Clinica Chimica Acta 450 (2015) 203–209
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Serum microRNA-210 levels in different groups of chronic hepatitis B patients Fujun Yu a, Jianhuan Yang b, Jinsheng Ouyang c, Yihu Zheng d, Bicheng Chen e, Guojun Li f, Zhongqiu Lu g, Peihong Dong a,⁎,1, Jianjian Zheng e,⁎⁎,1 a
Department of Infectious Diseases, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China Department of Children's internal medicine, The Second Affiliated Hospital & Yuying Children's hospital of Wenzhou Medical University, Wenzhou 325000, PR China Department of Respiratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China d Department of General Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China e Key Laboratory of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China f Department of Hepatology, Ningbo Yinzhou Second Hospital, Ningbo 315000, PR China g Emergency Department, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China b c
a r t i c l e
i n f o
Article history: Received 24 June 2015 Received in revised form 31 July 2015 Accepted 25 August 2015 Available online 28 August 2015 Keywords: MicroRNA-210 HBV carrier HBV DNA HBs antigen Chronic hepatitis B
a b s t r a c t Background: It has been reported that hepatitis B virus (HBV) replication can be suppressed by microRNA-210 (miR-210). However, whether serum miR-210 levels can serve as disease parameters in patients with chronic hepatitis B (CHB) remains unclear. Methods: Serum miR-210 levels were quantified in 115 CHB patients and 20 healthy controls by real-time PCR. Results: We found that serum miR-210 levels can discriminate the different groups of CHB patients from healthy control (P b 0.05), as well as patients with HBe antigen positive from those with HBe antigen negative (P b 0.05). Serum miR-210 levels correlated with HBV DNA and HBs antigen (r = 0.525, P b 0.001 and r = 0.348, P b 0.001). Notably, inactive carrier patients with high (N3500 IU/mL) or low (b 3500 IU/mL) levels of HBs antigen were differentiated by serum miR-210 levels (P b 0.05). Moreover, serum miR-210 levels correlated with liver inflammatory activity markers including alanine aminotransferase (ALT) and HAI score. However, there was no correlation of serum miR-210 levels with parameters of liver function including serum albumin, international normalized ratio and bilirubin, as well as the stages of liver fibrosis. Conclusions: Serum miR-210 can be used as an indicator of HBV replication and translation, and a potential marker of necroinflammation in patients with CHB. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chronic infection with the hepatitis B virus (HBV), accounting for 350–400 millions of patients worldwide, is a predominant etiological factor for liver disease in China [1,2]. In particular, about 7.8% of China population are HBV carriers (93 million, two-thirds of the world's total number of carriers) [3]. Once the infection becomes chronic, chronic hepatitis B (CHB) patients are at the risk of developing liver
Abbreviations: HBV, hepatitis B virus,; CHB, chronic hepatitis B; HCC, hepatocellular carcinoma; miRNA, microRNA; HAI, histological activity index; ALT, alanine aminotransferase; INR, international normalized ratio; cccDNA, covalently closed circular DNA; HIV, human immunodeficiency virus; HCV, hepatitis C virus; Ct, cycle threshold. ⁎ Correspondence to: P. Dong, Department of Infectious Diseases, The First Affiliated Hospital of Wenzhou Medical University, No. 2 FuXue lane, Wenzhou, Zhejiang, PR China. ⁎⁎ Correspondence to: J. Zheng, Key Laboratory of Surgery, The First Affiliated Hospital of Wenzhou Medical University, No. 2 FuXue lane, Wenzhou, Zhejiang, PR China. E-mail addresses: [email protected] (P. Dong), [email protected] (J. Zheng). 1 These authors share co-corresponding author.
http://dx.doi.org/10.1016/j.cca.2015.08.022 0009-8981/© 2015 Elsevier B.V. All rights reserved.
cirrhosis and hepatocellular carcinoma (HCC). The increased risks of liver cirrhosis and HCC associated with HBV DNA level and the persistence of HBe antigen have been well documented [4]. MicroRNAs (miRNAs) are evolutionarily highly conserved, small (18–24 nucleotides long) non-coding RNAs and play a vital role in many physiological processes, including cellular development, differentiation, metabolism, apoptosis and proliferation [5]. They are additionally implicated in pathogenesis of chronic inflammation, cardiovascular disease and cancer [6–8]. Notably, it has been demonstrated that miRNAs have entered the stage of virology. For example, Zhang et al. found that miR-210 negatively regulates HBV replication by targeting the HBV pre-S1 region in HepG2 2.2.15 cells under normoxic condition [9]. Coppola et al. reported that liver hsa-miR-125a-5p correlates with HBV replication and disease progression [10]. Recently, it has been demonstrated that cell-free miRNAs could circulate in the blood and serve as stable biomarkers for diseases [11–14]. Here, we aimed to study the significance of serum miR-210 levels in treatment-naive patients with different stages of CHB and compared them with surrogate markers of viral replication and translation.
