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Expression profiling of serum microRNA-101 in HBVassociated chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma a
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b
a
a
a
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Yun Xie , Qinwei Yao , Azeem Mehmood Butt , Jia Guo , Zhou Tian , Xuli Bao , Hongxia Li , a
Qinghua Meng & Jun Lu
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a
Hepatology and Cancer Biotherapy Ward; Beijing YouAn Hospital; Capital Medical University; Beijing, PR China b
Centre of Excellence in Molecular Biology (CEMB); University of the Punjab; Lahore, Pakistan Published online: 27 Jun 2014.
Click for updates To cite this article: Yun Xie, Qinwei Yao, Azeem Mehmood Butt, Jia Guo, Zhou Tian, Xuli Bao, Hongxia Li, Qinghua Meng & Jun Lu (2014) Expression profiling of serum microRNA-101 in HBV-associated chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, Cancer Biology & Therapy, 15:9, 1248-1255, DOI: 10.4161/cbt.29688 To link to this article: http://dx.doi.org/10.4161/cbt.29688
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Research Paper Research Paper
Cancer Biology & Therapy 15:9, 1248–1255; September 2014; © 2014 Landes Bioscience
Expression profiling of serum microRNA-101 in HBV-associated chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma Yun Xie1,†, Qinwei Yao1,†, Azeem Mehmood Butt2, Jia Guo1, Zhou Tian1, Xuli Bao1, Hongxia Li1, Qinghua Meng1,*, and Jun Lu1,* Hepatology and Cancer Biotherapy Ward; Beijing YouAn Hospital; Capital Medical University; Beijing, PR China; 2Centre of Excellence in Molecular Biology (CEMB); University of the Punjab; Lahore, Pakistan These authors contributed equally to this work.
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Keywords: microRNA-101; circulating microRNA; hepatitis B; hepatocellular carcinoma; expression profiling Abbreviations: CHB, chronic hepatitis B; LC, liver cirrhosis; HCC, hepatocellular carcinoma; ROC, receiver operating characteristic; AFP, α-fetoprotein
MicroRNAs (miRNAs) represent a class of evolutionarily conserved, non-coding small RNAs (18–25 nt) that have emerged as master regulators of several biological processes. Recently, circulating miRNAs have also been reported to be promising biomarkers for various pathological conditions. In the present study, we report the comparative expression profiling of microRNA-101 (miR-101) in serum and tissue samples from chronic hepatitis B (CHB), HBV-associated liver cirrhosis (HBV-LC), and HBV-associated hepatocellular carcinoma (HBV-HCC) patients and healthy controls. The serum miR-101 levels were found to be significantly downregulated in the HBV-HCC patients compared with the HBV-LC patients (P < 0.001), CHB patients (P < 0.001) and healthy controls but were upregulated in the HBV-LC patients compared with the CHB patients (P < 0.001) and healthy controls (P < 0.001). Consistent with the serum data, the expression of miR-101 was also upregulated and downregulated in the HBV-LC and HBV-HCC tissue samples, respectively. A receiver operating characteristic (ROC) analysis of serum miR-101 yielded an area under the ROC curve (AUC) of 0.976 with 95.5% sensitivity and 90.2% specificity when differentiating between HBV-HCC and HBV-LC. Our results suggest that the serum miR-101 level can serve as a potential non-invasive biomarker to differentiate HBV-HCC from HBV-LC.
Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and ranks as the third leading cause of cancer mortality.1 Although there are different viral and non-viral causes of HCC, it has been reported that almost 80% of disease cases are associated with hepatitis B virus (HBV) infection. HCC is often diagnosed at an advanced stage when curative resection of the tumor is no longer appropriate because of intrahepatic and extrahepatic metastases. At present, the diagnosis of HCC relies on the identification of a liver mass by radiological imaging tests, including ultrasonography, CT, and/or magnetic resonance imaging (MRI). These imaging techniques are often combined with the use of serum tumor markers, such as α-fetoprotein (AFP), which has been discontinued in several western countries due to its poor sensitivity and specificity, but this protein is still widely used as a serum tumor marker in many Asian countries including China.2,3 MicroRNAs (miRNAs) are small noncoding RNA (18–24 nucleotides) molecules that have been reported to play important
roles in the regulation of several biological processes, such as cell proliferation, differentiation and apoptosis.4,5 The ability of miRNAs to bind specific sites in the 3′-UTR of target mRNAs leads to translational repression and/or mRNA degradation.6 The deregulation of miRNA function can often lead to disorder of the internal environment and eventually to carcinogenesis. Many miRNA genes are located in cancer-associated genomic regions or in fragile sites that are frequently amplified or deleted in human cancers, suggesting that these miRNAs play an important role in malignant transformation.7 Apart from their tissue-specific origin and expression, it has recently been shown that miRNAs are also stable and detectable in various body fluids such as serum and plasma.8 Similar to the expression patterns in tissue specimens, the expression of circulating miRNAs has been shown to vary between disease cases and healthy controls for several types of cancers and diseases. This observation suggested that the circulating miRNAs could serve as useful, non-invasive biomarkers of disease stage and progression. In the case of HCC, the circulating levels of several miRNAs including miR-21, miR-122, miR-15b, and miR-130b have been reported as potential diagnostic markers.9,10
