The expression and clinical significance of circulating microRNA-21 in serum of five solid tumors

Abstract

Purpose

MicroRNA-21 (miR-21) was reported as being overexpressed in various human cancerous tissues, but its expression in cancerous serum was not unanimous in different laboratories. On the base of optimizing experimental design and improving trial protocol, we wanted to know whether the circulating microRNA-21 was dysregulated in the common solid cancers.

Methods

Using SYBR green real-time quantitative reverse transcription-PCR, we detected the expression of circulating miR-21 in 174 patients with solid cancers and 39 normal control subjects, including breast cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer. Furthermore, we analyzed the associations between miR-21 expression and clinical features of patients.

Results

miR-21 was significantly overexpressed in human solid cancerous serum relative to normal control (P < 0.001), and its sensitivity and specificity were significantly higher than the currently used tumor markers. High miR-21 expression was not correlated with gender, age, clinical stage, and lymph node metastasis status.

Conclusion

Circulating miR-21 could serve as a potential broad-spectrum serum-based biomarker for the detection of some solid cancers.

Introduction

Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries. According to the statistics, about 12.7 million cancer cases and 7.6 million cancer deaths are estimated to have occurred in 2008 worldwide, with 56 % of the cases and 64 % of the deaths in the economically developing world (Jemal et al. 2011). Most of the cancers have non-specific clinical presentations and are difficult to diagnose. Although imaging examination and biopsy have greatly improved diagnosis, the invasive, unpleasant, and inconvenient nature of current diagnostic procedures as well as radiation damage limits their applications (Duffy 2007; Roulston 1990). There is still a great need for simple and reliable biomarker detection for objective diagnostic testing of cancers. In addition, the current serum biomarkers, such as CEA, CA153 in clinical use, are lack of sufficient sensitivity, specificity, and accuracy. Hence, we wanted to explore novel non-invasive serum-based biomarkers with higher sensitivity and specificity for tumor detection.

MicroRNAs (miRNAs, miRs) are a new class of small, single-stranded non-coding RNA genes whose final products are ~22 nt functional RNA molecules. They play important roles in the regulation of target genes by binding to 3′-UTR of target mRNA to repress their translation or regulate degradation (Bartel 2004; Griffiths-Jones et al. 2006). It is currently believed that between 10 and 30 % of all human genes are targets for miRNA regulation (Lewis et al. 2005). Genome-wide studies have demonstrated that miRNA genes are frequently located at cancer-associated genomic regions or in fragile sites, and in minimal regions of loss of heterozygosity or of amplifications, or in common breakpoint regions, indicating the potential roles of miRNAs in tumorigenesis (Calin et al. 2004; Zamore et al. 2005). It has been identified that miR-21 targets the tumor suppressor genes tropomyosin 1 (TPM1), programmed cell death 4 (PDCD4), maspin, phosphatase and tensin homolog (PTEN), and reversion-inducing cysteine-rich protein with Kazal motifs (RECK) (Asangani et al. 2008; Meng et al. 2007; Zhang et al. 2008; Zhu et al. 2007, 2008). It shows that miR-21 has an oncogenic function (Esquela-Kerscher et al. 2006). And in recent years, miR-21 has been extensively studied in different types of cancers. Although many researches have proved that miR-21 is significant higher in tissue and cells of various cancers than normal control group. The researches of miR-21 in serum were not as many as the tissue, and the results of miR-21 expression in the same cancer from different laboratories were inconsistent (Asaga et al. 2011; Heneghan et al. 2010; Shen et al. 2011; Wang et al. 2010 Yan et al. 2008; Zheng et al. 2011). We did not know whether this difference was due to random error or miR-21 expression in serum was really different from the tissue. So the aim of our study was to assess the expression of circulating miR-21 in serum of some solid cancers as well as its relationship to clinicopathological features.