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2. Methods
2.2. Liver histology
2.1. Patient selection
Liver biopsy was performed using a 16-gauge Menghini needle. Each liver biopsy case was advised by physicians in care, and liver specimens at least 2.0 cm in length were obtained. Samples were fixed in formalin, embedded in paraffin, and stained with hematoxylin–eosin. Results were reviewed by experienced hepatopathologists [16]. In addition, at least 8–10 portal tracts in samples are required to admit patients. Histological activity index (HAI) and fibrosis stages (F0 = no fibrosis–F6 = cirrhosis) were assessed according to the Ishak scoring system [17].
Serum samples were obtained from patients attending the First Affiliated Hospital of Wenzhou Medical University in Wenzhou and Ningbo Yinzhou Second Hospital in Ningbo from 2007.1 to 2012.8 (Table 1). 115 treatment-naive patients with CHB were included in this study and underwent liver biopsy. Inclusion criteria were CHB defined by detectable serum HBs antigen and serum HBV DNA for more than six months. Exclusion criteria were: (i) patients aged less than 16 years, (ii) coinfection with human immunodeficiency virus (HIV), (iii) coexistence of liver injury caused by other etiologies, including hepatitis C virus (HCV) infection, drug intake, alcohol consumption and auto-immune hepatitis, (iv) severe systematic diseases, and (v) pregnancy and lactation [15]. Demographic and clinical information was obtained, and blood samples collected from all patients on the day of diagnostic liver biopsy. 20 healthy controls (with normal liver biochemistry, no history of liver disease or alcohol abuse and no viral hepatitis) served as control patients. Written informed consent for the use of blood samples was obtained from all participants. The project was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). All procedures were performed in accordance with the current international guidelines, standards on human experimentation of the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China), and the Helsinki Declaration of 1975, revised in 1983.
Table 1 Patient characteristics. Parameter
CHB patients
Healthy controls
Epidemiology Gender, m/f (%) Age, years, median (range)
69/46 (60.0/40.0) 53 (41.7, 61.2)
11/9 (55.0/45.0) 50 (40.3, 59.6)
Virology HBs antigen (log10 IU/mL), median (range) HBV DNA (log10 IU/mL), median (range) HBe antigen positive, n (%) HBe antigen negative Inactive carriers, n (%) Low replicative, elevated ALT n (%) High replicative, normal ALT n (%) High replicative, elevated ALT n (%)
3.60 (1.50, 5.00) 3.88 (1.69, 8.91) 16 (13.9) 20 (17.4) 9 (7.8) 31 (26.9) 39 (33.9)
HAI 2, n (%) 3, n (%) 4, n (%) 5, n (%) 6, n (%) 7, n (%) 8, n (%) 9, n (%) ≥11, n (%)
13 (11.3%) 25 (21.7%) 8 (7.0%) 10 (8.7%) 12 (10.4%) 16 (13.9%) 12 (10.4%) 9 (7.8%) 10 (8.7%)
Fibrosis F0, n (%) F1, n (%) F2, n (%) F3, n (%) F4, n (%) F5, n (%) F6, n (%)
15 (13.0%) 19 (16.5%) 24 (20.9%) 20 (17.4%) 21 (18.3%) 6 (5.2%) 10 (8.7%)
Inactive carriers: patients with low replicative (HBV DNA b 2000 IU/mL) and normal ALT (b40 U/L); low replicative HBe antigen negative elevated ALT: patients with low replicative (HBV DNA b2000 IU/mL) and elevated ALT (N40 U/L); high replicative HBe antigen negative normal ALT: patients with high replicative (HBV DNA N2000 IU/mL) and normal ALT (b40 U/L); high replicative HBe antigen negative elevated ALT: patients with high replicative (HBV DNA N2000 IU/mL) and elevated ALT (N40 U/L); HBe antigen positive: patients with HBe antigen-positive hepatitis.
2.3. Blood sample processing Blood samples were centrifuged at 3400 g for 7 min at room temperature within 4 h of acquisition. Sera were transferred into Eppendorf tubes and additionally centrifuged at 12,000 g for 10 min at 4 °C to remove the remaining cells. Serum samples were stored at − 80 °C pending RNA extraction.
