MicroRNA (miRNA) opens up a new field for molecular diagnosis of cancer. However, the role of circulating miRNAs in plasma/serum in cancer diagnosis is not clear. The aim of this study was to investigate whether plasma miRNAs can be used as biomarkers for the early detection of colorectal carcinoma (CRC). We measured the levels of 12 miRNAs (miR-134, −146a, −17-3p, −181d, −191, −221, −222, −223, −25, −29a, −320a and −92a) in plasma samples from patients with advanced colorectal neoplasia (carcinomas and advanced adenomas) and healthy controls using real-time RT-PCR. We found that plasma miR-29a and miR-92a have significant diagnostic value for advanced neoplasia. MiR-29a yielded an AUC (the areas under the ROC curve) of 0.844 and miR-92a yielded an AUC of 0.838 in discriminating CRC from controls. More importantly, these 2 miRNAs also could discriminate advanced adenomas from controls and yielded an AUC of 0.769 for miR-29a and 0.749 for miR-92a. Combined ROC analyses using these 2 miRNAs revealed an elevated AUC of 0.883 with 83.0% sensitivity and 84.7% specificity in discriminating CRC, and AUC of 0.773 with 73.0% sensitivity and 79.7% specificity in discriminating advanced adenomas. Collectively, these data suggest that plasma miR-29a and miR-92a have strong potential as novel noninvasive biomarkers for early detection of CRC.
Colorectal carcinoma (CRC) is one of the leading causes of cancer-related death worldwide.1 The CRC incidence and mortality in China increase rapidly in the past several decades.2 Detection of early-stage cancer and precancerous lesions appears to be a key measure to reduce its mortality, and most CRC-related deaths can be preventable through early detection and removal of early-stage cancer and precancerous lesions. The advanced adenoma (a size of at least 10 mm or histologically having high grade dysplasia or significant villous components) is associated with a high risk of progression to an invasive lesion, and represents the optimal target lesion for strategies to prevent CRC.3 Thus, most CRC screening studies evaluate the detection rate of invasive CRC and advanced adenomas.3, 4 Several CRC screening tests, including fecal occult-blood testing (FOBT), colonoscopy, and stool DNA test, have been available for years.4, 5 However, none of these methods has been established as a well-accepted screening tool, because of their low adherence rates, high cost or low sensitivity. An ideal screening method should have a high sensitivity and specificity for early-stage cancers and precancerous lesions; it should be also safe and affordable so that it can be broadly accepted by patients.
MicroRNAs (miRNAs) are ∼22 nucleotide noncoding RNA molecules that regulate a variety of cellular processes including cell differentiation, cell cycle progression and apoptosis. MiRNAs have been demonstrated to play an important role in the multistep processes of carcinogenesis either by oncogenic or tumor suppressor function. Study of miRNA has been extended into many kinds of tumors, including CRC.6, 7 Those studies have revealed that miRNAs may be potential diagnostic or prognostic tools for cancer.8, 9
Tumor-associated RNAs have been described in the serum/plasma of cancer patients for more than a decade.10 More recently, several reports also showed that circulating miRNAs existed in serum/plasma.11 Accordingly, several subsequent studies have proved that miRNAs can serve as potential biomarkers for various diseases including cancer.12–14 For example, Ng et al.13 revealed that miR-92 is significantly elevated in plasma of CRC patients and can be a potential noninvasive molecular marker for CRC detection. However, the early diagnostic value of circulating miRNAs has not been reported to date. In this study, we evaluated the feasibility of using plasma miRNAs as a noninvasive diagnostic test for early detection of CRC.
