http://informahealthcare.com/bmk ISSN: 1354-750X (print), 1366-5804 (electronic) Biomarkers, 2014; 19(7): 590–596 ! 2014 Informa UK Ltd. DOI: 10.3109/1354750X.2014.955059
RESEARCH ARTICLE
Circulating microRNA-21 (MIR-21) and phosphatase and tensin homolog (PTEN) are promising novel biomarkers for detection of oral squamous cell carcinoma WenHao Ren1*, Cui Qiang1*#, Ling Gao1,2*, Shao-Ming Li1, Lin-Mei Zhang1, Xiao-Long Wang1, Jian-Wei Dong1, Cheng Chen1, Chang-Yang Liu1, and Ke-Qian Zhi1
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1
Department of Oral and Maxillofacial Surgery, College of Stomatology, Xi’an Jiaotong University Xi’an, Shaanxi, P. R. China and 2Key Laboratory of Environment and Genes Related to Diseases, College of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi, P. R. China Abstract
Keywords
The purpose of this study was to investigate the potential of the blood levels of MIR-21 and PTEN as novel biomarkers for oral squamous cell carcinoma (OSCC). We initially detected MIR-21 and PTEN using real-time RT-PCR from 90 blood samples and then compared their results with expression in cancer tissues from 10 OSCC patients. Finally, we examined the relationship between these markers and clinical parameters. Blood MIR-21 and PTEN had significant diagnostic value for OSCC and, to an extent, correlated with the expression level of tumour MIR21 and PTEN. In addition, they were associated with differentiation and nodal status. Thus circulating MIR-21 and PTEN might represent new complementary tumour markers for OSCC.
Circulating biomarkers, MIR-21, OSCC, PTEN
Introduction Oral squamous cell carcinoma (OSCC) is a predominant type of head and neck squamous cell carcinoma (HNSCC), which is an aggressive and lethal malignancy (Chiu et al., 2013). OSCC is traditionally considered a major problem worldwide due to its extreme high prevalence (Maryam et al., 2012; O-Charoenrat et al., 2001; van der Waal et al., 2011) and the 5-year survival rate of patients with OSCC is poor (Ching-Yu et al., 2013; Markopoulos, 2012). It is known to be a devastating disease with treatment frequently leaving patients disfigured, and with diminished quality of Life (compromised speech, swallowing difficulties) and has debilitating adverse effects of radiation and chemotherapy (Denaro et al., 2011; Nagalakshmi, et al., 2014). The lack of early diagnostic methods is the main reason for poor prognosis in oral cancer patients, with over 60% of patients presenting in stages III and IV (Lingen et al., 2008). Thus, new approaches that can complement and improve current strategies for oral cancer diagnosis are urgently needed. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the translation of coding mRNAs by imperfectly acting on the 30 -untranslated regions (30 UTRs) of
*These authors contributed equally to this work. #Cui Qiang is responsible for statistical design/analysis. E-mail: [email protected] Address for correspondence: Ke-Qian Zhi, MD, PhD, Department of Oral and Maxillofacial Surgery, College of Stomatology Xi’an Jiaotong University, Number 98, Xiwu Road, Xi’an, ShaanXi 710004, P.R. China. Tel: 86-15309239634. E-mail: [email protected]
History Received 13 June 2014 Revised 12 August 2014 Accepted 12 August 2014 Published online 1 September 2014
the mRNA target (Esquela-Kerscher & Slack, 2006) and are stably detectable in plasma and serum (Calin et al., 2006; Chen et al., 2008; Mitchell et al., 2008). Several investigations have demonstrated expression of miRNAs was altered in a variety of cancers and have suggested that miRNAs have the potential to be useful diagnostic and prognostic biomarkers for multiple cancers such as lung cancer (Hu et al., 2010), leukaemia (Lawrie et al., 2008), pancreatic cancer (Wang et al., 2009), prostate cancer (Mitchell et al., 2008), colorectal cancer (Ng et al., 2009), ovarian cancer (Resnick et al., 2009), breast cancer (Heneghan et al., 2010). MIR-21, encoded on the X chromosome, is an important member of the miRNA family. It has been reported that MIR-21 is overexpressed in breast cancer (Asaga et al., 2011), lung cancer (Liu et al., 2012), prostate cancer (Zhang et al., 2011), ovarian cancer (Resnick et al., 2009), hepatocellular carcinoma (Li et al., 2011), lymphoma (Lawrie et al., 2008). To date, the expression of MIR-21 in OSCC and whether it can be used for a potential non-invasive molecular marker have not been evaluated. We hypothesised that circulating MIR-21 concentrations could act as a potentially useful biomarker in patients with OSCC. If this hypothesis holds true, it would signify a major breakthrough in OSCC management, bringing us ever closer to finding a novel, sensitive, and non-invasive biomarker for OSCC. Accordingly, we established a procedure to measure the circulating MIR-21 concentration and assessed whether it could screen cancer patients and monitor tumour dynamics and whether the levels were associated with clinic-pathological factors in OSCC patients (Li et al., 2011). In addition, the level of phosphatase and tensin homologue (PTEN), a
MIR-21 and PTEN biomarkers for OSCC
DOI: 10.3109/1354750X.2014.955059
direct target gene of MIR-21, was measured from the same blood samples to investigate whether PTEN levels are inversely related with MIR-21 levels in blood.