2.4. Detection of miR-210 with real-time quantitative RT-PCR According to the manufacturer's instructions for liquid samples, total RNA was extracted with the miRNeasy Mini Kit (Qiagen, Carlsbad, California, USA). Then DNase treatment (Qiagen, Carlsbad, California, USA) was performed to remove DNA contaminants in total RNA and final elution volume was 20 μL. In addition, serum RNA preparations were quantified using NanoDrop 1000 (Nanodrop, Wilmingtion, Delaware, USA) before the reverse transcription reaction. Reverse transcription was performed in a 20 μL reaction volume using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). For cDNA synthesis, reaction mixtures were sequentially incubated at 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. miR-210 was quantified in triplicate via qRT-PCR using the TaqMan MicroRNA Assay Kit (Applied Biosystems, Foster City, CA). According to the standard TaqMan MicroRNA assay protocol, real-time PCR was performed on an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA) under the following conditions: 95 °C for 10 min, followed by 35 cycles of 95 °C for 15 s and 60 °C for 60 s. Each PCR mixture (20 μL) included the reverse transcription products, TaqMan 2X Universal PCR Master Mix without UNG Amperase, miRNA-specific TaqMan probes, and primers supplied by Applied Biosystems. The cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to cross the threshold in qPCR. Ct values were calculated with SDS 2.0.1 software (Applied Biosystems, Foster City, CA). The formula 2−ΔCt was used to calculate the miRNA levels in serum, where ΔCt = mean (Ct of internal references) − Ct of target miRNA. The relative expression levels of miR210 were calculated and normalized to miR-16 (Applied Biosystems, Foster City, CA) using the comparativeΔCt method and the equation 2−ΔCt, as described previously [18]. 2.5. Serum HBV DNA and serum hepatitis marker analysis For all patients, serum HBV DNA was quantified using an Artus HBV QS-RGQ Kit (Qiagen, Hilden, Germany), with a lower detection limit of 10.2 IU/mL. HBsAg, HBeAg, and antibodies against HBsAg (anti-HBs), HBeAg (anti-HBe) and hepatitis B core antigen (anti-HBc) were determined using the Roche Modular E170 Immunoassay Analyzer (Roche, Basel, Switzerland). HBsAg titres were additionally determined with the HBsAg quantitative assay (Abbott Laboratories, Abbott Park, IL, USA) based on an automated chemiluminescent microparticle immunoassay (Abbott Architect i2000SR analyzer; Abbott Laboratories), according to the manufacturer's instructions. Architect HBsAg QT measures a range of HBsAg from 0.05 to 250 IU/mL. Therefore, the dilution for samples with higher HBsAg titre is required.
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2.6. Statistical analysis
3.2. The levels of serum miR-210 and HBV DNA in CHB patients
Data were analyzed using SPSS 13.0 (IBM, Armonk, NY). Statistical significance was determined using nonparametric Kruskal–Wallis or nonparametric Wilcoxon–Mann–Whitney test. Correlation coefficients (r) were calculated using Spearman correlation. In the box plots, vertical lines indicate the range, and horizontal boundaries of the boxes represent the first and third quartiles. Data were considered significant at P b 0.05.
To assess whether serum miR-210 level was associated with viral replication, the correlation between serum miR-210 concentrations and the viral load in CHB patients was analyzed. Serum miR-210 was significantly correlated with HBV DNA in all sera from CHB patients (r = 0.525, P b 0.001, Fig. 2A). According to HBe antigen status, CHB patients were divided into two subgroups: HBe antigen-positive and HBe antigen-negative. Then, the correlation between serum miR-210 and HBV DNA was further investigated in HBe antigen-positive and HBe antigen-negative patients. Accordingly, there was a significant correlation between serum miR-210 and HBV DNA in HBe antigen-positive patients (r = 0.365, P b 0.001, Fig. 2B). Similar result was also observed in HBe antigen-negative patients (r = 0.459, P b 0.001, Fig. 2C). These data suggest that serum miR-210 correlates with the viral load in CHB patients.