Materials and methods

Serum collection

Whole blood samples of various cancers were derived from patients at Peking University Cancer Hospital (Beijing, China) in 2011. All patients, including 50 patients with breast cancer, 31 with esophageal cancer, 30 with gastric cancer, 32 with colorectal cancer, 31 with lung cancer, had been histopathologically confirmed as cancers, and blood samples were taken at the time of initial consultation before definitive surgical intervention and/or adjuvant therapy. As the number of some cancers specimens such as prostate cancer, pancreatic cancer was less than ten cases; they were not included in our study. Disease staging was performed in accordance with the AJCC/UICC stage classification (7th edition) (Edge et al. 2010). Select characteristics of study subjects were summarized in Table 1.

Table 1.

Clinical characteristics of patients for serum miR-21 analysis

Characteristic	Breast cancer	Gastric cancer	Lung cancer	Esophageal cancer	Colorectal cancer
No of patients	50	30	31	31	32
Age (years)
Mean (range)	53 (28–89)	58 (36–84)	61 (47–78)	61 (46–82)	63 (45–80)
<55	27	13	8	6	6
≥55	23	17	23	25	25
Gender
Male	0	22	22	23	17
Female	50	8	9	8	15
Clinical stage
I–II	48	11	4	10	19
III–IV	2	19	27	21	13
Nodal status
Positive	26	18	4	19	13
Negative	24	12	27	12	19

Thirty-nine serum samples from healthy individuals (30 women, 9 men, median age 46) were used as controls. None of them had previously been diagnosed with a malignancy. Ethical permission and informed consent were obtained for the use of all samples. Blood samples were centrifuged at 3,000 rpm for 10 min at 4 °C to completely remove cellular components, and the supernatant (serum) was collected. Then the sera were immediately frozen to −80 °C until they were used.

RNA isolation and reverse transcription

RNA was extracted from 350 μl serum using TRIzol^® LS Reagent (Invitrogen, Carlsbad, CA, USA) according to the protocol provided by the manufacturer. Briefly, 1.0 ml TRIzol^® LS reagent was added to the serum sample, and the mixture was incubated for 5 min at room temperature. Then 200 μl chloroform was added and shook tube vigorously by hands for 15 s and incubated at room temperature for 15 min. After centrifugation at 12,000g for 15 min at 4 °C, the supernatant was transferred to a fresh tube, and 600 μl isopropanol was added. After incubation at −20 °C for 30 min, the mixture was centrifuged at 12,000g for 10 min at 4 °C to remove the supernatant, and the RNA pellet was washed with 75 % ethanol. After removal of ethanol by centrifugation at 7,500g for 5 min at 4 °C, RNA was air-dried for 20 min and then dissolved in 60 μl RNase-free water and stored at −80 °C until further processing.

20 μl Reverse transcriptase reactions contained 12 μl purified serum RNA, 4 μl 5× RT buffer, 1 μl 10 mM each of dNTPs (TianGen, China), 1 μl 200 U/μl M-MLV reverse transcriptase (Promega, Madison, WI, USA), and 0.3 μl 40 U/μl RNase Inhibitor (TianGen), 0.3 μl 1 M DTT (TianGen), and 1 μl antisense looped primer. The mix was incubated at 16 °C for 15 min, 42 °C for 30 min, and 85 °C for 5 min. No purified RNA control (replaced by RNase-free water) was used as reverse transcription negative controls (RT-NTC).

Quantification of serum miR-21

Subsequently, real-time quantification was performed using LightCycler^® 480 Real-Time PCR System (Roche, USA) with FastStart Universal SYBR Green Master (Rox) mix kit (Roche, USA). The 20 μl PCR included 5 μl RT product, 0.5 μl reverse primer, 0.5 μl sense primer, 10 μl SYBR green. The reactions were incubated in a 96-well optical plate at 95 °C for 10 min, followed by 42 cycles of 95 °C for 15 s and 60 °C for 1 min. The fluorescence signal was acquired at 60 °C. Melting curve analysis (60-95 °C with a heating rate of 0.1 °C per second and a continuous fluorescence measurement) as well as electrophoresis of the products on 2 % agarose gels containing ethidium bromide (0.5 mg/ml) was performed at the end of PCR cycles in order to validate the specificity of the PCR product. All reactions were run in triplicate, including blank controls without cDNA. The cycle threshold (Ct) was defined as the number of cycles required for the fluorescent signal to cross the threshold in real-time quantification PCR (qPCR). After reaction, the Ct data were determined using default threshold settings, and the mean Ct was determined from the triplicate PCRs.