Material and Methods
One hundred and fifty-seven patients with newly diagnosed advanced colorectal neoplasia, including 120 CRC and 37 advanced adenomas, were recruited in this study. Plasma samples were collected prior to definitive surgical management. Patients were excluded if they had any of the following: clinical diagnosis of familial adenomatous polyposis or hereditary nonpolyposis CRC, diagnosis of cancer at any site at the time of selection, undergoing chemotherapy or radiotherapy before blood sampling. Tumors were staged according to the tumor-node-metastasis (TNM) staging system of UICC. Fifty-nine age-matched healthy subjects were collected as the control based on their negative results of health examination including blood test, chest X-ray, abdominal ultrasound examination, fecal occult-blood testing, rectal touch, CT scan and colonoscopy. None of these controls had previously been diagnosed with any types of malignancy previously.
Informed consent was obtained from all participants for the use of their blood samples in this study. This project was approved by the Clinical Research Ethics Committee of Fudan University Cancer Hospital.
Samples processing and RNA extraction
Up to 8 ml of whole blood from each participant was collected in EDTA tube. Blood samples were centrifuged at 1,200g for 10 min at 4°C to spin down the blood cells, and the supernatant was transferred into microcentrifuge tubes, followed by second centrifugation at 12,000g for 10 min at 4°C to completely remove cellular components. Plasma was then aliquoted and stored at −80°C until use. Blood samples were processed and plasma was frozen within 4 hr of the blood draw. RNA was isolated from 400 μL plasma using the mirVana PARIS kit (Ambion, USA) following the manufacturer's protocol. To allow for normalization of sample-to-sample variation in the RNA isolation step, synthetic cel-miR-39 was added to each sample as described by Mitchell et al.11 RNAs were eluted with 100 μL of RNase-free water, and were concentrated in a final volume of 20 μL by using Eppendorf Concentrator Plus 5301 (Eppendorf, Germany). The concentration of all RNA samples was quantified by NanoDrop ND-1000 (Nanodrop, USA).
MiRNA quantification by real-time quantitative RT-PCR
Total RNA from 200 μL plasma or 100 ng total RNA (only for the evaluation test of internal control) was polyadenylated and reverse transcribed to cDNA in a final volume of 20 μL using miScript Reverse Transcription kit (Qiagen, Germanny). Real-time PCR was performed in duplicate measurements using miScript SYBR Green PCR kit (Qiagen) on the DNA Engine Opticon II system (Bio-Rad). The miRNA-specific primer sequences were designed based on the miRNA sequences obtained from the miRBase database (http://microrna.sanger.ac.uk/) (Supporting Information Table 1). Each amplification reaction was performed in a final volume of 20 μL containing 1 μL of the cDNA, 0.25 mM of each primer and 1x SYBR Green PCR Master mix. At the end of the PCR cycles, melting curve analyses as well as electrophoresis of the products on 3.0% agarose gels were performed in order to validate the specific generation of the expected PCR product. Each sample was run in duplicates for analysis. The expression levels of miRNAs were normalized to miR-16, and were calculated utilizing the 2-▵▵Ct method.15
The evaluation of internal controls for quantification of plasma miRNAs
To select a reliable internal control, we evaluated 3 candidate targets (miR-16, RNU6B and miR-191)16–19 in 48 plasma samples from 24 CRCs and 24 controls. These samples were processed under the exactly same conditions. The levels of these 3 candidates in the same mass of RNA or fixed volume of RNA elute from a given volume of starting plasma (10 μL) were separately evaluated. To further evaluate the stability of these genes in plasma by prolonged incubation at room temperature, 3 plasma specimens were randomly selected, and each one was divided into 4 parts. These sample aliquots were maintained at room temperature for 0, 1, 2 and 4 hr in nuclease-free tubes before being processed for RNA isolation. Expression of endogenous targets and spiked-in cel-miR-39 was measured in duplicates by using real-time qRT-PCR. PCR products were randomly selected for DNA sequencing.
