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Serum MicroRNAs as Biomarkers for Hepatocellular Carcinoma in Chinese Patients with Chronic Hepatitis B Virus Infection
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Serum MicroRNAs as Biomarkers for Hepatocellular Carcinoma in ...
www.plosone.org/.../info%3Adoi%2F10.1371%2Fjournal.pone.0028...
by P Qi - 2011
(2011) Serum MicroRNAs as Biomarkers for Hepatocellular Carcinoma in Chinese Patients with Chronic Hepatitis B Virus Infection. PLoS ONE 6(12): e28486
Peng Qi1#, Shu-qun Cheng2#, Hao Wang3, Nan Li2, Yue-feng Chen1, Chun-fang Gao1*
1 Department of Laboratory Medicine, Second Military Medical University, Eastern Hepatobiliary Hospital, Shanghai, China, 2 Department of Oncology Comprehensive Treatment, Second Military Medical University, Eastern Hepatobiliary Hospital, Shanghai, China, 3 Department of Laboratory Medicine, Second Military Medical University, Changzheng Hospital, Shanghai, China
Abstract
Background
MicroRNAs (miRNAs) have been shown to anticipate great cancer diagnostic potential. Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions. The objective of this study was to investigate the potential of serum miRNAs as novel biomarkers for hepatocellular carcinoma (HCC).
Methodology/Principal Findings
This study was divided into four phases: (I) Ten candidate serum miRNAs were detected by using real-time RT-PCR, corresponding 10 HCC patients with chronic hepatitis B virus (HBV) infection and 10 age- and sex-matched healthy subjects. (II) Marker validation by real-time RT-PCR on HBV patients with (n = 48) or without HCC (n = 48), and healthy subjects (n = 24). (III) Marker detection by real-time RT-PCR in sera from another 14 HCC patients before and 1 month after surgical resection. (IV) We examined the correlation between the expressions of candidate serum miRNAs with clinical parameters of HCC patients. Although miR-222, miR-223 or miR-21 were significantly up- or down-regulated between HCC patients and healthy controls, no significant difference was observed in the levels of these miRNAs between HBV patients without and with HCC. MiR-122 in serum was significantly higher in HCC patients than healthy controls (p<0.001). More importantly, it was found that the levels of miR-122 were significantly reduced in the post-operative serum samples when compared to the pre-operative samples. Although serum miR-122 was also elevated in HBV patients with HCC comparing with those without HCC, the difference was at the border line (p = 0.043).
Conclusions/Significance
Our results suggest that serum miR-122 might serve as a novel and potential noninvasive biomarker for detection of HCC in healthy subjects, moreover, it might serve as a novel biomarker for liver injury but not specifically for detection of HCC in chronic HBV infection patients.
Introduction
Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancers and it represents the third most common cause of death from cancer worldwide, with an increasing incidence expected in the next decades [1]. The major risk factors are chronic viral hepatitis B and C (HBV, HCV), alcohol abuse, primary biliary cirrhosis, xenobiotics, diabetes, non-alcoholic fatty liver disease and genetic disorders like haemochromatosis and α1-antitrypsin deficiency [2], [3]. In China, HCC is the second highest cancer killer since the 1990s [4] and HBV infection is highly endemic. The high mortality rate is due to its detection at late stage with limited therapeutic options. Indeed, the clinical heterogeneity of HCC and the lack of good diagnostic markers and treatment strategies have rendered the disease a major challenge.
The search for biomarkers for the diagnosis of diseases has become a rapidly growing area of clinical research. Ideally, biomarkers should be easily accessible such that they can be sampled non-invasively. Therefore biomarkers that can be sampled from body fluids, such as serum or urine, are particularly desirable. Circulating nucleic acids (CNAs) are extracellular nucleic acids found in cell-free serum, plasma and other body fluids from healthy subjects as well as from patients. The ability to detect and quantitate specific DNA and RNA sequences has opened up the possibility of diagnosis and monitoring of diseases, especially in the field of cancer [5]. Furthermore, in some recent studies it has been suggested a kind of non-coding RNA-microRNA (miRNA), also exist in cell-free serum and plasma, highlighting the field of using CNAs to diagnose cancer.
