Abstract
The Aim of this study was to evaluate the expression levels of miR-21, miR-221, miR-150, let-7a and miR-126a in peripheral blood of 71 patients with colorectal cancer and 80 matched healthy control individuals. We determined expression levels of these microRNAs in peripheral blood samples and used small nucleolar RNA (RNU48) as an internal control. Expression levels of miR-21 (p<0.0001) and miR-221 (p<0.0001) were significantly higher, whereas expression levels of miR-150 (p=0.0054) were significantly lower in the blood samples of patients with colorectal cancer in comparison to the control group. The combination of these three microRNAs enabled us to distinguish patients with colorectal cancer from healthy donors with a sensitivity of 80% and specificity of 74% (p<0.0001). We did not observe any correlation of the studied microRNAs with clinicopathological features of colorectal cancer, indicating that expression of these microRNAs is more likely related to the host response to the tumour than the tumour itself.
Colorectal cancer (CRC) represents a significant global health problem. Despite prevention and screening, this disease has an increasing incidence and high mortality. It is the most frequently occurring malignancy in the world, accounting for more than one million cases and 500,000 deaths per year (1, 2).
The invasive nature of current diagnostic procedures for CRC and the relatively high percentage of false-positive results limit the application of modern detection tools. The discovery and clinical evaluation of new specific and robust non-invasive biomarkers for diagnosis of CRC at an early stage, as well as for better prognosis and prediction of response to therapy, are very challenging (3). It is necessary to introduce a new non-invasive, but sufficiently sensitive method for detecting this disease. Numerous studies indicate that microRNA (miRNAs) can play an important role as reliable biomarkers for cancer detection and prognostic prediction, and even as novel targets for cancer therapy (4, 5).
The study of miRNA molecules is rapidly expanding the field of molecular biology and knowledge of their regulatory competence is becoming one of the basic building blocks of tumour biology. miRNAs are small, single-stranded, non-coding RNA molecules with a length of 19-25 nucleotides that regulate gene expression, significantly contribute to the management of the proteosynthesis, regulate the activation of genes at the post-transcriptional level, and participate in the regulation of the cell cycle. Their expression is different in healthy and disease-affected individuals (6, 7).
The detection of miRNAs circulating in blood has enormous potential because miRNAs serve as non-invasive biomarkers, not only for the diagnosis and prognosis of the disease, but also as novel predictors of response and sensitivity to cancer treatment (8).
In this study, we delt with CRC and identified types of miRNA molecules that have diagnostic relevance for this type of cancer. miRNAs, as new markers, could contribute to the improvement of existing CRC classification and lead to earlier detection and diagnosis. This could also enhance accurate prediction of the disease course.
The aim of the present study was to analyse the expression of selected microRNAs (miR-21, miR-221, miR-150, let-7a and miR-126a) and to determine whether the given values of analysed miRNAs could serve as relevant predictors for determination of disease.
Materials and Methods
Characteristics of the patients and controls. The study was approved by the Ethics Committee (EK 1010/2012) of Jessenius Faculty of Medicine in Martin and all individuals signed informed consent before participation. The study consisted of 71 patients (aged 40-86 years) with diagnosed CRC and 80 controls (aged 44-86 years). The patient group consisted of 46 men (mean age±SD= 66.54±9.25 years) and 25 women (mean age±SD=66.36±10.66 years). The control group was formed by 58 men (mean age±SD= 59.36±11.05 years) and 22 women (mean age±SD=67.64±12.28 years). There was no significant difference between the age of patients and control s (p=0.0075).
Methods. Peripheral blood (2.5 ml) was collected before therapy using PAXgene Blood RNA Tubes (Qiagen, Hilden, Germany) for in vitro diagnostic purposes. After blood collection, the tubes were kept for 2 h at room temperature. The RNA concentration and purity were confirmed by the spectrophotometric ratio using absorbance measurements at wavelengths of 260 nm and 280 nm on a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, MA, USA). Isolated RNA was stored at −80°C.
Reverse transcription (RT) was performed with 2 ng total RNA in a 10-μl reaction using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. RT primers were included in TagMan MicroRNA Assay Kit (Applied Biosystems). cDNA samples were stored at −20°C until subsequent quantitative polymerase chain reaction (qPCR).