Materials
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Patients and samples We collected blood samples from 58 patients with histologic diagnosis of OSCC at the Department of Surgery, Stomatology Hospital of Xi’an Jiaotong University College of Medicine and from 32 matched cancer-free donor volunteers enrolled at the same institute with no previous history of any cancer for more than 2 years. To examine the relationship between MIR-21 and PTEN, we initially investigated the level of blood MIR-21 and PTEN in all cases. Thereafter, we designed an experiment to investigate whether the blood MIR-21 and PTEN could reflect tumour MIR-21 and PTEN. Fresh tissue samples were acquired from 10 patients whose MIR-21 concentrations exceeded the highest level of the donor volunteers and whose PTEN concentrations were below the lowest value of the donor volunteers. We compared the MIR-21 and PTEN expression in primary lesions with those in the blood samples. All subjects were of the same ethnicity (the Han nationality). Relevant demographic and clinicpathological details were obtained from our prospectively maintained database. A total of 90 participants were recruited into this study. No significant differences in age or gender were found between OSCC patients and normal controls (p ¼ 0.791 and p ¼ 0.883, respectively, 2 test). The differentiation of tumours was assessed by one experienced pathologist. All blood samples were collected before any treatment in EDTA-coated blood collection tubes and stored at 80 C until further processing; tissue samples were also kept at 80 C. The aim of the study was explained in detail to all participants, and written informed consent was obtained from all patients prior to their samples in the present study. The study protocol has been approved by the Hospital Ethical Committee of Stomatology Hospital of Xi’an Jiaotong University College of Medicine. RNA extraction Total RNA was isolated from 1 ml of whole blood using the E.Z.N.A.Ô blood RNA kit (AmbionÕ , Life Technologies, Paisley, UK). The extracted RNA was ultimately eluted in 30 ll of DEPC-treated water according to the manufacturer’s directions. Tissue total RNA was extracted with Trizol (Invitrogen, Life Technologies, Carlsbad, CA) following the manufacturer’s instructions. NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE) was used to determine the purity and concentration of RNA by measuring the optical density (A260/28042.0; A260/ 23041.8). The samples were either preserved at 80 C or further processed. Protocols for the detection of MIR-21 and PTEN The concentration of MIR-21 and PTEN in blood was determined with quantitative real-time reverse transcriptionpolymerase chain reaction (RT-PCR). For PTEN analyses, the
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first-strand cDNA was generated using the PrimeScriptÔ RT reagent Kit following the manufacturer’s directions, while the mature MIRNA was reverse transcribed using MIRNA-specific primers for quantification of MIR-21. Quantitative real-time RT-PCR was performed using SYBR Premix Ex Taq II on an FTC-3000Ô System (Funglyn Biotech Inc., Toronto, Canada). DNA was amplified by a cycling programme which comprised preliminary denaturation at 95 C for 5 min followed by 40 cycles of denaturation at 95 C for 10 s, annealing at 60 C for 30 s and elongation at 72 C for 1 s and by a final elongation step at 72 C for 10 min. All samples were processed in triplicate. Small nuclear RNA U6 (snRNA U6) was the internal control (Wang et al., 2012) and was used to normalise MIR-21 values, while -actin was used as an endogenous control to standardise PTEN expression in the PCR. Relative quantitation of MIR-21 and PTEN was calculated using the 2DDCt method (Schmittgen & Livak, 2008). Statistical analysis Statistical analysis was done using the software package SPSS 19.0 for Windows (SPSS Inc., Chicago, IL). Data were expressed as the mean ± SD. Student’s t-test was used to compare the difference in blood MIR-21 and PTEN concentration between the cancer group and the healthy group, and analysis of variance (ANOVA) was used to analyse the correlation between the expression of MIR-21 and PTEN and the clinic-pathological features of the patients. In addition, a regression analysis was used to evaluate possible risk factors such as age, sex, tumour size, smoking, histology, site, and nodal status involving the gene expression of biomarker genes. The association between blood MIR-21 and PTEN concentration was analysed using Pearson’s correlation coefficient. Receiver operating characteristic (ROC) curves were constructed and the area under the curve (AUC) was calculated to determine the ability of the blood biomarker levels to discriminate between patients with OSCC and healthy control subjects. The Youden index (sensitivity + specificity 1) was used to determine the optimal cut-off point (Akobeng, 2007). All tests were two-tailed and a p value 50.05 denoted the presence of a statistically significant difference.
Results The expression and correlation between MIR-21 and PTEN in blood To explore the potential value of circulating MIR-21 and PTEN in OSCC, we first investigated the levels of MIR-21 and PTEN, in blood samples of OSCC patients (n ¼ 58) and donor volunteers (n ¼ 32). No significant difference was discovered in terms of the levels of snRNA U6 and b-actin between controls and OSCC patients. The expression levels of MIR-21 in blood were significantly higher in OSCC patients than in donor volunteers (p50.001) (Figure 1A), while PTEN concentration tend to be higher in controls (p50.001) (Figure 1B). Next, we examined the correlation between circulating MIR-21 levels and PTEN levels using Pearson’s correlation coefficient, and the results showed that there was
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Figure 1. Circulating levels of MIR-21 and PTEN in blood from OSCC patients and donor volunteers. Increased MIR-21 (A) and MIR-21/PTEN (C) levels in blood in OSCC patients compared with controls (Student’s t-test, p50.0001), while PTEN concentration (B) tend to be higher in controls (Student’s t-test, p50.0001). (D) The correlation between MIR-21 and PTEN in blood from 58 patients with OSCC.
Table 1. Expression of MIR-21 and PTEN in oral cancer tissue versus those in normal tissue.