*Correspondence to: Qinghua Meng; Email: [email protected]; Jun Lu; Email: [email protected] Submitted: 04/13/2014; Revised: 06/17/2014; Accepted: 06/22/2014; Published Online: 06/27/2014 http://dx.doi.org/10.4161/cbt.29688 1248
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Results Expression of miR-101 in liver tissues First, we measured the expression of miR-101 in CHB, HBVLC, HBV-HCC, and control tissue samples via in situ hybridization. The patients used for in situ hybridization were independent from those used for serum miRNA profiling, and their characteristics are given in Table S1. The expression levels of miR-101 in the CHB tissue samples were not significantly different from the levels in the normal liver tissue samples (P = 0.999). On the other hand, compared with the normal samples, the expression levels of miR-101 were upregulated in the HBV-LC tissue samples (P = 0.011) yet downregulated in the HBV-HCC tissue samples (P = 0.014) (Fig. 1A–E). Expression profile of circulating miRNA-101 To investigate whether levels of circulating miR-101 are also altered in patients with CHB, HBV-LC and HBV-HCC, the concentrations of miR-101 were measured in the sera from patients and healthy controls using quantitative real-time PCR. Consistent with our in situ hybridization data, although no significant difference (P = 1.000) was observed in the miR-101 serum levels between the control and CHB samples (Fig. 2), we found that serum miR-101 was significantly upregulated in the HBV-LC group compared with the CHB patients (P < 0.001) and control subjects (P < 0.001) (Fig. 2). We next investigated the levels of serum miR-101 in the HBV-HCC patients compared with the HBV-LC, CHB, and healthy subjects. The results showed that serum miR-101 was significantly downregulated in the HBVHCC patients compared with the HBV-LC (P < 0.001), CHB (P < 0.001), and healthy subjects (P = 0.003) (Fig. 2). Next, to explore the possible association between miR-101 and the risk of HBV-HCC derived from HBV-LC, binary logistic regression was used controlling for gender, age, alcohol status, and ALT at
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Figure 1. The expression levels of miR-101 in liver tissues. In situ hybridization was performed using locked nucleic acid (LNA)-modified probes for miR-101. Brown yellow staining in the cytoplasm represents positive staining for miR-101. (A) Normal liver tissues were graded positive for miR-101. (B) CHB liver tissues were graded positive for miR-101. (C) HBV-LC tissues were graded strong positive for miR-101. (D) HBV-HCC tissues were graded negative for miR-101. (E) The level of miR-101 expression in HBV-LC patients is higher than in healthy subjects and CHB patients, while it is lower in HBV-HCC patients. (* represents P < 0.05.)
the same level. A significantly decreased risk of HBV-HCC was found to be associated with upregulated miR-101. The odds ratio of developing HBV-HCC with upregulated miRNA-101 was 0.088 (95% CI = 0.032–0.244, P < 0.001). The correct percentage of the logistic regression model was 92.1%. The diagnostic potential of serum miR-101 To evaluate whether serum miR-101 can be used as a diagnostic marker for CHB, HBV-LC, and HBV-HCC, ROC curve analysis was performed. In the case of CHB compared with healthy controls, the AUC was 0.635 (95% CI: 0.548–0.716) with a sensitivity/specificity ratio of 84.8/40.4%, indicating that serum miR-101 may not be an optimal marker for CHB diagnosis (data not shown). This result parallels the lack of a
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Recently, genome-wide miRNA expression profiling has revealed overexpression of miR-101 in HCC tissues compared with normal/healthy controls.11 The aberrant expression and therapeutic potential of miR-101 was also demonstrated in another study showing that miR-101 is downregulated by the HBV X protein and induces epigenetic modifications in HBV-associated HCC (HBV-HCC) tissues.12 Although aberrant expression of circulating miR-101 has been reported previously in HBV-HCC, parallel expression profiling of miR-101 from the serum and tissues of patients at different stages of HBV-associated liver diseases have not yet been reported.13 The development of HCC is a multi-step process, and it is also unknown if serum miR-101 could be used as a marker for differentiating the different stages of HBV-associated liver disease. Therefore, in the present study, we performed serum miR-101 expression profiling of HBVassociated chronic HBV (CHB), HBV-associated liver cirrhosis (HBV-LC), and HBV-HCC patients to determine this marker’s diagnostic potential. Moreover, the hepatic miR-101 expression was also analyzed. For simplicity, the miR-101 that we analyzed in the present study will be referred to as miR-101 throughout the manuscript.
96.6/87.9% (Fig. 4E). This result is similar to the ROC analysis of miR-101 alone for differentiating HBV-LC from HBV-HCC (Fig. 4C). There was no correlation between the serum miR-101 expression levels and the patients’ clinical features, such as age, gender, ALT, T-Bil, PT, viral load, tumor size, Child–Pugh grade, AFP, or TNM classification (data not shown).
Figure 2. Serum miR-101 levels in healthy subjects, patients with CHB, patients with HBV-LC and patients with HBV-HCC. The median level of serum miR-101 was significantly higher in patients with HBV-LC than in patients with CHB or healthy subjects (P < 0.001), whereas the median level was significantly lower in patients with HBV-HCC than in healthy subjects (P = 0.012) or patients with HBV-LC (P < 0.001). The lines represent the range, median and quartiles of relative miRNA expression (ΔCt values) measured by qRT-PCR.
