut Universitari Parc Taulí-UAB for their continued technical assistance and collaboration. Finally, we thank Sarah Bonnin of the CRG Genomics Unit for the excellent technical assistance. This study was supported by ‘Fundació Benèfica Privada Barnola Vallribera Sant Josep’ from Sabadell and by ‘Fundació Parc Taulí - Institut Universitari UAB’ of the Sabadell Hospital (CIR2010029).
Unsupervised hierarchial clustering of 12 paired stage III colorectal tumor and non-tumor tissues. Using a 1.5-fold expression difference (Padj <0.05) as a cut-off level, 43 miRNAs were found to be differentially expressed in tumor vs. normal tissue. The relative miRNA expression changes are expressed by a color gradient intensity scale, as shown at the top. The lightest blue color indicates a maximal decrease and the lightest red color indicates a maximal increase in gene expression. Each column represents a separate CRC sample and each row a single miRNA. Samples are grouped into two main clusters: green cluster represents tumor tissue samples (T) and the yellow cluster represents normal colorectal tissue (N).
Differential expression of miR-18a and miR-29a in serum samples of colorectal cancer patients (CRC) and healthy individuals (CONT). The normalized Ct values (show as ΔCt values with respect to reference miR-103, miR-16 and miR-let-7a) indicate higher expression levels in CRC cases when compared with the controls. The horizontal line represents the mean value.
Demographic and clinicopathological characteristics of the colorectal cancer patients.
Variables | Tissue samples | Serum samples |
---|---|---|
No. of patients | 12 | 30 |
Median age (years ± SD) | 78.3±8 | 68.1±11 |
Gender | ||
Male | 9 | 18 |
Female | 3 | 12 |
Tumour size (cm) | ||
<4 | 2 | 12 |
≥4 | 10 | 18 |
Degree of differentiation | ||
Poor | 7 | 23 |
Moderate-Well | 5 | 7 |
Vascular invasion | ||
Yes | 4 | 10 |
No | 8 | 20 |
Perineural invasion | ||
Yes | 4 | 14 |
No | 8 | 16 |
T stage | ||
T3 | 5 | 20 |
T4 | 7 | 10 |
N stage | ||
N1 | 6 | 18 |
N2 | 6 | 12 |
M stage | ||
M0 | 12 | 30 |
Clinical stage | ||
III | 12 | 30 |
Localization | ||
Left | 6 | 9 |
Right | 5 | 17 |
Rectum | 1 | 4 |
List of 13 deregulated miRNAs identified by array profiling in stage III CRC and selected for experimental validation by qRT-PCR.
miRNA | Chromosome localization | FC | Experimentally observed target genes |
---|---|---|---|
hsa-miR-21 | 17q23 | 3.56 | ACTA2, APAF1, BMPR2, BTG2, CDC25A, CDK6, CDKN1A, E2F1, FAS, FASLG, IL6R, JAG1, MARCKS, NFIB, PIK3R1, PTEN, SERPINB5, SOCS5, SPRY2, TGFBR2, TIMP3, TPM1 |
hsa-miR-135b | 1q32.1 | 2.76 | APC, JAK2, RUNX2, SMAD5 |
hsa-miR-20a | 13q31.3 | 2.48 | BAMBI, BCL2, BCL2L11, BMPR2, CCND1, CDKN1A, E2F1, ESR1, IL8, JAK1, MICA, NCOA3, PPARG, PTEN, RB1, RBL2, RUNX1, S1PR1, STAT3, TGFBR2, TP63, VEGFA, VIM |
hsa-miR-223 | Xq22 | 2.40 | IRS1, MEF2C, RHOB, STMN1, VIM |
hsa-miR-224 | Xq28 | 2.18 | AP2M1, KLK1 |
hsa-miR-92a | 13q31.3 |
2.08 | BCL2L11, BMPR2, CDKN1A, CDKN1C, ITGA5, MAP2K4 |
hsa-miR-96 | 1q32.2 |
2.05 | FOXO1, IRS1, MITF, RYK |
hsa-miR-142-3p | 17q22 | 2.01 | BCL2L1, LIFR |
hsa-miR-34a | 1p36.22 | 1.92 | AXIN2, BCL2, CCND1, CD47, CDK6, CREB1, DLL1, E2F3, HDAC1, JAG1, MAP2K1, MET, MYC, MYCN, NOTCH1, NOTCH2, VEGFA, WNT1 |
hsa-miR-29a | 7q32.3 | 1.90 | ACVR2A, CAV2, CDC42, CDK6, COL1A1, COL1A2, COL3A1, DNMT3A, DNMT3B, DUSP2, HDAC4, MCL1, PIK3R1, PPM1D, TCL1A, TGFB3, TPM1, YY1 |
hsa-miR-141 | 12p13.31 | 1.76 | BAP1, CTNNB1, CYP1B1, EPHA2, EXOC5, MAP2K4, PRKACB, TGFB2 |
hsa-miR-31 | 9p21.3 | 1.57 | HIF1A, ITGA5, PDGFB, PPP2R2A, RDX, RHOA |
hsa-miR-18a | 13q31.3 | 1.36 | BCL2L11, CDKN1A, CTGF, E2F1, ESR1, THBS1, TPG3 |
miRNA, microRNA; CRC, colorectal cancer; qRT-PCR, quantitative real-time polymerase chain reaction; FC, fold change. Observed predicted targets of differentially expressed miRNAs were assessed using Ingenuity Pathway Analysis (IPA; Ingenuity Systems, Redwood City, CA, USA).
Mean fold change of the 7 miRNAs identified in the serum samples of CRC patients and healthy individuals.
miRNA | Type | Mean FC | P-value |
---|---|---|---|
miR-18a | Target | 2.229 | 0.038 |
miR-20a | Target | 1.378 | 0.593 |
miR-21 | Target | 2.081 | 0.087 |
miR-29a | Target | 2.811 | 0.038 |
miR-34a | Target | 3.205 | 0.055 |
miR-92a | Target | 0.444 | 0.087 |
miR-96 | Target | 2.267 | 0.107 |
miR-103 | Selected control | 1.324 | 0.496 |
miR-16 | Selected control | 0.829 | 0.593 |
miR-let-7a | Selected control | 0.910 | 0.702 |
CRC, colorectal cancer; FC, fold-change. p-values were adjusted using the Benjamini-Hochberg false discovery rate.