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Case 10 Rate of higher expression level in oral cancer tissue Rate of lower expression level in oral cancer tissue
MIR-21
PTEN
1.0446 1.3495 1.0823 1.8139 1.0803 1.2224 1.0205 1.3289 1.1021 0.5650 90%
0.3850 0.2952 0.1507 0.1732 0.3850 0.2857 0.1460 0.2133 0.2808 10.5692 10%
10%
90%
negative significant correlation between them (r ¼ 0.555, p50.001) (Figure 1D). Relationship of the expression of MIR-21 and PTEN between blood and primary oral cancer tissue The levels of MIR-21 and PTEN in cancer tissues compared with adjacent normal tissues were measured in 10 patients who had higher levels of blood MIR-21 and lower levels of blood PTEN than the donor volunteers. MIR-21 and PTEN obtained from fresh tissue were amplified, and found to be of good quality for amplification through a quality verification process (data not shown). The level of MIR-21 in primary oral cancer
tissues was higher than that in normal tissues in nine of the 10 patients analysed (90%), whereas PTEN was lower in nine patients (90%) (Table 1). These results indicated that similar profiles of MIR-21 and PTEN were found in primary cancer tissues and blood samples from most patients. Therefore, the level of blood MIR-21 and PTEN might reflect the expression level of tumour MIR-21 and PTEN to a certain extent. Differentiating power of MIR-21, PTEN and the combination of MIR-21 and PTEN for OSCC To further identify the potential diagnostic value of the blood levels of MIR-21 and PTEN for OSCC patients, the levels of MIR-21 and PTEN were measured for a total of 90 blood samples, including 58 OSCC and 32 normal controls. ROC curve analyses revealed that blood MIR-21 and PTEN were valuable biomarkers for distinguishing OSCC patients from controls, and the area under the curve (AUC) for MIR-21 and PTEN was 0.788 (95% CI: 0.692–0.883) (Figure 2A) and 0.731 (95% CI: 0.624–0.838) (Figure 2B), respectively. At a cut-off value of 9.646 for MIR-21, the optimal sensitivity and specificity were 62.1% and 90.6%, respectively. The odds ratio for MIR-21 9.646 being associated with OSCC patients was 15.818 (95% CI: 4.304–58.317). As for PTEN, at a cut-off value of 8.875, the optimal sensitivity and specificity were 65.5% and 71.9%, respectively. The odds ratio for PTEN 8.875 being associated with patient was 4.856 (95% CI: 1.893–12.453). To examine a more sensitive diagnostic biomarker in blood, we analysed the ratio of MIR-21/PTEN as a combined
MIR-21 and PTEN biomarkers for OSCC
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DOI: 10.3109/1354750X.2014.955059
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Figure 2. Receiver operating characteristics (ROC) curve analysis using blood MIR-21 (A), PTEN (B) and MIR-21/PTEN (C) for discriminating OSCC patients from healthy controls. Blood MIR-21 showed the greatest AUC (the areas under the ROC curve) of 0.788 (95% CI: 0.692–0.883) with 62.1% sensitivity and 90.6% specificity (A), and blood PTEN yielded AUC of 0.731 (95% CI: 0.624–0.838) with 65.5% sensitivity and 71.9% specificity of (B), while blood MIR-21/PTEN yielded AUC of 0.774 (95% CI: 0.677–0.872) with 84.5% sensitivity and 62.5% specificity of (C).
biomarker. The ratio of MIR-21/PTEN in blood showed a significantly elevated level when compared to those in normal controls (p50.001) (Figure 1C). The ROC analysis for the ratio of MIR-21/PTEN did not improve the stratification power characterised, as it was characterised by an AUC of 0.774 (Figure 2C) (95% CI: 0.677–0.872) for the ratio of MIR-21/PTEN. In this model, an optimal cut-off point was found to be 1.332 and showed a better sensitivity of 84.5% with a specificity of 62.5%. These results suggest that MIR-21 and PTEN in blood are new complementary tumour markers for OSCC. Relationship between blood level of MIR-21 and PTEN and clinical characteristics The results of the correlation analysis between MIR-21 and PTEN blood levels and clinic-pathological features in 58 OSCC patients are shown in Table 2. The expression of MIR21 and PTEN is displayed as the mean ± SD. No significant association was observed between the expression of MIR-21 and PTEN and age, sex, tumour size, smoking history, and
tumour location (p40.05). Relative MIR-21 blood levels showed a trend toward elevation in patients with poormoderately differentiated tumours compared with welldifferentiated tumours (p ¼ 0.001) and in patients with lymph node involvement compared with patients without lymph node involvement (p50.001). However, the levels of PTEN demonstrated a downward trend in tumours with decreased differentiation (p ¼ 0.01) or positive nodal status (p ¼ 0.003). Using regression analysis, as displayed in Table 3, the possible risk factors affecting gene expression of the two biomarkers were assessed. These results suggest that only the histology and nodal status for OSCC may be significant risk factors for gene expression of MIR-21 and PTEN.
Discussion To our knowledge, this is the first reported investigation that directly obtained total RNA from whole blood. There are many advantages, including streamlining the procedure, reducing time and overall cost, minimising human and
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Table 2. Correlation between the level of MIR-21 and PTEN in the blood of patients with oral squamous cell carcinoma (OSCC) and clinicopathological factors. Blood MIR-21 Level (DCt)
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Variable Age(years) Mean(range) 550 50 Sex Male Female Tumour size 2 25 Smoking Non-smokers Current smokers Histology Well Poor-moderate Site Gingiva Buccal Tongue Others Nodal status Negative Positive
Blood PTEN level (DCt) a
Patients (n ¼ 58)
Mean ± SD
p value
Mean ± SD
p valuea
61 (25–92) 16 (27.6%) 42 (72.4%)
9.36 ± 1.18 9.49 ± 1.15
0.700
9.92 ± 1.49 9.16 ± 1.45
0.086
39 (67.2%) 19 (32.8%)
9.49 ± 1.24 9.41 ± 0.99
0.806
9.45 ± 1.56 9.30 ± 1.27
0.720
19 (32.8%) 39 (67.2%)
9.68 ± 1.14 9.35 ± 1.16
0.313
9.10 ± 1.34 9.55 ± 1.51
0.271
31 (53.4%) 27 (46.6%)
9.36 ± 1.23 9.58 ± 1.20
0.477
9.24 ± 1.54 9.60 ± 1.36
0.356
30 (51.7%) 28 (48.3%)
9.94 ± 0.99 8.94 ± 1.12
0.001*
8.94 ± 1.40 9.91 ± 1.44
0.01*
16 9 18 15
(27.6%) (15.5%) (31.0%) (25.9%)
9.58 ± 1.07 9.73 ± 1.37 9.15 ± 1.03 9.53 ± 1.28
0.582
9.13 ± 1.33 8.98 ± 1.26 10.04 ± 1.10 9.19 ± 1.91
0.166
33 (56.9%) 25 (43.1%)
9.61 ± 1.20 8.85 ± 0.90
50.001*
8.92 ± 1.52 10.04 ± 1.12
0.003*
a p values are from Student’s t-test or one-way ANOVA. *Indicates a significant difference (p50.05).
Table 3. Regression analysis to assess the possible risk factors affecting gene expression of the two biomarkers in blood samples of OSCC patients and donor volunteers.