The 20-μl reaction mixture required for the qPCR included RT product, TaqMan Universal PCR Master Mix no UNG (Applied Biosystems), Nuclease-free water and probe mixture of the TaqMan MicroRNA Assay Protocol (Applied Biosystems). All reactions were performed in duplicate.
The qPCRs were performed using an IQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Relative quantification (RQ) was carried out using the delta delta CTcomparative method.
Normalization and data analysis. In the context of finding appropriate endogenous controls, 60 samples were tested. This group consisted of 30 patients and 30 controls. In order to select the appropriate endogenous control for data normalization, three internal reference genes (three small nucleolar RNAs: RNU6B, RNU44, and RNU48) were tested. Obtained data were evaluated using GenEx software (MultiD Analyses AB, Munich, Germany) consisting of geNorm and NormFinder algorithms.
Analysis and statistical evaluation of the results were performed with Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA). Differences between miRNA levels and clinicopathological parameters were evaluated using two tests: for testing of two groups, the Mann–Whitney U-test was used, and for testing of three or more groups, the Kruskal–Wallis test was used. A value of p<0.05 was considered significant.
The rate of expression of statistically significant miRNAs in relation to CRC was evaluated by receiver operating characteristic (ROC) analysis.
Results
The results of comparing endogenous controls suitable for data normalization are shown in Figure 1. RNU48 gene was selected as a suitable reference for the normalization of miRNA expression data.
The expression levels of three analyzed miRNA were significantly different between patients and control group; miR-21 (p<0.0001), miR-221 (p<0.0001) were up-regulated, and miR-150 (p=0.0145) was found down-regulated in patients (Figure 2).
Statistically significant differences were not found in expression levels of let-7a (p=0.1404) and miR-126a (p=0.2739). However, a slightly increased level of let-7a and slightly reduced level of miR-126a in patients with CRC were observed.
By ROC analysis, it was confirmed that miR-21 and miR-221, and a combination of miR-21, miR-221 and miR-150 was able to serve as a potential diagnostic biomarker of CRC (summarized in Table I, Figure 3), although no correlation between analysed miRNA and clinicopathological characteristics of patients was found. Results are presented as the median relative expression values, with ranges defined by 25th-75th percentiles, and p-values in Table II.
Discussion
Although significant results have recently been achieved in the field of clinical diagnosis of CRC, and the mortality rate due to some malignant diseases in developed countries has decreased, the cancer incidence has remained the same. A significant factor which influences mortality rate is an early and accurate diagnosis (1). The first step in cancer treatment should be a proper characterization of an individual's tumour; this means analysis of tumour cells that are removed during surgery. In order to avoid the need for such strong intervention, new suitable non-invasive alternatives have been introduced, such as the fluid biopsy (circulating tumour cells) (9) and miRNA-based biomarkers (10). Both methods allow for early detection of various cancer types and their proper differential diagnostics.
miRNAs that circulate freely or exist in exosomal shells are less vulnerable to RNase-mediated degradation, and can be extracted from a wide variety of biological and clinical materials, collected in clinical settings (archival formalin-fixed paraffin-embedded) tissues and body fluids. Endogenous miRNAs in the blood tend to remain stable for a long time, and can withstand several repeated freeze-thaw cycles. Growing evidence indicates that cancer cells secrete miRNAs into the systemic circulation (11, 12). This fact is one of the main reasons for the study of miRNAs as biomarkers in the field of cancer research (11).
Selected suitable endogenous control using algorithm geNorm (A) and NormFinder (B).
One of the major concerns of using miRNA expression analysis in body fluids, such as blood, is an inadequate selection of internal controls for the normalization of miRNA expression (13). Consequently, there is no established housekeeping miRNA gene for normalization of the expression of circulating miRNAs in body fluids (14, 15). MicroRNA with stable expression for specific experimental conditions is most often used as a suitable endogenous control for data normalisation of miRNA expression. Reference genes of small nuclear RNAs, for example, RNU6B (16, 17, 18), RNU48 (19) or a 5S rRNA (20) are frequently used for the analysis of tissue miRNA. Mitchell et al. (21) reported that synthetic versions of miRNA from other organisms (Caenorhabditis elegans) can be beneficially used for the normalization of data for circulating miRNA and identification of an appropriate endogenous control is required prior to each experiment. Based on literature data (17, 18, 22, 23), we analysed a combination of three reference genes RNU48, RNU6B, RNU44, and the gene RNU48 was found to be an appropriate endogenous control.