significant difference in the serum miR-101 levels between CHB and healthy controls described above. In the case of HBV-LC compared with healthy controls, the AUC was 0.884 (95% CI: 0.800–0.942), and the sensitivity and specificity calculated at the cut-off value of 10.50 were 80.3% and 80.0%, respectively (Fig. 3A). For HBV-HCC compared with healthy controls, the AUC was 0.788 (95% CI: 0.693–0.865), and at the cut-off value of 8.99, the sensitivity and specificity were 76.1% and 70.0%, respectively (Fig. 3B). We next investigated whether serum miR-101 has the potential to serve as a marker of disease progression or to differentiate between CHB, HBV-LC, and HBV-HCC patients. For the discrimination between patients with CHB and either HBV-LC or HBV-HCC, miR-101 had an AUC of 0.861 (95% CI: 0.792– 0.913) (Fig. 4A) and 0.777 (95% CI: 0.701–0.842), respectively (Fig. 4B). At the cut-off values of 10.91 (CHB compared with HBV-LC) and 9.70 (CHB compared with HBV-HCC), serum miR-101 had sensitivity/specificity ratios of 75.4/83.5% and 88.1/62.0%, respectively. In the case of HBV-LC compared with HBV-HCC, the AUC was 0.976 (95% CI: 0.931–0.995), and at a cut-off value of 10.08, the sensitivity and specificity of serum miR-101 were 95.5% and 90.2%, respectively (Fig. 4C). Next, the predicted probability of being diagnosed with HBVHCC from a stepwise logistic regression model based on AFP and miR-101 was used to construct the ROC curve. The AFP alone yielded an AUC of 0.762 (95% CI: 0.674–0.836) with 44.8% sensitivity and 96.6% specificity when differentiating HBVLC from HBV-HCC (Fig. 4D). The AUC of serum miR-101 combined with AFP was also calculated and found to be 0.973 (95% CI: 0.925–0.994) with a sensitivity/specificity ratio of
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The aberrant expression of miRNA-101 has been recently reported in several cancers, including ovarian, renal, prostate, gastric, and lung cancer,14-20 highlighting miR-101 as a bona fide tumor suppressor miRNA. Downregulation of miRNA-101 in association with HCC has also been reported in different studies that have collectively shown that miRNA-101 expression has the potential to inhibit HCC cell proliferation, suppress tumorigenicity, and promote apoptosis by modulating the expression of multiple transcription factors and cell cycle-related genes in HCC.21-23For instance, Su et al. reported the downregulation of miR-101 in HBV-HCC tissues and cell lines compared with controls. This study suggested that miR-101 downregulation is a frequent event in human HCC tissues that leads to hepatocarcinogenesis through the overexpression of Mcl-1, an antiapoptotic member of the Bcl-2 family, which has a complementary binding site for the miR-101 in its 3′-UTR that is a direct target of miR-101.19 Similarly, Zhang et al. reported the downregulation of miR-101 and parallel overexpression of SOX9, another direct target of miR-101, in clinical HCC tissues and the correlation of miR-101 downregulation with tumor aggressiveness and poor prognosis.22 As the association between miR-101 and HCC has been strengthened by the available data, it has been suggested that miR-101 could serve as a potential prognostic marker and therapeutic target for HCC.21-23 We were interested in determining the differences in miR-101 expression at different stages of HBVassociated liver diseases (CHB, HBV-LC, and HBV-HCC) and, moreover, whether miR-101 could serve as a non-invasive diagnostic biomarker. For this purpose, we performed expression analysis of miR-101 in the liver tissue and serum samples from CHB, HBV-LC, HBV-HCC, and control groups by in situ hybridization and real-time PCR, respectively. Two independent study groups were used for tissue and serum miRNA-101 analysis, and collectively, the results from each group were in agreement. We showed that the miRNA-101 levels in the sera and liver tissues were down- regulated in HBV-HCC patients but upregulated in HBV-LC patients compared with CHB patients and healthy controls. To evaluate the diagnostic potential of serum miR-101, ROC curves were generated. Our data showed that serum miR-101 is superior to AFP for diagnosing HBVHCC derived from HBV-LC, as combining serum miR-101 with AFP conferred no advantage over serum miR-101 alone for detecting HBV-HCC derived from HBV-LC. Combining the data from the tissue and serum miRNA levels, ROC curves
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Discussion
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and binary logistic analysis, we propose that serum miRNA-101 could serve as a non-invasive biomarker to monitor HBV-HCC that is derived from HBVLC and also the progression of CHB infection to HBV-LC. The progression of HBV-associated liver disease to HCC consists of several stages. Additionally, the expression of several miRNAs is stage-dependent in many diseases; some miRNAs show early expression, whereas some are expressed only during the advanced stages of disease. For instance, the liverspecific miRNA-122 has been reported Figure 3. ROC curve analysis of serum miR-101 for discriminating; (A) HBV-LC patients and healthy to be upregulated in the tissue and serum subjects, (B) HBV-HCC patients and healthy subjects. samples of CHB patients, yet downregulated in the tissue and serum samples of HBV-HCC patients.24 Other examples include miR-25 and monitoring the development of HBV-HCC from HBV-LC and let-7f, which were altered significantly in HBV-HCC patients but the development of HBV-LC from CHB. unchanged in CHB patients compared with controls.25 Based on our current analysis, we also suggest that miR-101 is a potential disease progression marker; while we observed no significant difMaterials and Methods ference in the expression of miR-101 in tissue and serum samples from CHB patients compared with healthy controls, a significant Study subjects and clinical characteristics expression difference emerged when comparing these samples to The study protocol was approved by the Ethics Committhose from HBV-LC patients (upregulation) and HBV-HCC tee of Beijing YouAn Hospital, Capital Medical University and patients (downregulation). In our opinion, however, the lack of a adhered to the tenets of the Declaration of Helsinki. Written significant difference between the CHB and control samples in informed consent was obtained from the participants for the use the present study can be attributed to the following factors. First, of their blood, tissue samples and clinical records in this study. the group used for serum analysis in this study consisted of CHB The study population consisted of 67 HBV-HCC patients, 61 patients with low staging and grading scores, which therefore