Risk factors Age Sex Tumour size Smoking Histology Site Nodal status
MIR-21
PTEN
OSCC patients donor volunteers volunteers
OSCC patients donor volunteers
0.286 0.997 0.949 0.846 0.003* 0.704 0.002*
0.454 0.097 0.147
0.268 0.918 0.943 0.900 0.008* 0.748 0.002*
0.158 0.742 0.110
*The p values of the differences of DCt values in blood samples of OSCC patients and donor volunteers were shown and donor volunteers do not have information for tumour size, histology, site, and nodal status.
mechanical errors, and avoiding nucleic acid loss. As we hypothesised, the up-regulation of MIR-21 and down-regulation of PTEN expression between the blood of OSCC patients and controls reached statistical significance in our study. Previous findings have indicated that elevated levels of the circulating cancer-associated nucleic acids in pre-operative samples were potentially due to the release by cancer cells (Sidransky et al., 1997; Stroun et al., 1989) and Li et al. have reported that the level of MIR-21 was inversely correlated with PTEN expression in OSCC cancer tissues (Li et al., 2009). These two points led us to speculate that there was negative relationship between circulating MIR-21 and PTEN in OSCC patients. We found that the overall
correlation coefficient between MIR-21 and PTEN levels for all patients was 0.555 (p50.001), in accordance with our expectation. It has been verified that MIR-21 modifies N-methyl-N-nitro-N-nitrosoguanidine-induced gastric tumourigenesis by targeting FASLG and BTG2 (Yang et al., 2014) and promotes cholangiocarcinoma growth by targeting 15-PGDH (Lu et al., 2014). Moreover, MIR-21 inhibits the PI3K/Akt-NF-kB signalling pathway and promotes apoptosis of gastric cancer cells induced by celastrol (Sha et al., 2014). Additionally, MIR-21 can contribute to tumour initiation, progression, metastasis, and chemo- or radio-resistance as an oncomiR by modulating PTEN and PDCD4 expression and PTEN-dependent pathways in several human cancers, including human hepatocellular cancer, ovarian epithelial carcinomas, and non-small cell lung cancer cells (Liu et al., 2013; Lou et al., 2010; Meng et al., 2007). Our findings provide a firm basis for further investigation regarding their roles in OSCC. To confirm whether circulating MIR-21/PTEN could be released from primary oral tumours, we performed a comparison between expression of MIR-21 in blood and primary tumour tissues. The results indicated that blood and primary OSCC tissue samples showed profiles with respect to expression of MIR-21/PTEN in almost all cases. One patient, however, showed an opposite pattern of nucleic acid levels, high blood PTEN with a low expression in OSCC tissue and low blood MIR-21 with a high expression in cancer tissue. Although the reason for this discrepancy is not clear at present, one possible explanation for this finding may be the heterogeneity of the primary tumours. In the present study, the expression correlation of MIR-21 and PTEN between blood
MIR-21 and PTEN biomarkers for OSCC
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DOI: 10.3109/1354750X.2014.955059
and tumour tissue has not been absolutely elucidated. Some researchers have assumed that circulating nucleic acid levels might not always directly relate to changes occurring in tumour tissues but may also reflect indirect effects and secretion by non-tumour cells. Thus, further investigation is needed to clarify this relationship and evaluate whether circulating MIR-21 and PTEN could be useful for clinical diagnosis for OSCC. To evaluate the utility of MIR-21 and PTEN as novel biomarkers, ROC analysis was conducted to evaluate the discriminating power for patients with OSCC. The AUC for MIR-21 and PTEN were 0.788 and 0.731, respectively, by ROC analysis. Furthermore, we discovered that the ratio of the circulating levels of MIR-21/PTEN did increase the ability of nucleic acids to discriminate cancer cases from controls with a sensitivity of 84.5%, which was above the sensitivity of either MIR-21 or PTEN alone and could be satisfactory for clinical application. Previous studies have reported that determining the expression ratios of genes or miRNAs may be a useful technique to improve diagnostic potential (Avissar et al., 2009; Gordon et al., 2002). So far, there have been few published studies that analyse the ratio of circulating miRNAs levels and gene levels to improve the sensitivity and specificity of diagnostic biomarkers. Surprisingly, in our study, we confirmed that MIR-21 and PTEN levels in blood were misregulated and that their ratio had important value for characterising OSCC. MIR-21 has grounds for serious investigation because it has increased expression in many cancers, such as breast cancer (Avissar et al., 2009) and lung cancer (Keller et al., 2011), and has been considered to have an important role in diagnosis, metastasis, progression, treatment, and prognosis of malignancies. Li et al. reported that MIR-21 was overexpressed in tongue squamous cell carcinoma and indicated poor prognosis (Li et al., 2009). Consistent with their results, our data showed that there was a higher expression of MIR-21 in OSCC patients than that in control cases. In addition, our data show a correlation between the MIR-21 level and both lymph node metastasis and advanced clinical stage, which is consistent with the study by Liu et al. (2012) in lung cancer. Thus, blood MIR-21 could be used as a potential biomarker in the diagnosis of OSCC patients with lymph node metastasis or advanced clinical stage. In diagnostic studies, one key issue is how to choose appropriate controls, and it is practically difficult to ensure that the control group is indeed truly healthy; therefore, detailed information on personal health should be obtained when selecting controls. An expanded number of samples may also be useful to eliminate potential sampling error and increase the confidence in these findings.
Conclusions In summary, circulating levels of MIR-21 and PTEN might provide new complementary tumour markers for OSCC. Our study serves as a basis for further investigation, preferably in large prospective studies that would enable application as a non-invasive screening tool for OSCC in routine clinical practice.
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Declaration of interest This work was supported in part by the Project of Technological Innovation Planning in Shannxi Province (grant 2012KTCL03-17), the Specialised Research Fund for the Doctoral Program of Higher Education of China (grants 20120201110063 and 20130201120054), the Natural Science Foundation of China (grant 81272957), and the Fundamental Research Funds for the Central Universities (grant xjj2013057). The authors declare that there is no conflict of interest.