Current research suggests that there is a relationship between the levels of miRNA in peripheral blood and in the tumour tissue (24, 25).
In our study, the expression of selected miRNAs was determined in samples of whole peripheral blood from patients with CRC and healthy controls.
One of the most studied miRNAs is miR-21. Its deregulation and increased expression was demonstrated in a wide variety of tumours, such as breast, pancreas, lung, liver, chronic lymphocytic leukemia, glioblastoma, and CRC (26, 27). It was found that miR-21 participates in the multistep process of colorectal tumorigenesis by regulation of the mitogen-activated protein kinase pathway, as well as in association with wingless-type protein/β-catenin signaling by targeting phosphatase and tensin homolog (PTEN), programmed cell death (PDCD), and dickkopf WNT signaling pathway inhibitor 2 (DKK2) genes (11). Kannan et al. dealt with free circulating miR-21 in the plasma and their study demonstrated that plasma miR-21 could be used to identify patients with CRC and differentiate them from healthy volunteers with high sensitivity (90%) and specificity (90%) (28).
Our analysis showed that there was a statistically significant increase in the expression of miR-21 in peripheral blood of patients with CRC, compared to a control group. This corresponds to the data found in the literature. We also found that increased expression of miR-21 differentiated patients with CRC from the control group with 71.8% sensitivity and 67.5% specificity.
Pu et al. analyzed the miR-221 levels in 103 patients with CRC and 37 healthy controls (29). The plasma miR-221 expression was increased and differentiated patients from the controls with 86% sensitivity and 41% specificity. The results showed that plasma miR-221 correlated with the expression of p53 in formalin-fixed paraffin-embedded tissue. An increased level of miRNA in this patient group was also confirmed in serum, together with miR-134, miR-146a, miR-222 and miR-23. Tsunoda et al. in their study described that kirsten rat sarcoma viral oncogene homolog (KRAS) can increase the expression of oncogenic cluster miR-221/222 and thereby contributes to overall disease progression in patients with CRC (30).
Different expressions of miR-21 (A), miR-221 (B) and miR-150 (C) in samples from patients with colorectal cancer (CRC). and controls. miR-21 and miR-221 showed significantly higher expression in patients, whereas miR-150 was significantly lower in patients.
Combined receiver operating characteristic curve for miR-21, miR-221 and miR-150.
Results of receiver operating characteristic (ROC) analysis for miR-21, miR-221, miR-150 and their combination.
The current findings suggest that miR-150 may be a key regulator of tumorigenesis and progression of CRC. Based on in vitro as well as in vivo studies, Feng et al. reported that miR-150 acts as a tumour suppressor (31). miR-150 is involved in the mechanism of the CRC at the molecular level by inhibiting the target gene proto-oncogene sequences (c-MYB). Kent et al. analysing HT29 cell line of colorectal adenocarcinoma demonstrated that miR-150 down-regulates expression of c-MYB and b-cell leukemia/lymphoma 2 (BCL2) genes (32).
Our analysis showed a decrease in miR-150 level in the peripheral blood of patients compared to controls. This finding is in accordance with previously published data dealing with miR-150 targets and suggests that this miRNA could play a key role in both inhibition (if normally expressed) and progression (if its expression is decreased) of CRC tumorigenesis.
The more we understand the etiological and biological nature of CRC, the better equipped we will be for designing effective preventive, diagnostic and therapeutic tools to help reduce the burden of this disease.
Peripheral blood miRNAs differentially expressed between patients with colorectal cancer (CRC) and healthy donors and correlation of miRNAs with clinicopathological features of CRC.
Defining the specific miRNA profiles of a patient's blood in the future could contribute not only to early diagnosis of the disease but could also enable determination of predictive factors that would lead to the most appropriate personalized and targeted treatment of patients with CRC.
Acknowledgements
This work was supported by the Ministry of Health of the Slovak Republic under the contract 2012/25-UKMA-2, by the project Competence Center for Research and Development in the Field of Diagnostics and Therapy of Oncological Diseases, ITMS: 26220220153 and by the project “Biomedical Center Martin“ ITMS code: 26220220187, the project is co-financed from EU sources.
Footnotes
Conflicts of Interest
The Authors confirm that there are no conflicts of interest in regard to this study.
- Received August 9, 2016.
- Revision received September 12, 2016.
- Accepted September 21, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