did HBV-LC patients, 79 CHB patients and 30 healthy subjects. not have high hepatic activity index (HAI) scores. However, we The patients’ characteristics are summarized in Table 1. All also suspect that the inclusion of patients with higher inflam- patients were positive for HBsAg and did not have any other mation and fibrosis scores in the CHB group may bias the data liver diseases, such as CHC, alcoholic liver disease, autoimmune toward a false-positive result, as patients with liver cirrhosis usu- liver disease, or metabolic liver disease, based on clinical reports. ally have higher HAI scores and the inclusion of these patients in The degree of inflammation of all of the patients was between the CHB group may lead to an increase in the total level of miR- G1 and G2. The fibrosis staging of the CHB patients was 101 expression in this group. Another possible explanation is that between S0 and S2. The fibrosis staging of the HBV-LC and the transcription factors associated with liver inflammation may HBV-HCC patients was between S3 and S4. The blood samples not be targets of miR-101; the previously reported targets of miR- from the HBV-HCC patients were obtained before the surgical 101 that are liver-associated transcription factors have mostly resections were performed. The data on all of the subjects were been those involved in hepatocarcinogenesis and advanced-stage obtained from medical records, pathology reports, and personal liver diseases.21,22 However, these speculations warrant further interviews with the subjects. The data collected included age, investigations with a larger sample size. Similarly, the inclusion gender, serum albumin (ALB) level, total bilirubin level (T-Bil), of matched liver samples before and after HCC development alanine aminotransferase (ALT) level, prothrombin time (PT), could lead to a better understanding of the dynamics of miR-101 HBV DNA viral load, AFP, tumor number and size, tumor difexpression in HCC progression as miRNA-101 has been linked ferentiation, tumor stage, relapse time, and time of death. The to tumor migration and recurrence.16,26,27 Finally, whether miR- clinical stage of HBV-HCC was evaluated based on the TNM 101 has the same function in other liver disease and HCC etiolo- classification system. Child–Pugh scoring was performed to catgies such as HCV and alcoholic cirrhosis remains unclear. egorize the LC and HCC patients at Child–Pugh grades A, B, Taken together, the findings from our present study demon- or C. strate that miR-101 levels are elevated in the sera and liver tisSerum preparation sues of HBV-LC patients and decreased in HBV-HCC patients, Ten milliliters of peripheral blood was collected from each which suggests that serum miR-101 is a potential biomarker for individual directly into serum tubes at the time of liver biopsy
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RNA extraction and reverse transcription Total RNA was extracted from 200 μL of serum using a QuantoBio Total RNA Isolation Kit (QuantoBio Co.) following the manufacturer’s protocol. Briefly, 200 μL of lysis buffer and 40 μL of lysis enhancer solution were added to each sample and vortexed for 15 s. For the normalization of sampleto-sample variation during RNA isolation and as an internal control, 25 fmol of synthetic Caenorhabditis elegans miRNA-39 (cel-miR-39-3p) was added to each lysed sample as described previously.8 This step was followed by the addition of 440 μL of acidified phenol:chloroform and vigorous shaking for 30 s. The samples were centrifuged at 16 000 g in a microcentrifuge at 4 °C for 10 min to separate the aqueous phase (upper phase) and the organic phase (lower phase). The aqueous phase was then transferred to new RNase-free microcentrifuge DNA LoBind tubes (Eppendorf). Ethanol was added to the aqueous phase to a final concentration of 30%, and this mixture was vigorously shaken for 15 s. Then, the 650 μL mixtures were centrifuged at 10 000 g at room temperature for 30 s to pellet the RNA. After the washing steps, the total RNA including miRNAs was resuspended in 50 μL RNase-free water and stored at −80 °C until further use. The concentration and purity of the total RNA was determined using a NanoDrop 8000 (Thermo Fisher Scientific). cDNA was reverse transcribed from the total RNA via the polyadenylation method. Briefly, 1 ng of the total RNA template was mixed with 2 μL 10× buffer, 2 μL dATP (10 mM), 0.5 μL poly(A) polymerase (NEB), Figure 4. ROC curve analysis of serum miR-101 for discriminating; (A) CHB patients from HBV-LC patients, (B) CHB patients from HBV-HCC patients, (C) HBV-LC patients from HBV-HCC patients, (D) AFP 0.5 μL RNase inhibitor (Promega), alone for discriminating HBV-LC from HBV-HCC patients. (E) AFP in combination with miR-101 for disand RNase-free water (Promega) to a criminating HBV-HCC from HBV-LC patients. final volume of 20 μL and incubated at 37 °C for 1 h. Then, 1 μL of 0.5 μg/μL or before surgery. The tubes were initially centrifuged at 1500 g RT primer was added, and the reactions were incubated at 70 °C for 10 min. The serum was then aliquoted and additionally cen- for 5 min followed by immediate incubation on ice for at least trifuged at 13 000 g for 15 min at 4 °C to completely remove the 2 min to disrupt the secondary structures of the RNA and the cell debris and any remaining cells. The supernatant was then primer. In the final step, the 20 μL reaction mixture from the transferred to new 2.0-mL tubes without disturbing the pelleted above step was mixed with 4 μL 5× buffer, 1 μL dNTP (10 mM) debris. Any samples with signs of hemolysis were excluded from (Sigma), 0.5 μL M-MLV reverse transcriptase (Promega), 0.5 μL the study. The serum samples were then stored at −80 °C until RNase inhibitor (Promega), 10 μL A-Plus reaction mix, and further use. 4 μL RNase free water (Promega), and incubated at 42 °C for
Table 1. Summary of clinical characteristics of CHB, HBV-LC, HBV-HCC and healthy subjects
Gender n (%)
Group II: HBV-L (n = 61)
Group III: HBVHCC (n = 67)
Group IV: Control (n = 30)
P values
Mean ± SD
32.38 ± 11.93
51.19 ± 9.57
51.69 ± 10.43
37.26 ± 10.79
Cancer Biology & Therapy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kcbt20
Expression profiling of serum microRNA-101 in HBVassociated chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma a
a
b
a
a
a
a
Yun Xie , Qinwei Yao , Azeem Mehmood Butt , Jia Guo , Zhou Tian , Xuli Bao , Hongxia Li , a
Qinghua Meng & Jun Lu
a
a
Hepatology and Cancer Biotherapy Ward; Beijing YouAn Hospital; Capital Medical University; Beijing, PR China b
Centre of Excellence in Molecular Biology (CEMB); University of the Punjab; Lahore, Pakistan Published online: 27 Jun 2014.