The purpose of the internal reference gene is to normalize the PCRs for the amount of RNA added to the reactions. As there is no current consensus on the use of house-keeping miRNAs for qPCR analysis, based on previously published results, we used miR-16 as the internal reference for quantification (Heneghan et al. 2010; Huang et al. 2010; Lawrie et al. 2008; Liu et al. 2010; Song et al. 2011; Wang et al. 2009; Wong et al. 2008; Xiao et al. 2011). The relative amount of miR-21 was normalized to miR-16. The fold change for miR-21 from patients relative to the control was calculated using the 2^−ΔΔCt method (Livak et al. 2001), where ΔΔCt = ΔCt (patient) −ΔCt (calibrator) and ΔCt = Ct (miR-21)-Ct (miR-16). A serum pool of 80 healthy donors was packed in 500-μl eppendorf tube and stored at −80 °C. In every experiment, we took one as the calibration sample.

Amplification efficiency and Pearson’s correlation coefficient

Amplification efficiency of real-time PCR was analyzed according to the protocols of Schmittgen et al. (2004). Briefly, a ten-fold dilution series of cDNA containing the tested miR-21 and the reference miR-16 were used as the template for real-time PCR to generate a plot of log concentration of the tested miRNA at different dilutions versus the corresponding cycle threshold (Ct). The slope of the linear plot is defined as  −(1/log E), where E is the amplification efficiency, and its value should approach 2 if the efficiency reaches the maximum (Livak et al. 2001).

Correlation is a technique for investigating the relationship between two quantitative, continuous variables. The nearer the scatter of points is to a straight line, the higher the strength of association between the variables. The Pearson’s correlation coefficient (R) may take any value from  −1 to +1.

Statistical analysis

Statistical analyses were performed with SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Data shown were presented as mean ± SE, and the differences of miR-21 expression levels between groups were determined by the Student’s t test. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were used to assess the feasibility of using serum miR-21 as a diagnostic tool in discriminating patients from normal control. We used the Youden index for identification of the optimal cut-off point. All P values were two-sided, and less than 0.05 was considered statistically significant.

Results

Real-time PCR amplification efficiency and linearity

The real-time PCR amplification efficiencies (E) in the exponential phase were calculated according to the equation: E = 10[ −(1/slope)]. The results showed that the amplification efficiencies of the target and reference gene (miR-21, 1.87; miR-16, 1.84) approached the maximum value, and the difference between them was less than 5 %. The best fit line also demonstrated the excellent linearity between the RNA input and the Ct values for qRT-PCR (Pearson’s correlation coefficient r > 0.99, Fig. 1).

Fig. 1.

Scatter plot of log concentration of the miR-21 at different dilutions versus the corresponding cycle threshold (Ct). Ten-fold serial dilution of miRNA was used to generate the scatter plot. Linearity was confirmed within these concentrations. Pearson correlation analysis showed correlation between the RNA input, and the Ct values was good

Intra- and inter-assay variation

To confirm the accuracy and reproducibility of real-time PCR, the intra-assay precision was determined in six repeats within one lightcycler run. Inter-assay variation was investigated in all experimental runs performed on different days. Test variability of target gene miR-21 was low in inter-test experiments (0.21 %) and in intra-test experiments (1.94 %). And the variability of reference gene miR-16 was also small (Table 2). These data fully proved that our study had a good repeatability and reliability.

Table 2.