Selection and validation of plasma miRNA markers
At first, a panel of 12 miRNAs (miR-134, −146a, −17-3p, −181d, −191, −221, −222, −223, −25, −29a, −320a and −92a) were selected based on previous reports,11–13, 20 and were measured using qRT-PCR on a small set of plasma samples (20 CRCs and 20 controls, Supporting Information Table 2). Those up-regulated markers in cancer plasma were further validated in an independent large-scale set of plasma from 80 CRC patients, 37 advanced adenomas and 39 healthy controls by using qRT-PCR. Lastly, plasma samples from another 20 CRC patients (Supporting Information Table 2) were collected before and after the tumor resection, and these samples were used to determine whether those up-regulated markers in cancer plasma were reduced after the tumor resection.
Expression levels of plasma miRNAs were compared using the Mann-Whitney U test or the Kruskall-Wallis test. Multivariate logistic regression model was established and leave-one-out cross validation to find the best logistic model. Receiver-operating characteristics (ROC) curves were established to evaluate the diagnostic value of plasma miRNAs for differentiating between tumors and controls. A p value of less than 0.05 was considered statistically significant. All statistical analysis was performed with SPSS 13.0 software (SPSS, Inc., Chicago, IL).
A total of 216 participants including 120 CRC patients, 37 advanced adenomas and 59 normal subjects were recruited into this study (Table 1 and Supporting Information Table 2). No significant differences of age or gender were found between CRCs and normal controls (p = 0.083, ANOVA; p = 0.851, χ2 test), or between advanced adenomas and normal controls (p = 0.226, ANOVA; p = 0.710, χ2 test).
Table 1. Patient information
The evaluation of the potential internal control for quantification of plasma miRNA
To reliably select an internal control for quantification of plasma miRNA, we examined the levels of miR-16, miR-191 and RNU6B by using qRT-PCR in 48 plasma samples (24 CRCs and 24 controls). To normalize the difference of extraction efficiency and reverse transcription efficiency among different samples, the plasma levels of miR-16, miR-191 and RNU6B were compared with spiked-in cel-miR-39. No significant difference was found in terms of the levels of miR-16 (p = 0.138) and RNU6B (p = 0.592) between CRCs and controls; while the levels of miR-191 in CRC plasma were higher than those in controls (p = 0.021). When normalized by matching the amount of input RNA, similar results were observed (Supporting Information Fig. 1). Then, we compared the stability of RNU6B and miR-16 in plasma, and found that miR-16 levels did not change after prolonged incubation time at room temperature (p = 0.631, Supporting Information Fig. 2A). However, RNU6B appeared to be less stable than miR-16; and both the RNU6B levels in 2 hr and 4 hr groups were significant lower than that of 0-hr group (p = 0.025 and p = 0.006) (Supporting Information Fig. 2B). In addition, sequence analysis also revealed that RNU6B molecules were polyadenylated at different bases in the 3 terminal of the RNA. Those results suggested that RNU6B was rapidly degraded at random sites after prolonged incubation at room temperature (Supporting Information Fig. 3a-b). In contrast to RNU6B, miRNAs existed in plasma with intact molecules (Supporting Information Fig. 3d-f). Therefore, miR-16 was selected as the normalization control as it displayed higher stability and abundance than RNU6B.
Preliminary marker selection on a small set of plasma samples
To identify the miRNAs which are up-regulated in CRC plasma, we first examined the expression levels of 12 miRNAs (miR-134, −146a, −17-3p, −181d, −191, −221, −222, −223, −25, −29a, −320a and −92a) based on previous reports,11–13, 20 by using qRT-PCR in 40 plasma samples (20 CRCs and 20 controls), and found that miR-29a and −92a were significantly elevated in CRC plasma when compared with normal controls (p < 0.001). The miR-17-3p levels in plasma were too low to be quantificated accurately by PCR with the mean Ct (cycle threshold) value of less than 34; while miR-221, −222 and −181d displayed poor results of melting curve analysis. No significant difference was observed in the levels of miR-134, −146a, −181d, −191, −223, −320a between CRC patients and controls (p = 0.922 for miR-134, p = 0.146 for miR-146a, p = 0.937 for miR-191, p = 0.089 for miR-223, p = 0.813 for miR-320a); while miR-25 was down-regulated in CRC plasma when compared to controls (p = 0.027).