Click for updates To cite this article: Yun Xie, Qinwei Yao, Azeem Mehmood Butt, Jia Guo, Zhou Tian, Xuli Bao, Hongxia Li, Qinghua Meng & Jun Lu (2014) Expression profiling of serum microRNA-101 in HBV-associated chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, Cancer Biology & Therapy, 15:9, 1248-1255, DOI: 10.4161/cbt.29688 To link to this article: http://dx.doi.org/10.4161/cbt.29688
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Research Paper Research Paper
Cancer Biology & Therapy 15:9, 1248–1255; September 2014; © 2014 Landes Bioscience
Expression profiling of serum microRNA-101 in HBV-associated chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma Yun Xie1,†, Qinwei Yao1,†, Azeem Mehmood Butt2, Jia Guo1, Zhou Tian1, Xuli Bao1, Hongxia Li1, Qinghua Meng1,*, and Jun Lu1,* Hepatology and Cancer Biotherapy Ward; Beijing YouAn Hospital; Capital Medical University; Beijing, PR China; 2Centre of Excellence in Molecular Biology (CEMB); University of the Punjab; Lahore, Pakistan These authors contributed equally to this work.
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Keywords: microRNA-101; circulating microRNA; hepatitis B; hepatocellular carcinoma; expression profiling Abbreviations: CHB, chronic hepatitis B; LC, liver cirrhosis; HCC, hepatocellular carcinoma; ROC, receiver operating characteristic; AFP, α-fetoprotein
MicroRNAs (miRNAs) represent a class of evolutionarily conserved, non-coding small RNAs (18–25 nt) that have emerged as master regulators of several biological processes. Recently, circulating miRNAs have also been reported to be promising biomarkers for various pathological conditions. In the present study, we report the comparative expression profiling of microRNA-101 (miR-101) in serum and tissue samples from chronic hepatitis B (CHB), HBV-associated liver cirrhosis (HBV-LC), and HBV-associated hepatocellular carcinoma (HBV-HCC) patients and healthy controls. The serum miR-101 levels were found to be significantly downregulated in the HBV-HCC patients compared with the HBV-LC patients (P < 0.001), CHB patients (P < 0.001) and healthy controls but were upregulated in the HBV-LC patients compared with the CHB patients (P < 0.001) and healthy controls (P < 0.001). Consistent with the serum data, the expression of miR-101 was also upregulated and downregulated in the HBV-LC and HBV-HCC tissue samples, respectively. A receiver operating characteristic (ROC) analysis of serum miR-101 yielded an area under the ROC curve (AUC) of 0.976 with 95.5% sensitivity and 90.2% specificity when differentiating between HBV-HCC and HBV-LC. Our results suggest that the serum miR-101 level can serve as a potential non-invasive biomarker to differentiate HBV-HCC from HBV-LC.
Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and ranks as the third leading cause of cancer mortality.1 Although there are different viral and non-viral causes of HCC, it has been reported that almost 80% of disease cases are associated with hepatitis B virus (HBV) infection. HCC is often diagnosed at an advanced stage when curative resection of the tumor is no longer appropriate because of intrahepatic and extrahepatic metastases. At present, the diagnosis of HCC relies on the identification of a liver mass by radiological imaging tests, including ultrasonography, CT, and/or magnetic resonance imaging (MRI). These imaging techniques are often combined with the use of serum tumor markers, such as α-fetoprotein (AFP), which has been discontinued in several western countries due to its poor sensitivity and specificity, but this protein is still widely used as a serum tumor marker in many Asian countries including China.2,3 MicroRNAs (miRNAs) are small noncoding RNA (18–24 nucleotides) molecules that have been reported to play important
roles in the regulation of several biological processes, such as cell proliferation, differentiation and apoptosis.4,5 The ability of miRNAs to bind specific sites in the 3′-UTR of target mRNAs leads to translational repression and/or mRNA degradation.6 The deregulation of miRNA function can often lead to disorder of the internal environment and eventually to carcinogenesis. Many miRNA genes are located in cancer-associated genomic regions or in fragile sites that are frequently amplified or deleted in human cancers, suggesting that these miRNAs play an important role in malignant transformation.7 Apart from their tissue-specific origin and expression, it has recently been shown that miRNAs are also stable and detectable in various body fluids such as serum and plasma.8 Similar to the expression patterns in tissue specimens, the expression of circulating miRNAs has been shown to vary between disease cases and healthy controls for several types of cancers and diseases. This observation suggested that the circulating miRNAs could serve as useful, non-invasive biomarkers of disease stage and progression. In the case of HCC, the circulating levels of several miRNAs including miR-21, miR-122, miR-15b, and miR-130b have been reported as potential diagnostic markers.9,10
*Correspondence to: Qinghua Meng; Email: [email protected]; Jun Lu; Email: [email protected] Submitted: 04/13/2014; Revised: 06/17/2014; Accepted: 06/22/2014; Published Online: 06/27/2014 http://dx.doi.org/10.4161/cbt.29688 1248