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Resnick KE, Alder H, Hagan JP, et al. (2009). The detection of differentially expressed microRNAs from the serum of ovarian cancer patients using a novel real-time PCR platform. Gynecol Oncol 112: 55–9. Schmittgen TD, Livak KJ. (2008). Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–8. Sha M, Ye J, Zhang LX, et al. (2014). Celastrol induces apoptosis of gastric cancer cells by miR-21 inhibiting PI3K/Akt-NF-kappaB signaling pathway. Pharmacology 93:39–46. Sidransky D. (1997). Nucleic acid based methods for the detection of cancer. Science 278:1054–59. Stroun M, Anker P, Maurice P, et al. (1989). Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology 46:318–22. van der Waal I, de Bree R, Brakenhoff R, Coebergh JW. (2011). Early diagnosis in primary oral cancer: is it possible? Med Oral Patol Oral Cir Bucal 16:e300–5 Wang J, Chen J, Chang P, et al. (2009). MicroRNAs in plasma of pancreatic ductal adenocarcinoma patients as novel blood-based biomarkers of disease. Cancer Prev Res (Phila) 2:807–13. Wang M, Gu H, Wang S, et al. (2012). Circulating miR-17-5p and miR20a: molecular markers for gastric cancer. Mol Med Rep 5:1514–20. Yang Q, Xu E, Dai J, et al. (2014). miR-21 regulates N-methyl-N-nitroN0 -nitrosoguanidine-induced gastric tumorigenesis by targeting FASLG and BTG2. Toxicol Lett 228:147–56. Zhang HL, Yang LF, Zhu Y, et al. (2011). Serum miRNA-21: elevated levels in patients with metastatic hormone-refractory prostate cancer and potential predictive factor for the efficacy of docetaxel-based chemotherapy. Prostate 71:326–31.
RESEARCH ARTICLE
Circulating microRNA-21 (MIR-21) and phosphatase and tensin homolog (PTEN) are promising novel biomarkers for detection of oral squamous cell carcinoma WenHao Ren1*, Cui Qiang1*#, Ling Gao1,2*, Shao-Ming Li1, Lin-Mei Zhang1, Xiao-Long Wang1, Jian-Wei Dong1, Cheng Chen1, Chang-Yang Liu1, and Ke-Qian Zhi1
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1
Department of Oral and Maxillofacial Surgery, College of Stomatology, Xi’an Jiaotong University Xi’an, Shaanxi, P. R. China and 2Key Laboratory of Environment and Genes Related to Diseases, College of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi, P. R. China Abstract
Keywords
The purpose of this study was to investigate the potential of the blood levels of MIR-21 and PTEN as novel biomarkers for oral squamous cell carcinoma (OSCC). We initially detected MIR-21 and PTEN using real-time RT-PCR from 90 blood samples and then compared their results with expression in cancer tissues from 10 OSCC patients. Finally, we examined the relationship between these markers and clinical parameters. Blood MIR-21 and PTEN had significant diagnostic value for OSCC and, to an extent, correlated with the expression level of tumour MIR21 and PTEN. In addition, they were associated with differentiation and nodal status. Thus circulating MIR-21 and PTEN might represent new complementary tumour markers for OSCC.
Circulating biomarkers, MIR-21, OSCC, PTEN
Introduction Oral squamous cell carcinoma (OSCC) is a predominant type of head and neck squamous cell carcinoma (HNSCC), which is an aggressive and lethal malignancy (Chiu et al., 2013). OSCC is traditionally considered a major problem worldwide due to its extreme high prevalence (Maryam et al., 2012; O-Charoenrat et al., 2001; van der Waal et al., 2011) and the 5-year survival rate of patients with OSCC is poor (Ching-Yu et al., 2013; Markopoulos, 2012). It is known to be a devastating disease with treatment frequently leaving patients disfigured, and with diminished quality of Life (compromised speech, swallowing difficulties) and has debilitating adverse effects of radiation and chemotherapy (Denaro et al., 2011; Nagalakshmi, et al., 2014). The lack of early diagnostic methods is the main reason for poor prognosis in oral cancer patients, with over 60% of patients presenting in stages III and IV (Lingen et al., 2008). Thus, new approaches that can complement and improve current strategies for oral cancer diagnosis are urgently needed. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the translation of coding mRNAs by imperfectly acting on the 30 -untranslated regions (30 UTRs) of
*These authors contributed equally to this work. #Cui Qiang is responsible for statistical design/analysis. E-mail: [email protected] Address for correspondence: Ke-Qian Zhi, MD, PhD, Department of Oral and Maxillofacial Surgery, College of Stomatology Xi’an Jiaotong University, Number 98, Xiwu Road, Xi’an, ShaanXi 710004, P.R. China. Tel: 86-15309239634. E-mail: [email protected]
History Received 13 June 2014 Revised 12 August 2014 Accepted 12 August 2014 Published online 1 September 2014
the mRNA target (Esquela-Kerscher & Slack, 2006) and are stably detectable in plasma and serum (Calin et al., 2006; Chen et al., 2008; Mitchell et al., 2008). Several investigations have demonstrated expression of miRNAs was altered in a variety of cancers and have suggested that miRNAs have the potential to be useful diagnostic and prognostic biomarkers for multiple cancers such as lung cancer (Hu et al., 2010), leukaemia (Lawrie et al., 2008), pancreatic cancer (Wang et al., 2009), prostate cancer (Mitchell et al., 2008), colorectal cancer (Ng et al., 2009), ovarian cancer (Resnick et al., 2009), breast cancer (Heneghan et al., 2010). MIR-21, encoded on the X chromosome, is an important member of the miRNA family. It has been reported that MIR-21 is overexpressed in breast cancer (Asaga et al., 2011), lung cancer (Liu et al., 2012), prostate cancer (Zhang et al., 2011), ovarian cancer (Resnick et al., 2009), hepatocellular carcinoma (Li et al., 2011), lymphoma (Lawrie et al., 2008). To date, the expression of MIR-21 in OSCC and whether it can be used for a potential non-invasive molecular marker have not been evaluated. We hypothesised that circulating MIR-21 concentrations could act as a potentially useful biomarker in patients with OSCC. If this hypothesis holds true, it would signify a major breakthrough in OSCC management, bringing us ever closer to finding a novel, sensitive, and non-invasive biomarker for OSCC. Accordingly, we established a procedure to measure the circulating MIR-21 concentration and assessed whether it could screen cancer patients and monitor tumour dynamics and whether the levels were associated with clinic-pathological factors in OSCC patients (Li et al., 2011). In addition, the level of phosphatase and tensin homologue (PTEN), a
MIR-21 and PTEN biomarkers for OSCC
DOI: 10.3109/1354750X.2014.955059
direct target gene of MIR-21, was measured from the same blood samples to investigate whether PTEN levels are inversely related with MIR-21 levels in blood.