Intra-assay and inter-assay variation of real-time PCR

	miR-21	miR-16
Inter-assay
Mean ± SE	25.665 ± 0.022	23.013 ± 0.023
CV	0.21 %	0.25 %
Intra-assay
Mean ± SE	27.812 ± 0.144	21.787 ± 0.091
CV	1.94 %	1.56 %

Confirmation of real-time PCR products specificity

DNA melting curve analysis is a relatively new analytical technique that can be applied post-PCR to provide information on the characteristics of the amplification products. A lightcycler melting curve analysis was performed, which resulted in single product-specific melting temperature (Fig. 2a). No specific PCR product was generated during the applied 42 real-time PCR amplification cycles. And the specificity of PCR products was further validated by electrophoresis using 2 % agarose with the expected size (miR-21, 64 bp; mir-16, 58 bp, Fig. 2b). Both methods confirmed the high specificity of the products.

Fig. 2.

Specificity of amplifying miR-21 by real-time reverse transcription-polymerase chain reaction. a SYBR green melting curve of miR-21 assay. b 2 % Agarose gel electrophoresis of the RT-PCR amplification products. Marker is a 100 bp DNA ladder (Life Technologies, Grand Island, New York, USA). 1–3, different patient samples; RT-NTC, reverse transcription no RNA template control; NTC, negative controls contained no template cDNA. The Ct value of RT-NTC was 35.74, more 8–10 cycles than the samples values, so it could be regarded as background interference, and the signal to noise ratio (SNR) was 256-1024:1. Therefore, it could proof the background did not affect the specificity and accuracy of real-time PCR. Single melting curve and the single band located in the desired size demonstrated the high specificity of each pair of primers

Increased levels of serum miR-21 in patients with cancers

We first analyzed the expression of miR-21 in five solid tumors, including breast cancer (BC), colorectal cancer (CRC), lung cancer (LC), esophageal cancer (EC), and gastric cancer (GC), and compared the different expression of miR-21 between these tumors and normal control group. The mean levels of serum miR-21 in these cancers were significantly higher than in healthy control (6.88, 6.10, 6.76, 4.92, 6.34 vs. 3.05, respectively, all P < 0.001, Fig. 3a). Subsequently, we evaluated whether there was a correlation between the level of miR-21 and clinical characteristics of patients. The results showed that miR-21 expression had nothing to do with gender, age, clinical stage, and lymph node metastasis status (all P > 0.05, Fig. 3b). Furthermore, the relative expression of mir-21 was not associated with other clinicopathologic features of breast cancer, such as estrogen receptor (ER), progesterone receptor (PR), menopause and KI-67 status (data not shown).

Fig. 3.

Column charts of serum levels of miR-21. Values shown were normalized to miR16. a Comparison of serum miR-21 level in cancers and normal control. The median levels of serum miR-21 in patients with cancers were significantly higher than the normal group (all *P < 0.001). b Comparison of serum miR-21 level in five cancers with differential lymph node metastasis status (positive or negative). Although the miR-21 levels in lymphatic metastasis positive group were slightly higher than that of lymphatic metastasis negative group in colorectal, esophageal and lung cancer, but there was no significant difference between them (all *P > 0.05)

Evaluation of serum miR-21 as a potential diagnostic marker

To evaluate whether the serum miR-21 can be used as a potential diagnostic marker for cancers, ROC curve analyses were performed. It was revealed that the level of serum miR-21 was a potential marker for discriminating cancer patients from healthy controls, with ROC curve areas of 0.88 (95 % CI: 0.81–0.96) for breast cancer, 0.85 (95 % CI: 0.76–0.94)for colorectal cancer, 0.88 (95 % CI: 0.80–0.96) for lung cancer, 0.74 (95 % CI: 0.62–0.85) for esophageal cancer, and 0.81 (95 % CI: 0.70–0.91) for gastric cancer. At the cut-off values of 4.58, 3.59, 3.63, 3.37, and 5.63, the sensitivity and the specificity for miR-21 in diagnosis the above five cancers were 80.0 and 87.7 %, 87.5 and 74.4 %, 87.1 and 74.4 %, 71.0 and 69.2 %, 56.7 and 94.9 %, respectively (Fig. 4). The results suggested the circulating miR-21 in serum could serve as a promising diagnostic tumor marker with high sensitivity and specificity.