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Results Expression of miR-101 in liver tissues First, we measured the expression of miR-101 in CHB, HBVLC, HBV-HCC, and control tissue samples via in situ hybridization. The patients used for in situ hybridization were independent from those used for serum miRNA profiling, and their characteristics are given in Table S1. The expression levels of miR-101 in the CHB tissue samples were not significantly different from the levels in the normal liver tissue samples (P = 0.999). On the other hand, compared with the normal samples, the expression levels of miR-101 were upregulated in the HBV-LC tissue samples (P = 0.011) yet downregulated in the HBV-HCC tissue samples (P = 0.014) (Fig. 1A–E). Expression profile of circulating miRNA-101 To investigate whether levels of circulating miR-101 are also altered in patients with CHB, HBV-LC and HBV-HCC, the concentrations of miR-101 were measured in the sera from patients and healthy controls using quantitative real-time PCR. Consistent with our in situ hybridization data, although no significant difference (P = 1.000) was observed in the miR-101 serum levels between the control and CHB samples (Fig. 2), we found that serum miR-101 was significantly upregulated in the HBV-LC group compared with the CHB patients (P < 0.001) and control subjects (P < 0.001) (Fig. 2). We next investigated the levels of serum miR-101 in the HBV-HCC patients compared with the HBV-LC, CHB, and healthy subjects. The results showed that serum miR-101 was significantly downregulated in the HBVHCC patients compared with the HBV-LC (P < 0.001), CHB (P < 0.001), and healthy subjects (P = 0.003) (Fig. 2). Next, to explore the possible association between miR-101 and the risk of HBV-HCC derived from HBV-LC, binary logistic regression was used controlling for gender, age, alcohol status, and ALT at
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Figure 1. The expression levels of miR-101 in liver tissues. In situ hybridization was performed using locked nucleic acid (LNA)-modified probes for miR-101. Brown yellow staining in the cytoplasm represents positive staining for miR-101. (A) Normal liver tissues were graded positive for miR-101. (B) CHB liver tissues were graded positive for miR-101. (C) HBV-LC tissues were graded strong positive for miR-101. (D) HBV-HCC tissues were graded negative for miR-101. (E) The level of miR-101 expression in HBV-LC patients is higher than in healthy subjects and CHB patients, while it is lower in HBV-HCC patients. (* represents P < 0.05.)
the same level. A significantly decreased risk of HBV-HCC was found to be associated with upregulated miR-101. The odds ratio of developing HBV-HCC with upregulated miRNA-101 was 0.088 (95% CI = 0.032–0.244, P < 0.001). The correct percentage of the logistic regression model was 92.1%. The diagnostic potential of serum miR-101 To evaluate whether serum miR-101 can be used as a diagnostic marker for CHB, HBV-LC, and HBV-HCC, ROC curve analysis was performed. In the case of CHB compared with healthy controls, the AUC was 0.635 (95% CI: 0.548–0.716) with a sensitivity/specificity ratio of 84.8/40.4%, indicating that serum miR-101 may not be an optimal marker for CHB diagnosis (data not shown). This result parallels the lack of a
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Recently, genome-wide miRNA expression profiling has revealed overexpression of miR-101 in HCC tissues compared with normal/healthy controls.11 The aberrant expression and therapeutic potential of miR-101 was also demonstrated in another study showing that miR-101 is downregulated by the HBV X protein and induces epigenetic modifications in HBV-associated HCC (HBV-HCC) tissues.12 Although aberrant expression of circulating miR-101 has been reported previously in HBV-HCC, parallel expression profiling of miR-101 from the serum and tissues of patients at different stages of HBV-associated liver diseases have not yet been reported.13 The development of HCC is a multi-step process, and it is also unknown if serum miR-101 could be used as a marker for differentiating the different stages of HBV-associated liver disease. Therefore, in the present study, we performed serum miR-101 expression profiling of HBVassociated chronic HBV (CHB), HBV-associated liver cirrhosis (HBV-LC), and HBV-HCC patients to determine this marker’s diagnostic potential. Moreover, the hepatic miR-101 expression was also analyzed. For simplicity, the miR-101 that we analyzed in the present study will be referred to as miR-101 throughout the manuscript.
96.6/87.9% (Fig. 4E). This result is similar to the ROC analysis of miR-101 alone for differentiating HBV-LC from HBV-HCC (Fig. 4C). There was no correlation between the serum miR-101 expression levels and the patients’ clinical features, such as age, gender, ALT, T-Bil, PT, viral load, tumor size, Child–Pugh grade, AFP, or TNM classification (data not shown).
Figure 2. Serum miR-101 levels in healthy subjects, patients with CHB, patients with HBV-LC and patients with HBV-HCC. The median level of serum miR-101 was significantly higher in patients with HBV-LC than in patients with CHB or healthy subjects (P < 0.001), whereas the median level was significantly lower in patients with HBV-HCC than in healthy subjects (P = 0.012) or patients with HBV-LC (P < 0.001). The lines represent the range, median and quartiles of relative miRNA expression (ΔCt values) measured by qRT-PCR.