Materials
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Patients and samples We collected blood samples from 58 patients with histologic diagnosis of OSCC at the Department of Surgery, Stomatology Hospital of Xi’an Jiaotong University College of Medicine and from 32 matched cancer-free donor volunteers enrolled at the same institute with no previous history of any cancer for more than 2 years. To examine the relationship between MIR-21 and PTEN, we initially investigated the level of blood MIR-21 and PTEN in all cases. Thereafter, we designed an experiment to investigate whether the blood MIR-21 and PTEN could reflect tumour MIR-21 and PTEN. Fresh tissue samples were acquired from 10 patients whose MIR-21 concentrations exceeded the highest level of the donor volunteers and whose PTEN concentrations were below the lowest value of the donor volunteers. We compared the MIR-21 and PTEN expression in primary lesions with those in the blood samples. All subjects were of the same ethnicity (the Han nationality). Relevant demographic and clinicpathological details were obtained from our prospectively maintained database. A total of 90 participants were recruited into this study. No significant differences in age or gender were found between OSCC patients and normal controls (p ¼ 0.791 and p ¼ 0.883, respectively, 2 test). The differentiation of tumours was assessed by one experienced pathologist. All blood samples were collected before any treatment in EDTA-coated blood collection tubes and stored at 80 C until further processing; tissue samples were also kept at 80 C. The aim of the study was explained in detail to all participants, and written informed consent was obtained from all patients prior to their samples in the present study. The study protocol has been approved by the Hospital Ethical Committee of Stomatology Hospital of Xi’an Jiaotong University College of Medicine. RNA extraction Total RNA was isolated from 1 ml of whole blood using the E.Z.N.A.Ô blood RNA kit (AmbionÕ , Life Technologies, Paisley, UK). The extracted RNA was ultimately eluted in 30 ll of DEPC-treated water according to the manufacturer’s directions. Tissue total RNA was extracted with Trizol (Invitrogen, Life Technologies, Carlsbad, CA) following the manufacturer’s instructions. NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE) was used to determine the purity and concentration of RNA by measuring the optical density (A260/28042.0; A260/ 23041.8). The samples were either preserved at 80 C or further processed. Protocols for the detection of MIR-21 and PTEN The concentration of MIR-21 and PTEN in blood was determined with quantitative real-time reverse transcriptionpolymerase chain reaction (RT-PCR). For PTEN analyses, the
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first-strand cDNA was generated using the PrimeScriptÔ RT reagent Kit following the manufacturer’s directions, while the mature MIRNA was reverse transcribed using MIRNA-specific primers for quantification of MIR-21. Quantitative real-time RT-PCR was performed using SYBR Premix Ex Taq II on an FTC-3000Ô System (Funglyn Biotech Inc., Toronto, Canada). DNA was amplified by a cycling programme which comprised preliminary denaturation at 95 C for 5 min followed by 40 cycles of denaturation at 95 C for 10 s, annealing at 60 C for 30 s and elongation at 72 C for 1 s and by a final elongation step at 72 C for 10 min. All samples were processed in triplicate. Small nuclear RNA U6 (snRNA U6) was the internal control (Wang et al., 2012) and was used to normalise MIR-21 values, while -actin was used as an endogenous control to standardise PTEN expression in the PCR. Relative quantitation of MIR-21 and PTEN was calculated using the 2DDCt method (Schmittgen & Livak, 2008). Statistical analysis Statistical analysis was done using the software package SPSS 19.0 for Windows (SPSS Inc., Chicago, IL). Data were expressed as the mean ± SD. Student’s t-test was used to compare the difference in blood MIR-21 and PTEN concentration between the cancer group and the healthy group, and analysis of variance (ANOVA) was used to analyse the correlation between the expression of MIR-21 and PTEN and the clinic-pathological features of the patients. In addition, a regression analysis was used to evaluate possible risk factors such as age, sex, tumour size, smoking, histology, site, and nodal status involving the gene expression of biomarker genes. The association between blood MIR-21 and PTEN concentration was analysed using Pearson’s correlation coefficient. Receiver operating characteristic (ROC) curves were constructed and the area under the curve (AUC) was calculated to determine the ability of the blood biomarker levels to discriminate between patients with OSCC and healthy control subjects. The Youden index (sensitivity + specificity 1) was used to determine the optimal cut-off point (Akobeng, 2007). All tests were two-tailed and a p value 50.05 denoted the presence of a statistically significant difference.
Results The expression and correlation between MIR-21 and PTEN in blood To explore the potential value of circulating MIR-21 and PTEN in OSCC, we first investigated the levels of MIR-21 and PTEN, in blood samples of OSCC patients (n ¼ 58) and donor volunteers (n ¼ 32). No significant difference was discovered in terms of the levels of snRNA U6 and b-actin between controls and OSCC patients. The expression levels of MIR-21 in blood were significantly higher in OSCC patients than in donor volunteers (p50.001) (Figure 1A), while PTEN concentration tend to be higher in controls (p50.001) (Figure 1B). Next, we examined the correlation between circulating MIR-21 levels and PTEN levels using Pearson’s correlation coefficient, and the results showed that there was
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Figure 1. Circulating levels of MIR-21 and PTEN in blood from OSCC patients and donor volunteers. Increased MIR-21 (A) and MIR-21/PTEN (C) levels in blood in OSCC patients compared with controls (Student’s t-test, p50.0001), while PTEN concentration (B) tend to be higher in controls (Student’s t-test, p50.0001). (D) The correlation between MIR-21 and PTEN in blood from 58 patients with OSCC.
Table 1. Expression of MIR-21 and PTEN in oral cancer tissue versus those in normal tissue.