Large-scale validation of miRNA markers on plasma samples
To further evaluate the diagnostic value of miR-29a and miR-92a identified in the stage of preliminary marker selection, the levels of these 2 miRNAs were measured on a total of 159 plasma samples including 100 CRC and 59 normal controls (Table 1). For limited sample size (especially controls) in this study, these 40 cases in preliminary marker selection were also been included in the large-scale validation. Both miR-29a and miR-92a levels were significantly up-regulated in CRC plasma than those in controls (p < 0.0001, Fig. 1, Supporting Information Table 3). ROC curve analyses revealed that both plasma miR-29a and miR-92a were valuable biomarkers for differentiating CRC from controls with an AUC (the areas under the ROC curve) of 0.844 (95% CI: 0.786–0.903) and 0.838 (95% CI: 0.775–0.900), respectively (Figs. 2a and 2b). At the cutoff value of 1.330 for miR-29a, the optimal sensitivity and specificity were 69.0% and 89.1%, respectively. At the cutoff value of 1.231 for miR-92a, the sensitivity and the specificity were 84.0% and 71.2%, respectively. Multivariate logistic regression analyses on variables including age, gender and plasma miRNAs revealed that both plasma miR-29a and miR-92a were potential biomarkers for CRC diagnosis (p < 0.0001). The odds ratio for cases with miR29a >1.330 being associated with CRC was 16.5 (95% CI: 6.752–40.693), and for cases with miR-92a >1.231 being associated with CRC was 13.0 (95% CI: 5.965–28.202). More importantly, addition of miR-92a could improve the differentiation power of miR-29a between CRC patients and controls, resulting in an increased AUC of 0.883 (95% CI: 0.830–0.937) with 83.0% sensitivity and 84.7% specificity, indicating the additive effect in the diagnostic value of these 2 miRNAs (Fig. 2e).
To prove circulating miR-29a and miR-92a in plasma are of tumor origin, their levels were measured in an independent set of 20 CRC patients (before and 7–10 days after surgical removal of the tumors. It was found that these levels of both miR-29a and miR-92a were significantly reduced in the postoperative plasma samples when compared to the preoperative samples (p < 0.05, Wilcoxon test; Fig. 3).
The diagnostic value of miR-29a and miR-92a for advanced adenomas
To test the diagnostic value of miR-29a and miR-92a in early lesion in the development of CRC, we measured the expression of these miRNAs in plasma samples of 37 advanced adenomas, and found a significant elevated expression when compared to those in normal controls (p < 0.0001 for miR-29a, p < 0.0001 for miR-92a). ROC curve analyses also showed that both of the 2 miRNAs could differentiate advanced adenomas from normal controls with an AUC of 0.769 for miR-29a (95% CI: 0.669–0.869) and 0.749 for miR-92a (95% CI: 0.642–0.856), respectively (Figs. 2c and 2d). At the cutoff value of 1.210 for miR-29a, the sensitivity and the specificity were 62.2% and 84.7%, respectively. At the cutoff value of 1.682 for miR-92a, the sensitivity and the specificity were 64.9% and 81.4%, respectively. Combination ROC analyses also revealed a litter increased AUC value of 0.773 (95% CI: 0.669–0.877) with 73.0 % sensitivity and 79.7% specificity. Multivariate logistic regression analyses revealed that both plasma miR-29a and miR-92a were potential molecular markers for advanced adenomas (p < 0.0001) after adjusted for patients' age and gender. The odds ratio for cases with miR29a >1.210 being associated with advanced adenomas was 12.20 (95% CI: 4.350–34.237), and for cases with miR-92a >1.682 was 4.56 (95% CI: 1.893–10.988).