MiRNAs are a group of tiny RNAs with a fundamental role in the regulation of gene expression. Aberrant expression of several miRNAs was found to be involved in a large variety of neoplasms [6], including HCC [7]-[12]. A relevant and important feature of miRNAs is their remarkable stability. They are known to be well preserved in tissue samples even after years of formalin-fixation and paraffinembedding, and can be efficiently extracted from and quantified in such specimens [13]. Investigation of cancer-specific miRNAs in the circulation is an emerging and exciting field of study. One of the first studies measuring miRNA levels in serum was reported by Lawrie et al. [14], who showed that sera levels of miR-21 were associated with relapse-free survival in patients with diffuse large B-cell lymphoma. Subsequently, circulating miRNAs have been postulated as novel biomarkers for cancer, and other disease processes [15]-[27]. To date, there have been no systematical reports on the role of circulating miRNAs in HCC, and it is not fully understood whether serum miRNAs have a clinicopathological influence in HCC. We hypothesized that levels of specific cancer-associated miRNAs in circulation would differ between HCC patients and chronic HBV infection patients without HCC or healthy individuals. If this hypothesis held truth, it would signify a major breakthrough in HCC management, bringing us ever closer to finding a novel, sensitive, and noninvasive biomarker for this common disease.
The primary aim of this study was to investigate whether cancer-specific miRNAs are detectable and altered in serum of HCC patients compared with age- and sex-matched disease and healthy controls. We also collected serum samples from HCC patients before and after the tumor resection, and these samples were used to determine whether those up-regulated markers in cancer serum were reduced after the tumor resection. Finally, a potential relationship between circulating miRNAs levels and existing clinicopathological features of HCC, such as tumor number, size, growth phase, stage, Child-pugh grade and overall survival, was investigated.
Results
Patient Characteristics
A total of 152 participants including 70 HBV-positive HCC patients, 48 chronic HBV infection patients without HCC, and 34 normal subjects were recruited into this study (Table 1). There were no significant differences of age (t-test) and sex (Pearson χ2 test) between cases and controls. In addition, the HCC group and the other two controls groups had statistically different laboratory results for ALB, T-Bil and ALT (p<0.001).
With regard to clinicopathologic characteristics of HCC patients, single tumor was found in 48 patients (68.6%), tumor diameter was <5 cm in 17 patients (24.3%), 51 patients were also with hepatic cirrhosis. The histologic grade of HCC was grade I-II in 14 cases, grade III-IV in 51 cases. Tumor stage was obtained according to the TNM criteria, 8, 36 and 20 patients was in stage I, II, III, and only 6 patients was in stage IV. According to the Child classification [28], 59, 8 and 3 patients was with mild (grade A), moderate (grade B) and severe (grade C) liver damage, respectively. All HCC patients had completed follow-up. Forty-one patients who survived more than 20 months (average, 25.65 months; range, 21.3 to 31.3 months) on the last follow-up were classified as the longer-survival group, whereas the rest twenty-nine patients who had survival times less than 20 months (average, 10.93 months; range, 5.3 to 19.2 months) were classified as the shorter-survival group.
Identification of HCC-associated MiRNAs in Serum
The goal of the present study was to explore the potential use of serum miRNAs as biomarkers for HCC. In this marker discovery phase, a panel of 10 cancer associated miRNAs was chosen on the basis of their reported relevance to HCC [7]-[12] and detected by RT-qPCR among 10 HCC patients and 10 healthy subjects. Using miR-16 as normalization control, 3 significantly up-regulated miRNAs (miR-122, miR-222 and miR-223) and 1 significantly down-regulated miRNA (miR-21) in serum in HCC patients were identified (Figure 1 A-,B, C, D), while expression levels of miR-221 and miR-301 in serum were non-significantly higher in HCC patients than in healthy subjects (Figure 1 E, F). As for miR-224, let-7a and miR-199a, the detection rates were <50% in serum samples by RT-qPCR, thus, these miRNAs were not chosen in further analytic studies.
Marker Selection and Validation in Serum Samples
To validate the 4 putative markers identified from the marker discovery phase, RT-qPCR assays were developed to quantify miRNAs in serum among 48 HCC patients, 48 HBV patients without HCC and 24 healthy subjects. Our data indicated that expression levels of miR-122 in serum were significantly higher in HCC patients than disease controls (p<0.05) or healthy controls (p<0.001) (Figure 2 A). Although levels of miR-222 and miR-223 were also significantly elevated in HCC patients than in healthy controls (p<0.05), no significant difference was observed for these two miRNAs between HBV subjects with and without HCC (p>0.05) (Figure 2 B, C). Interestingly, the levels of miR-223 in serum of HBV patients without HCC were non-significantly higher than those in patients with HCC. In addition, levels of miR-21 were reduced in HCC patients than in healthy controls (p<0.05), no significant difference was observed for miR-21 between HBV subjects with and without HCC (p>0.05) (Figure 2 D).