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Case 10 Rate of higher expression level in oral cancer tissue Rate of lower expression level in oral cancer tissue
MIR-21
PTEN
1.0446 1.3495 1.0823 1.8139 1.0803 1.2224 1.0205 1.3289 1.1021 0.5650 90%
0.3850 0.2952 0.1507 0.1732 0.3850 0.2857 0.1460 0.2133 0.2808 10.5692 10%
10%
90%
negative significant correlation between them (r ¼ 0.555, p50.001) (Figure 1D). Relationship of the expression of MIR-21 and PTEN between blood and primary oral cancer tissue The levels of MIR-21 and PTEN in cancer tissues compared with adjacent normal tissues were measured in 10 patients who had higher levels of blood MIR-21 and lower levels of blood PTEN than the donor volunteers. MIR-21 and PTEN obtained from fresh tissue were amplified, and found to be of good quality for amplification through a quality verification process (data not shown). The level of MIR-21 in primary oral cancer
tissues was higher than that in normal tissues in nine of the 10 patients analysed (90%), whereas PTEN was lower in nine patients (90%) (Table 1). These results indicated that similar profiles of MIR-21 and PTEN were found in primary cancer tissues and blood samples from most patients. Therefore, the level of blood MIR-21 and PTEN might reflect the expression level of tumour MIR-21 and PTEN to a certain extent. Differentiating power of MIR-21, PTEN and the combination of MIR-21 and PTEN for OSCC To further identify the potential diagnostic value of the blood levels of MIR-21 and PTEN for OSCC patients, the levels of MIR-21 and PTEN were measured for a total of 90 blood samples, including 58 OSCC and 32 normal controls. ROC curve analyses revealed that blood MIR-21 and PTEN were valuable biomarkers for distinguishing OSCC patients from controls, and the area under the curve (AUC) for MIR-21 and PTEN was 0.788 (95% CI: 0.692–0.883) (Figure 2A) and 0.731 (95% CI: 0.624–0.838) (Figure 2B), respectively. At a cut-off value of 9.646 for MIR-21, the optimal sensitivity and specificity were 62.1% and 90.6%, respectively. The odds ratio for MIR-21 9.646 being associated with OSCC patients was 15.818 (95% CI: 4.304–58.317). As for PTEN, at a cut-off value of 8.875, the optimal sensitivity and specificity were 65.5% and 71.9%, respectively. The odds ratio for PTEN 8.875 being associated with patient was 4.856 (95% CI: 1.893–12.453). To examine a more sensitive diagnostic biomarker in blood, we analysed the ratio of MIR-21/PTEN as a combined
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Figure 2. Receiver operating characteristics (ROC) curve analysis using blood MIR-21 (A), PTEN (B) and MIR-21/PTEN (C) for discriminating OSCC patients from healthy controls. Blood MIR-21 showed the greatest AUC (the areas under the ROC curve) of 0.788 (95% CI: 0.692–0.883) with 62.1% sensitivity and 90.6% specificity (A), and blood PTEN yielded AUC of 0.731 (95% CI: 0.624–0.838) with 65.5% sensitivity and 71.9% specificity of (B), while blood MIR-21/PTEN yielded AUC of 0.774 (95% CI: 0.677–0.872) with 84.5% sensitivity and 62.5% specificity of (C).
biomarker. The ratio of MIR-21/PTEN in blood showed a significantly elevated level when compared to those in normal controls (p50.001) (Figure 1C). The ROC analysis for the ratio of MIR-21/PTEN did not improve the stratification power characterised, as it was characterised by an AUC of 0.774 (Figure 2C) (95% CI: 0.677–0.872) for the ratio of MIR-21/PTEN. In this model, an optimal cut-off point was found to be 1.332 and showed a better sensitivity of 84.5% with a specificity of 62.5%. These results suggest that MIR-21 and PTEN in blood are new complementary tumour markers for OSCC. Relationship between blood level of MIR-21 and PTEN and clinical characteristics The results of the correlation analysis between MIR-21 and PTEN blood levels and clinic-pathological features in 58 OSCC patients are shown in Table 2. The expression of MIR21 and PTEN is displayed as the mean ± SD. No significant association was observed between the expression of MIR-21 and PTEN and age, sex, tumour size, smoking history, and
tumour location (p40.05). Relative MIR-21 blood levels showed a trend toward elevation in patients with poormoderately differentiated tumours compared with welldifferentiated tumours (p ¼ 0.001) and in patients with lymph node involvement compared with patients without lymph node involvement (p50.001). However, the levels of PTEN demonstrated a downward trend in tumours with decreased differentiation (p ¼ 0.01) or positive nodal status (p ¼ 0.003). Using regression analysis, as displayed in Table 3, the possible risk factors affecting gene expression of the two biomarkers were assessed. These results suggest that only the histology and nodal status for OSCC may be significant risk factors for gene expression of MIR-21 and PTEN.
Discussion To our knowledge, this is the first reported investigation that directly obtained total RNA from whole blood. There are many advantages, including streamlining the procedure, reducing time and overall cost, minimising human and
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Table 2. Correlation between the level of MIR-21 and PTEN in the blood of patients with oral squamous cell carcinoma (OSCC) and clinicopathological factors. Blood MIR-21 Level (DCt)
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Variable Age(years) Mean(range) 550 50 Sex Male Female Tumour size 2 25 Smoking Non-smokers Current smokers Histology Well Poor-moderate Site Gingiva Buccal Tongue Others Nodal status Negative Positive
Blood PTEN level (DCt) a
Patients (n ¼ 58)
Mean ± SD
p value
Mean ± SD
p valuea
61 (25–92) 16 (27.6%) 42 (72.4%)
9.36 ± 1.18 9.49 ± 1.15
0.700
9.92 ± 1.49 9.16 ± 1.45
0.086
39 (67.2%) 19 (32.8%)
9.49 ± 1.24 9.41 ± 0.99
0.806
9.45 ± 1.56 9.30 ± 1.27
0.720
19 (32.8%) 39 (67.2%)
9.68 ± 1.14 9.35 ± 1.16
0.313
9.10 ± 1.34 9.55 ± 1.51
0.271
31 (53.4%) 27 (46.6%)
9.36 ± 1.23 9.58 ± 1.20
0.477
9.24 ± 1.54 9.60 ± 1.36
0.356
30 (51.7%) 28 (48.3%)
9.94 ± 0.99 8.94 ± 1.12
0.001*
8.94 ± 1.40 9.91 ± 1.44
0.01*
16 9 18 15
(27.6%) (15.5%) (31.0%) (25.9%)
9.58 ± 1.07 9.73 ± 1.37 9.15 ± 1.03 9.53 ± 1.28
0.582
9.13 ± 1.33 8.98 ± 1.26 10.04 ± 1.10 9.19 ± 1.91
0.166
33 (56.9%) 25 (43.1%)
9.61 ± 1.20 8.85 ± 0.90
50.001*
8.92 ± 1.52 10.04 ± 1.12
0.003*
a p values are from Student’s t-test or one-way ANOVA. *Indicates a significant difference (p50.05).