Relationship between plasma levels of miR-29a and miR-92a and clinical characteristics
Next, we examined the correlation between the expression of miR-29a and miR-92a with clinical parameters. No significant association was found between these 2 miRNAs and gender, age, nodal status, tumor location, tumor size or histology (p > 0.05, data not shown), while the levels of miR-29a demonstrated an elevation trend in patients with more advanced T-classification (p = 0.075) or positive nodal status (p = 0.052).
Patients were further stratified based on the diagnosis of advanced adenoma and the TNM staging of CRC, and advanced adenoma was considered the least advanced and TNM Stage IV was most advanced. As shown in Figure 4, any of the 5 groups has elevated plasma miR-29a and miR-92a when compared with the controls (p < 0.0001). Advanced adenomas expressed lower levels of plasma miR-29a and miR-92a as compared with CRC (p = 0.039 for miR-29a, p = 0.080 for miR-92a), and miR-29a is expressed at higher levels in more advanced tumors (p = 0.051). No significant difference was observed between the miR-92a levels among the 5 groups (p = 0.309).
In this study, we found that the levels of miR-29a and miR-92a in plasma samples from patients with advanced colorectal neoplasia were significantly higher than those in healthy controls. Both miR-29a and miR-92a had significant diagnostic value for CRC and yielded AUC of 0.844 and 0.838, respectively. Combined ROC analyses using these 2 targets could yield an increased AUC of 0.883 with 83.0% sensitivity and 84.7% specificity in discriminating CRC from normal controls. More importantly, these 2 miRNAs also discriminated advanced adenomas from controls and yielded an AUC of 0.773 with 73.0% sensitivity and 79.7% specificity, suggesting their potential value for early detection of CRC. To our knowledge, it is the first report to evaluate the diagnostic value of plasma miRNAs on early detection of cancer.
Results from recent studies revealed that miRNAs are potential diagnostic biomarkers and prognostic factors in cancers.6 Tumor-derived miRNAs was firstly described in plasma by Mitchell et al. in year 2008,11 which has attracted significant interest in the field.12, 13, 20–22 The first serum miRNA biomarker discovered was miR-21. Lawrie et al.23 found that patients with diffuse large B cell lymphoma had high serum levels of miR-21, which associated with increased relapse-free survival. Mitchell et al. found that plasma miR-141 could efficiently identify prostate cancer patients, and revealed for the first time that circulating miRNAs may have important value for cancer diagnosis. Around the same time, Chen et al. also demonstrated that specific serum miRNA profiles can be identified in serum from patients with lung cancer, CRC, and diabetes patients.12 Although the clinical significance of these findings has not been elucidated in detail, those findings demonstrated that circulating miRNAs could be noninvasive diagnostic or prognostic markers for cancer.
The first over-expressed miRNA in plasma from CRC patients was miR-92a, which is part of the mir-17-92 gene cluster, located on chromosome 13q13. As a known oncomir, mir-17-92 cluster could promote cell proliferation, suppressed apoptosis, induce tumor angiogenesis and accelerated tumor progression.24–27 The over-expression of miR-92a has been observed in CRC,6, 7, 27, 28 lung cancer,29 hepatocellular carcinoma,30 B-cell lymphoma,31 thyroid cancer32 and nasopharyngeal carcinoma,33 suggesting its important role in tumorigenesis. Recently, Ng et al. reported for the first time that plasma miR-92a was a potential marker for CRC diagnosis with 89% sensitivity and 70% specificity. Although a different normalization control was used and higher ratio of patients with TNM Stage I was enrolled (26% vs. 6.7%) in this study, our results were comparable with that of Ng et al. In addition, the up-regulation of miR-92a was also observed in the serum of ovarian cancer patients.20 Interestingly, contrary result was also reported in leukemia. Tanaka et al. revealed that miR-92a dramatically decreased in the plasmas of acute leukemia patients using miR-638 as the internal control, while their result showed that miR-92a was up-regulated in leukemia cells.21 The inconsistent results may due to the different quantification methods or different diseases.