Fig. 4.

Receiver-operator characteristic (ROC) curves of the miR-21 for discriminating cancer patients from normal control. a Breast cancer. b Colorectal cancer. c Lung cancer. d Esophageal cancer. e Gastric cancer

Discussion

Recently, the search for novel tumor markers for diagnosis is one of the most rapidly growing areas in cancer research (Chen et al. 2008; Duffy 2001; Thomas et al. 2001). The desired biomarker should be easily accessible such that it can be sampled relatively non-invasively, sensitive enough to detect early presence of tumors in almost all patients and absent or minimal in healthy individuals. Therefore, the discovery of circulating miRNAs in serum and plasma from cancer patients represented a new approach for diagnostic screening of tumors (Brase et al. 2010; Kosaka et al. 2010).

To our best knowledge, the present study is the first one to systematically research circulating miR-21 in so many solid cancers. The fold change was calculated using the equation 2^−ΔΔCt. Our research firmly support the notion that the circulating miR-21 is clearly detectable in serum samples, and its level is significantly higher in patients with cancers than in healthy controls. This data suggests that miR-21 has the potential to be a broad-spectrum clinically useful non-invasive serum-based biomarker in some cancers, including breast cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, offering higher sensitivity and specificity tests than those currently available for diagnosis of cancers. But no significant association was found between the levels of miR-21 and gender, age, clinical stage, and lymph node metastasis status in the five solid cancers. So at present, we can not demonstrate when the level of miR-21 increases in the process of cancer, and this area is worth our further study.

Our study is also the first using a serum pool of 80 normal human as a calibrator to calculate the ΔΔCt, so we can control the intra-precision effectively and avoid the test errors due to the random selection of the calibrator. In addition to the normal negative control that provides a control for external contamination or other factors that can result in a non-specific increase in the fluorescence signal, we also set up a reverse transcription negative control (RT-NTC) that can effectively demonstrate that the background interference caused by non-specific amplification of stem-loop primers did not affect the accuracy and reliability of the tests. Furthermore, the price of SYBR green real-time quantitative reverse transcription-PCR is significantly lower than TaqMan PCR analysis; it also provides a strong technical support and a suitable platform for circulating microRNA entering into clinical use. As most patients with pancreatic cancer, liver cancer, or prostate cancer have accepted radiation or chemotherapy before they enter into our hospital, it requires a longer time for us to collect the serum from non-treatment patients, and we will analyze the miR-21 expression in these tumors in the future study.

The limitations of our study were that our sample size was relative small, and we did not collect the long-term follow-up data. And the relationship of miR-21 with clinical characteristics should be explored by conducting further studies with larger sample numbers. Additionally, although the target genes of miR-21, such as RECK, PTEN, PDCD4, maspin, have been reported recently, the mechanism and the molecular pathway are unknown. This is an area that clearly needs further research. It would also be interesting to investigate miR-21 expression in other body fluids, such as urine and saliva, and we can also use a panel of circulating miRNAs to diagnose cancers (Hu et al. 2010). Importantly, the ability to use antagomirs to block miR-21 transcription opens avenues for the possible use of miRNAs in novel personal drug therapies for some cancers (Brown et al. 2009; Love et al. 2008).

In summary, the findings discussed above highlight the potential clinical utility of circulating miR-21 in cancer diagnosis. Our results encourage further studies on the use of miRNAs in serum samples as an easy and convenient method of cancer. Large-scale investigations and additional improvements are urgently needed to pave the way from basic research to routine clinical utilization.

Abbreviations

miR-21

MicroRNA-21

Ct

Cycle threshold

ROC

Receiver operating characteristic

RT-NTC

Reverse transcription negative controls

TPM1

Tumor suppressor genes tropomyosin 1

PDCD4

Programmed cell death 4

PTEN

Phosphatase and tensin homolog

RECK

Reversion-inducing cysteine-rich protein with Kazal motifs

BC

Breast cancer

CRC

Colorectal cancer

LC

Lung cancer

EC

Esophageal cancer

GC

Gastric cancer