Table 3. Regression analysis to assess the possible risk factors affecting gene expression of the two biomarkers in blood samples of OSCC patients and donor volunteers.
Risk factors Age Sex Tumour size Smoking Histology Site Nodal status
MIR-21
PTEN
OSCC patients donor volunteers volunteers
OSCC patients donor volunteers
0.286 0.997 0.949 0.846 0.003* 0.704 0.002*
0.454 0.097 0.147
0.268 0.918 0.943 0.900 0.008* 0.748 0.002*
0.158 0.742 0.110
*The p values of the differences of DCt values in blood samples of OSCC patients and donor volunteers were shown and donor volunteers do not have information for tumour size, histology, site, and nodal status.
mechanical errors, and avoiding nucleic acid loss. As we hypothesised, the up-regulation of MIR-21 and down-regulation of PTEN expression between the blood of OSCC patients and controls reached statistical significance in our study. Previous findings have indicated that elevated levels of the circulating cancer-associated nucleic acids in pre-operative samples were potentially due to the release by cancer cells (Sidransky et al., 1997; Stroun et al., 1989) and Li et al. have reported that the level of MIR-21 was inversely correlated with PTEN expression in OSCC cancer tissues (Li et al., 2009). These two points led us to speculate that there was negative relationship between circulating MIR-21 and PTEN in OSCC patients. We found that the overall
correlation coefficient between MIR-21 and PTEN levels for all patients was 0.555 (p50.001), in accordance with our expectation. It has been verified that MIR-21 modifies N-methyl-N-nitro-N-nitrosoguanidine-induced gastric tumourigenesis by targeting FASLG and BTG2 (Yang et al., 2014) and promotes cholangiocarcinoma growth by targeting 15-PGDH (Lu et al., 2014). Moreover, MIR-21 inhibits the PI3K/Akt-NF-kB signalling pathway and promotes apoptosis of gastric cancer cells induced by celastrol (Sha et al., 2014). Additionally, MIR-21 can contribute to tumour initiation, progression, metastasis, and chemo- or radio-resistance as an oncomiR by modulating PTEN and PDCD4 expression and PTEN-dependent pathways in several human cancers, including human hepatocellular cancer, ovarian epithelial carcinomas, and non-small cell lung cancer cells (Liu et al., 2013; Lou et al., 2010; Meng et al., 2007). Our findings provide a firm basis for further investigation regarding their roles in OSCC. To confirm whether circulating MIR-21/PTEN could be released from primary oral tumours, we performed a comparison between expression of MIR-21 in blood and primary tumour tissues. The results indicated that blood and primary OSCC tissue samples showed profiles with respect to expression of MIR-21/PTEN in almost all cases. One patient, however, showed an opposite pattern of nucleic acid levels, high blood PTEN with a low expression in OSCC tissue and low blood MIR-21 with a high expression in cancer tissue. Although the reason for this discrepancy is not clear at present, one possible explanation for this finding may be the heterogeneity of the primary tumours. In the present study, the expression correlation of MIR-21 and PTEN between blood
MIR-21 and PTEN biomarkers for OSCC
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and tumour tissue has not been absolutely elucidated. Some researchers have assumed that circulating nucleic acid levels might not always directly relate to changes occurring in tumour tissues but may also reflect indirect effects and secretion by non-tumour cells. Thus, further investigation is needed to clarify this relationship and evaluate whether circulating MIR-21 and PTEN could be useful for clinical diagnosis for OSCC. To evaluate the utility of MIR-21 and PTEN as novel biomarkers, ROC analysis was conducted to evaluate the discriminating power for patients with OSCC. The AUC for MIR-21 and PTEN were 0.788 and 0.731, respectively, by ROC analysis. Furthermore, we discovered that the ratio of the circulating levels of MIR-21/PTEN did increase the ability of nucleic acids to discriminate cancer cases from controls with a sensitivity of 84.5%, which was above the sensitivity of either MIR-21 or PTEN alone and could be satisfactory for clinical application. Previous studies have reported that determining the expression ratios of genes or miRNAs may be a useful technique to improve diagnostic potential (Avissar et al., 2009; Gordon et al., 2002). So far, there have been few published studies that analyse the ratio of circulating miRNAs levels and gene levels to improve the sensitivity and specificity of diagnostic biomarkers. Surprisingly, in our study, we confirmed that MIR-21 and PTEN levels in blood were misregulated and that their ratio had important value for characterising OSCC. MIR-21 has grounds for serious investigation because it has increased expression in many cancers, such as breast cancer (Avissar et al., 2009) and lung cancer (Keller et al., 2011), and has been considered to have an important role in diagnosis, metastasis, progression, treatment, and prognosis of malignancies. Li et al. reported that MIR-21 was overexpressed in tongue squamous cell carcinoma and indicated poor prognosis (Li et al., 2009). Consistent with their results, our data showed that there was a higher expression of MIR-21 in OSCC patients than that in control cases. In addition, our data show a correlation between the MIR-21 level and both lymph node metastasis and advanced clinical stage, which is consistent with the study by Liu et al. (2012) in lung cancer. Thus, blood MIR-21 could be used as a potential biomarker in the diagnosis of OSCC patients with lymph node metastasis or advanced clinical stage. In diagnostic studies, one key issue is how to choose appropriate controls, and it is practically difficult to ensure that the control group is indeed truly healthy; therefore, detailed information on personal health should be obtained when selecting controls. An expanded number of samples may also be useful to eliminate potential sampling error and increase the confidence in these findings.
Conclusions In summary, circulating levels of MIR-21 and PTEN might provide new complementary tumour markers for OSCC. Our study serves as a basis for further investigation, preferably in large prospective studies that would enable application as a non-invasive screening tool for OSCC in routine clinical practice.
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Declaration of interest This work was supported in part by the Project of Technological Innovation Planning in Shannxi Province (grant 2012KTCL03-17), the Specialised Research Fund for the Doctoral Program of Higher Education of China (grants 20120201110063 and 20130201120054), the Natural Science Foundation of China (grant 81272957), and the Fundamental Research Funds for the Central Universities (grant xjj2013057). The authors declare that there is no conflict of interest.
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