Although miR-29a may act as a tumor suppressor as suggested from a study on lung cancer,34 we demonstrated that miR-29a is significantly upregulated in plasma of patients with advanced colorectal neoplasia. Gebeshuber et al.35 revealed that miR-29a was up-regulated in mesenchymal, metastatic RasXT cells relative to epithelial EpRas cells, and could suppress the expression of tristetraprolin. They also observed enhanced miR-29a and reduced tristetraprolin levels in breast cancer patient samples. These results indicate that miR-29a can act as either oncogene or tumor suppressor, depending on the circumstances. In a recent study conducted by Resnick et al., miR-29a was also significantly over-expressed in the serum from ovarian cancer patients.20 Clearly, the exact role of miR-29a in cancer needs to be fully investigated in the future.
It has been demonstrated that timely recognition and removal of adenomatous polyps significantly decrease the risk of death from CRC in affected patients. Advanced adenomas, which have the greatest risk to develop into CRC, could be cured just by radical operation. We evaluated the value of plasma miRNAs on diagnosis of advanced adenomas, and our results revealed that both miR-29a and miR-92a have significant diagnostic value for advanced adenomas. Since these 2 circulating miRNAs are significantly elevated in cancer patients from the early stages of cancer and in relatively small tumors, it may be proposed as an early detection test for CRC.
Normalization is a key step for the accurate quantification of RNA levels with qRT-PCR. A common problem in the circulating miRNA research is that no consensus internal controls have been established. We evaluated 3 potential controls for miRNA, miR-16, RNU6B and miR-191,16–19 and found that miR-16 and RNU6B were identified as potential controls given consistent expression across all patients and controls. Given the small snRNAs are longer than miRNAs and have different function and structure, the existence pattern and stability of RNU6B in plasma may be different from miRNAs.11, 12 Our results proved our hypothesis and revealed that RNU6B is unstable at room temperature, so we use miR-16 as the internal control for plasma miRNA quantification. In other studies on different tumors, including CRC,13 breast cancer,22 ovarian cancer20 and leukemia21 miR-16 was also presented in plasma/serum at similar levels across normal controls and patients. However, given the early stage of circulating miRNA research, additional studies are necessary for the identification of an accurate normalization protocol, and empirical validation of stable endogenous control miRNAs for different diseases or tissues studied is also needed.
In addition to the normalization of qRT-PCR data, appropriate control is also a key issue for diagnostic studies. However, it is practically difficult to ensure that the control group is indeed truly healthy, so detailed information on personal health should be collected when selecting controls. Large sample size may also be helpful to eliminate the potential sampling error.
Several tests have been suggested for CRC screening. However, no single test or strategy for CRC screening can be endorsed on the basis of currently available data; and several approaches are included as options in the screening guidelines.4 Colonoscopy is currently the most reliable screening tool for CRC; the invasive nature brings about low adherence rates. FOBT, another one common used screening procedure, is limited by low sensitivity and specificity. Stool DNA test, showing acceptable sensitivity for CRC, has major limitations for its labor-intensive feature and high cost. Although the diagnostic efficiency of these 2 plasma miRNAs (miR-29a and miR-92a) may not be optimal, a panel of plasma miRNA markers may improve the sensitivity and specificity of this assay for CRC screening. Patients with increased plasma miRNAs might prompt more accurate and specific clinical examinations such as colonoscopy.
In conclusion, plasma miR-29a and miR-92a appear to be novel biomarkers for early detection of CRC. Our data serve as basis for further investigation, preferably in large prospective studies before these 2 miRNAs can be used as a noninvasive screening tool for colorectal neoplasia in routine clinical practice.
The authors thank for Dr. Jingsong Liu for critical reading of this article.