In order to prove circulating miR-122 in serum is of tumor origin, its levels were measured in an independent set of 14 HCC patients (before and one month after surgical removal of the tumors). It was found that the levels of miR-122 were significantly reduced in the post-operative serum samples when compared to the pre-operative samples, reaching levels comparable with healthy subjects (Figure 3).
The Diagnostic Value of MiR-122 for HCC
To evaluate whether serum miR-122 can be used as a potential diagnostic marker for HCC, ROC curve analyses were performed. It was revealed that serum miR-122 was a potential marker for discriminating HCC patients from healthy controls with an AUC (the areas under the ROC curve) of 0.869 (95% CI: 0.786-0.952) (Figure 4 A). At the cut-off value of 0.475, the sensitivity and specificity for this marker was 81.6% and 83.3%. However, the AUC of serum miR-122 for discriminating HBV patients with HCC from those without HCC was only 0.630 (95% CI: 0.516-0.743) (Figure 4 B). At the cut-off value of 0.651, the sensitivity and specificity for this marker were 77.6% and 57.8%.
Relationship of Circulating MiRNAs to Clinicopathological Parameters
It was reported that some unique miRNA signatures were associated with prognostic factors and disease progression in several cancers. Therefore, we examined the correlation between the expression of circulating miR-122, miR-222, miR-223 and miR-21 with clinical parameters. No significant association was found between the four miRNAs and tumor number, size, growth phase, stage, Child-pugh grade and overall survival (p>0.05), while the levels of miR-122 demonstrated an elevation trend in patients with small tumor (p = 0.055). In addition, large tumor patients were found to have non-significantly higher levels, on average, of serum miR-21 compared with those with small tumor (p = 0.051). A potential relationship between circulating miRNA levels and status of liver cirrhosis, was also investigated, but no statistically significant difference was identified for any of these parameters.
Discussion
HCC represents an extremely poor prognostic cancer that remains one of the most common and aggressive human malignancies worldwide. The early diagnosis of HCC is of great clinical desirable and the improved prognosis of HCC if the patients could get surgical treatment early. Up to now, alpha-fetoprotein (α-AFP) has mainly been used in clinic for diagnosis of primary HCC; however, its sensitivity and specificity are not satisfying [29], novel biomarkers for early HCC diagnosis are greatly needed.
Results from recent studies revealed that circulating miRNAs are potential diagnostic biomarkers and prognostic factors in various kinds of diseases, especially in the field of cancer. The first serum miRNA biomarker discovered was miR-21. Lawrie et al. found that patients with diffuse large B cell lymphoma had high serum levels of miR-21, which associated with increased relapse-free survival [14]. Mitchell et al. demonstrated the presence of circulating tumor-derived miRNAs in blood by using a mouse prostate cancer xenograft model system and showed that measurements obtained from plasma were strongly correlated with those obtained from sera, suggesting that both serum and plasma samples would be adequate for measuring specific miRNA levels [16]. In another study, Chen et al. demonstrated that by using serum directly or by extracting RNA from the serum they could identify unique miRNA expression profiles for lung cancer, colorectal cancer and diabetes patients compared with healthy subjects [15]. Circulating miRNAs have also been postulated as novel biomarkers for ovarian cancer [17], [21], pancreatic cancer [19] and colorectal cancer [23], [24]. Although the clinical significance of these findings has not been elucidated in detail, those findings demonstrated that circulating miRNAs could be noninvasive diagnostic or prognostic markers for cancer.
In this study, we confirm that some miRNAs can be measured from a relatively small amount of serum. In addition, as there is no current consensus on the use of house-keeping miRNAs for RT-qPCR analysis, based on previously published results [14], [23] and as recommended by the manufacturer (Applied Biosystems), we used miR-16 levels for normalization. We found that the levels of miR-122 in serum samples from HCC patients were significantly higher than healthy subjects (p<0.001). Although serum miR-122 was also elevated in HBV patients with HCC comparing with those without HCC, the difference was at the border line (p = 0.043). Serum miR-122 yielded an AUC of 0.869 (95% CI: 0.786-0.952) for discriminating HCC from healthy subjects and only 0.630 (95% CI: 0.516-0.743) for discriminating HBV patients with HCC from those without HCC. At a cut-off value of 0.474, the sensitivity was 81.6% and the specificity was 83.3% in discriminating HCC from healthy subjects, and 77.6% sensitivity and 57.8% specificity in discriminating HBV patients with HCC from those without HCC at the cut-off value of 0.651. More importantly, it was found that the levels of miR-122 were significantly reduced in the post-operative serum samples when compared to the pre-operative samples, reaching levels comparable with healthy subjects, indicating that the elevation of serum miR-122 is likely derived from HCC.