significant difference in the serum miR-101 levels between CHB and healthy controls described above. In the case of HBV-LC compared with healthy controls, the AUC was 0.884 (95% CI: 0.800–0.942), and the sensitivity and specificity calculated at the cut-off value of 10.50 were 80.3% and 80.0%, respectively (Fig. 3A). For HBV-HCC compared with healthy controls, the AUC was 0.788 (95% CI: 0.693–0.865), and at the cut-off value of 8.99, the sensitivity and specificity were 76.1% and 70.0%, respectively (Fig. 3B). We next investigated whether serum miR-101 has the potential to serve as a marker of disease progression or to differentiate between CHB, HBV-LC, and HBV-HCC patients. For the discrimination between patients with CHB and either HBV-LC or HBV-HCC, miR-101 had an AUC of 0.861 (95% CI: 0.792– 0.913) (Fig. 4A) and 0.777 (95% CI: 0.701–0.842), respectively (Fig. 4B). At the cut-off values of 10.91 (CHB compared with HBV-LC) and 9.70 (CHB compared with HBV-HCC), serum miR-101 had sensitivity/specificity ratios of 75.4/83.5% and 88.1/62.0%, respectively. In the case of HBV-LC compared with HBV-HCC, the AUC was 0.976 (95% CI: 0.931–0.995), and at a cut-off value of 10.08, the sensitivity and specificity of serum miR-101 were 95.5% and 90.2%, respectively (Fig. 4C). Next, the predicted probability of being diagnosed with HBVHCC from a stepwise logistic regression model based on AFP and miR-101 was used to construct the ROC curve. The AFP alone yielded an AUC of 0.762 (95% CI: 0.674–0.836) with 44.8% sensitivity and 96.6% specificity when differentiating HBVLC from HBV-HCC (Fig. 4D). The AUC of serum miR-101 combined with AFP was also calculated and found to be 0.973 (95% CI: 0.925–0.994) with a sensitivity/specificity ratio of
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The aberrant expression of miRNA-101 has been recently reported in several cancers, including ovarian, renal, prostate, gastric, and lung cancer,14-20 highlighting miR-101 as a bona fide tumor suppressor miRNA. Downregulation of miRNA-101 in association with HCC has also been reported in different studies that have collectively shown that miRNA-101 expression has the potential to inhibit HCC cell proliferation, suppress tumorigenicity, and promote apoptosis by modulating the expression of multiple transcription factors and cell cycle-related genes in HCC.21-23For instance, Su et al. reported the downregulation of miR-101 in HBV-HCC tissues and cell lines compared with controls. This study suggested that miR-101 downregulation is a frequent event in human HCC tissues that leads to hepatocarcinogenesis through the overexpression of Mcl-1, an antiapoptotic member of the Bcl-2 family, which has a complementary binding site for the miR-101 in its 3′-UTR that is a direct target of miR-101.19 Similarly, Zhang et al. reported the downregulation of miR-101 and parallel overexpression of SOX9, another direct target of miR-101, in clinical HCC tissues and the correlation of miR-101 downregulation with tumor aggressiveness and poor prognosis.22 As the association between miR-101 and HCC has been strengthened by the available data, it has been suggested that miR-101 could serve as a potential prognostic marker and therapeutic target for HCC.21-23 We were interested in determining the differences in miR-101 expression at different stages of HBVassociated liver diseases (CHB, HBV-LC, and HBV-HCC) and, moreover, whether miR-101 could serve as a non-invasive diagnostic biomarker. For this purpose, we performed expression analysis of miR-101 in the liver tissue and serum samples from CHB, HBV-LC, HBV-HCC, and control groups by in situ hybridization and real-time PCR, respectively. Two independent study groups were used for tissue and serum miRNA-101 analysis, and collectively, the results from each group were in agreement. We showed that the miRNA-101 levels in the sera and liver tissues were down- regulated in HBV-HCC patients but upregulated in HBV-LC patients compared with CHB patients and healthy controls. To evaluate the diagnostic potential of serum miR-101, ROC curves were generated. Our data showed that serum miR-101 is superior to AFP for diagnosing HBVHCC derived from HBV-LC, as combining serum miR-101 with AFP conferred no advantage over serum miR-101 alone for detecting HBV-HCC derived from HBV-LC. Combining the data from the tissue and serum miRNA levels, ROC curves
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Discussion
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and binary logistic analysis, we propose that serum miRNA-101 could serve as a non-invasive biomarker to monitor HBV-HCC that is derived from HBVLC and also the progression of CHB infection to HBV-LC. The progression of HBV-associated liver disease to HCC consists of several stages. Additionally, the expression of several miRNAs is stage-dependent in many diseases; some miRNAs show early expression, whereas some are expressed only during the advanced stages of disease. For instance, the liverspecific miRNA-122 has been reported Figure 3. ROC curve analysis of serum miR-101 for discriminating; (A) HBV-LC patients and healthy to be upregulated in the tissue and serum subjects, (B) HBV-HCC patients and healthy subjects. samples of CHB patients, yet downregulated in the tissue and serum samples of HBV-HCC patients.24 Other examples include miR-25 and monitoring the development of HBV-HCC from HBV-LC and let-7f, which were altered significantly in HBV-HCC patients but the development of HBV-LC from CHB. unchanged in CHB patients compared with controls.25 Based on our current analysis, we also suggest that miR-101 is a potential disease progression marker; while we observed no significant difMaterials and Methods ference in the expression of miR-101 in tissue and serum samples from CHB patients compared with healthy controls, a significant Study subjects and clinical characteristics expression difference emerged when comparing these samples to The study protocol was approved by the Ethics Committhose from HBV-LC patients (upregulation) and HBV-HCC tee of Beijing YouAn Hospital, Capital Medical University and patients (downregulation). In our opinion, however, the lack of a adhered to the tenets of the Declaration of Helsinki. Written significant difference between the CHB and control samples in informed consent was obtained from the participants for the use the present study can be attributed