MiR-122 not only is evolutionary conserved across species and but also was identified as the most abundant liver specific miRNA constituting 70% of total hepatic miRNAs while cloning small RNAs from different tissues in mice [30], [31]. MiR-122 facilitates replication [32] and translation [33] of hepatitis C viral RNA and positively regulates cholesterol and triglyceride level [34], [35]. Significantly, the down-regulation of miR-122 was detected in more than 70% of HCC [36]. It was shown that the level of miR-122 expression increases in the mouse liver throughout development, to reach the maximum just before birth. Thus, the loss of expression of miR-122 of HCC cells may represent either a differentiation reversion or a block to a less differentiated status of liver cells. In our study, it appears contrary and unexpected that the levels of miR-122 are elevated in serum of HCC patients. Our results showed that the elevated serum miR-122 is presented not only in HBV patients with HCC but also in HBV patients without HCC, suggesting that the elevated miR-122 in the serum of patients may also reflect liver injury [22], [37]. Hepatocytes contain abundant miR-122 and damage of hepatocytes caused by inflammation due to virus infection or cancer would be expected to release significant amount of this miRNA into the circulation. Because serum miRNAs have been shown to be very stable [16], miRNAs leaked from damaged hepatocytes would accumulate in blood to a high level. This might explain why miR-122 is down-regulated in HCC tissues but elevated in serum of HBV patients without or with HCC. Interestingly, our data indicated that expression levels of miR-122 in serum were significantly higher in HCC patients than disease controls or healthy controls, while Xu et al. showed that expression levels of serum miR-122 were significantly higher in HBV patients than HCC or healthy controls [26]. The reason may be that we and Xu et al. use the different normalization control (miR-16 vs miR-181a and miR-181c).
MiR-223 is one of the miRNAs that has been given much attention in the literature. This miRNA is usually regarded as a bone marrow specific miRNA that functions as an important modulator of cellular differentiation [38], [39]. In addition to this, a recent study observed that miR-223 was commonly repressed in HCC [11], suggesting a potential role of this miRNA in liver disease. In our study, levels of miR-223 were significantly elevated in HCC patients than in healthy controls, while no significant difference was observed for this miRNA between HBV subjects with and without HCC. Moreover, the levels of miR-223 in serum of HBV patients without HCC were higher than those in HCC patients or healthy subjects. This finding points out that elevated serum miR-223 could also come from tissue injury such as hepatitis. Since patients with chronic hepatitis may have more serious damage of hepatocytes than patients with HCC, it is reasonable to see much higher level of serum miR-223 in patients with chronic B hepatitis than in patients with HCC. For example, similar results have been obtained in the previous study, showing that elevation of serum miR-223 come from hepatic ischemia/reperfusion injury [40].
MiR-21 is one of the most prominent miRNAs implicated in the genesis and progression of human cancer. The earliest study showed that miR-21 is commonly and markedly up-regulated in human glioblastoma, and inhibition of miR-21 expression leads to caspase activation and associated apoptotic cell death in multiple glioblastoma cell lines [41]. Subsequently, there is a growing body of evidence to prove that miR-21 is overexpressed in a variety of tumors such as breast cancer [41], lung cancer [42], colon cancer [43], [44], and HCC [7], [8] with proproliferative and anti-apoptotic function. Recent studies have shown that serum miR-21 levels are significantly increased in patients with defused large B-cell lymphoma or ovarian cancer [14], [21]. In the present study, however, we found lower levels of serum miR-21 in HBV patients without or with HCC than in healthy controls. The role played by circulating miRNAs is also poorly understood. The detected circulating miRNAs might be derived from dying tumor cells, from tumor cells that have been lysed, from cells infiltrating the lymphomas, from other tissues affected by ongoing diseases, or because the tumor cells actively secrete miRNAs into the surrounding environment. These results indicate that miR-21 can act as either oncogene or tumor suppressor, depending on the targets they regulate and the upstream factors that can regulate dysfunction of miR-21. Clearly, the exact role of miR-21 in cancer needs to be fully investigated in the future.
Although the diagnostic efficiency of serum miR-122 may not be optimal, a panel of serum miRNA markers may improve the sensitivity and specificity of this assay for HCC screening. Patients with increased serum miRNAs might prompt more accurate and specific clinical examinations.
In conclusion, serum miR-122 might serve as a novel and potential biomarker for detection of HCC in healthy subjects. Moreover, it might serve as a novel biomarker for liver injury but not specifically for detection of HCC in chronic HBV infection patients. Our data serve as basis for further investigation, preferably in large prospective studies before miR-122 can be used as a noninvasive screening tool for HCC in routine clinical practice.
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