to the following factors. First, of their blood, tissue samples and clinical records in this study. the group used for serum analysis in this study consisted of CHB The study population consisted of 67 HBV-HCC patients, 61 patients with low staging and grading scores, which therefore did HBV-LC patients, 79 CHB patients and 30 healthy subjects. not have high hepatic activity index (HAI) scores. However, we The patients’ characteristics are summarized in Table 1. All also suspect that the inclusion of patients with higher inflam- patients were positive for HBsAg and did not have any other mation and fibrosis scores in the CHB group may bias the data liver diseases, such as CHC, alcoholic liver disease, autoimmune toward a false-positive result, as patients with liver cirrhosis usu- liver disease, or metabolic liver disease, based on clinical reports. ally have higher HAI scores and the inclusion of these patients in The degree of inflammation of all of the patients was between the CHB group may lead to an increase in the total level of miR- G1 and G2. The fibrosis staging of the CHB patients was 101 expression in this group. Another possible explanation is that between S0 and S2. The fibrosis staging of the HBV-LC and the transcription factors associated with liver inflammation may HBV-HCC patients was between S3 and S4. The blood samples not be targets of miR-101; the previously reported targets of miR- from the HBV-HCC patients were obtained before the surgical 101 that are liver-associated transcription factors have mostly resections were performed. The data on all of the subjects were been those involved in hepatocarcinogenesis and advanced-stage obtained from medical records, pathology reports, and personal liver diseases.21,22 However, these speculations warrant further interviews with the subjects. The data collected included age, investigations with a larger sample size. Similarly, the inclusion gender, serum albumin (ALB) level, total bilirubin level (T-Bil), of matched liver samples before and after HCC development alanine aminotransferase (ALT) level, prothrombin time (PT), could lead to a better understanding of the dynamics of miR-101 HBV DNA viral load, AFP, tumor number and size, tumor difexpression in HCC progression as miRNA-101 has been linked ferentiation, tumor stage, relapse time, and time of death. The to tumor migration and recurrence.16,26,27 Finally, whether miR- clinical stage of HBV-HCC was evaluated based on the TNM 101 has the same function in other liver disease and HCC etiolo- classification system. Child–Pugh scoring was performed to catgies such as HCV and alcoholic cirrhosis remains unclear. egorize the LC and HCC patients at Child–Pugh grades A, B, Taken together, the findings from our present study demon- or C. strate that miR-101 levels are elevated in the sera and liver tisSerum preparation sues of HBV-LC patients and decreased in HBV-HCC patients, Ten milliliters of peripheral blood was collected from each which suggests that serum miR-101 is a potential biomarker for individual directly into serum tubes at the time of liver biopsy
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RNA extraction and reverse transcription Total RNA was extracted from 200 μL of serum using a QuantoBio Total RNA Isolation Kit (QuantoBio Co.) following the manufacturer’s protocol. Briefly, 200 μL of lysis buffer and 40 μL of lysis enhancer solution were added to each sample and vortexed for 15 s. For the normalization of sampleto-sample variation during RNA isolation and as an internal control, 25 fmol of synthetic Caenorhabditis elegans miRNA-39 (cel-miR-39-3p) was added to each lysed sample as described previously.8 This step was followed by the addition of 440 μL of acidified phenol:chloroform and vigorous shaking for 30 s. The samples were centrifuged at 16 000 g in a microcentrifuge at 4 °C for 10 min to separate the aqueous phase (upper phase) and the organic phase (lower phase). The aqueous phase was then transferred to new RNase-free microcentrifuge DNA LoBind tubes (Eppendorf). Ethanol was added to the aqueous phase to a final concentration of 30%, and this mixture was vigorously shaken for 15 s. Then, the 650 μL mixtures were centrifuged at 10 000 g at room temperature for 30 s to pellet the RNA. After the washing steps, the total RNA including miRNAs was resuspended in 50 μL RNase-free water and stored at −80 °C until further use. The concentration and purity of the total RNA was determined using a NanoDrop 8000 (Thermo Fisher Scientific). cDNA was reverse transcribed from the total RNA via the polyadenylation method. Briefly, 1 ng of the total RNA template was mixed with 2 μL 10× buffer, 2 μL dATP (10 mM), 0.5 μL poly(A) polymerase (NEB), Figure 4. ROC curve analysis of serum miR-101 for discriminating; (A) CHB patients from HBV-LC patients, (B) CHB patients from HBV-HCC patients, (C) HBV-LC patients from HBV-HCC patients, (D) AFP 0.5 μL RNase inhibitor (Promega), alone for discriminating HBV-LC from HBV-HCC patients. (E) AFP in combination with miR-101 for disand RNase-free water (Promega) to a criminating HBV-HCC from HBV-LC patients. final volume of 20 μL and incubated at 37 °C for 1 h. Then, 1 μL of 0.5 μg/μL or before surgery. The tubes were initially centrifuged at 1500 g RT primer was added, and the reactions were incubated at 70 °C for 10 min. The serum was then aliquoted and additionally cen- for 5 min followed by immediate incubation on ice for at least trifuged at 13 000 g for 15 min at 4 °C to completely remove the 2 min to disrupt the secondary structures of the RNA and the cell debris and any remaining cells. The supernatant was then primer. In the final step, the 20 μL reaction mixture from the transferred to new 2.0-mL tubes without disturbing the pelleted above step was mixed with 4 μL 5× buffer, 1 μL dNTP (10 mM) debris. Any samples with signs of hemolysis were excluded from (Sigma), 0.5 μL M-MLV reverse transcriptase (Promega), 0.5 μL the study. The serum samples were then stored at −80 °C until RNase inhibitor (Promega), 10 μL A-Plus reaction mix, and further use. 4 μL RNase free water (Promega), and incubated at 42 °C for
Table 1. Summary of clinical characteristics of CHB, HBV-LC, HBV-HCC and healthy subjects
Gender n (%)
Group II: HBV-L (n = 61)
Group III: HBVHCC (n = 67)
Group IV: Control (n = 30)
P values
Mean ± SD
32.38 ± 11.93
51.19 ± 9.57
51.69 ± 10.43
37.26 ± 10.79