3. Results 3.1. Serum miR-210 levels in different groups of patients with CHB To investigate whether serum miR-210 levels were abnormally altered in CHB patients, serum miR-210 levels were detected in sera from 115 patients with CHB as well as 20 healthy controls. According to HBV DNA levels and HBe antigen status of EASL guidelines, CHB patients were classified to three groups including inactive carriers, HBe antigen-negative hepatitis and HBe antigen-positive hepatitis. Moreover, we added two additional groups of patients with HBe antigennegative: patients with low replicative (HBV DNA b2000 IU/mL) and elevated alanine aminotransferase (ALT), and patients with high replicative (HBV DNA N2000 IU/mL) and normal ALT. qRT-PCR analysis revealed that compared with healthy controls, serum miR-210 levels were increased in all subgroups of CHB patients, with the highest levels in HBe antigen positive hepatitis group (P b 0.05, Fig. 1A and Table 2). In the patients with HBe antigen negative, serum miR-210 levels were significantly higher in the high HBV replicative group than in the low HBV replicative group (P b 0.05, Fig. 1A and Table 2). We also observed that compared with HBe antigen positive hepatitis group, serum miR-210 levels were reduced in healthy controls, inactive carriers, low-replicative HBe antigen-negative patients with elevated ALT, high-replicative HBe antigen-negative patients with normal ALT and high-replicative HBe antigen-negative patients with elevated ALT, with the lowest levels in healthy controls (P b 0.05, Fig. 1A and Table 2). Our data suggest that serum miR-210 levels were elevated in CHB patients and might be associated with viral replication. In contrast, the Ct value of miR-16, an ubiquitous miRNA which is not specific for the liver, did not differ between healthy controls and all the patients (P N 0.05, Fig. 1B). Serum miR-16 level could be served as an internal control for normalization, which is consistent with the previous study [19].
3.3. The levels of serum miR-210 and HBs antigen in CHB patients To further confirm the correlation between viral replication and serum miR-210 in CHB patients, we correlated serum miR-210 levels with the viral activity. HBs antigen levels, correlating with the amount of HBV-infected cells and hepatic HBs antigen, were reported as markers of viral activity [20]. Next, the correlation between serum miR-210 and HBs antigen levels was analyzed in CHB patients. Our results showed that there was a significant correlation between serum miR-210 and HBs antigen levels (r = 0.348, P b 0.001, Fig. 3A). Notably, it was also found that serum miR-210 correlated with HBs antigen levels in HBe antigen-positive patients (r = 0.294, P b 0.001, Fig. 3B) and HBe antigen-negative patients (r = 0.284, P b 0.001, Fig. 3C). Combining the above data, our results suggest that serum miR-210 correlates with viral replication in CHB patients. 3.4. Serum miR-210 serves as a potential marker to discriminate inactive carriers with lower or higher risk for disease progression It is known that many inactive carriers are at risk for viral flare-up and HBs antigen level could serve as a useful marker for the identification of carriers with low and high risk of viral flare-up [21,22]. Next, to determine the correlation between serum miR-210 and HBs antigen level in patients with hepatitis B carrier status, serum miR-210 levels were analyzed in HBV carrier with high and low levels of HBs antigen (higher and lower than 3500 IU/mL). We found that serum miR-210
Fig. 1. Increased serum miR-210 levels in CHB patients. (A) Serum miR-210 levels in different groups of CHB patients and healthy controls. Vertical lines indicate the range, and horizontal boundaries of the boxes represent the first and third quartiles. *P b 0.05 compared with healthy controls, **P b 0.05 compared with inactive carrier, ***P b 0.05 compared with low replicative HBe antigen negative elevated ALT, #P b 0.05 compared with high replicative HBe antigen negative normal ALT and ##P b 0.05 compared with high replicative HBe antigen negative elevated ALT. (B) Serum miR-16 levels in all CHB patients and healthy controls. There is no significant difference between CHB patients and healthy controls (P N 0.05).
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Table 2 Serum miR-210 levels in different groups. Group
Number
Median
Data range (miR-210/miR-16) 25%
75%
Healthy controls Inactive carriers Low replicative HBe antigen negative elevated ALT High replicative HBe antigen negative normal ALT High replicative HBe antigen negative elevated ALT HBe antigen positive
20 20 9 31 39 16
0.115 0.490 0.690 0.780 1.120 2.215
0.072 0.382 0.590 0.650 0.880 1.673
0.177 0.687 0.770 1.002 1.420 2.733
Inactive carriers: patients with low replicative (HBV DNA b2000 IU/mL) and normal ALT (b40 U/L); low replicative HBe antigen negative elevated ALT: patients with low replicative (HBV DNA b2000 IU/mL) and elevated ALT (N40 U/L); high replicative HBe antigen negative normal ALT: patients with high replicative (HBV DNA N2000 IU/mL) and normal ALT (b40 U/L); high replicative HBe antigen negative elevated ALT: patients with high replicative (HBV DNA N2000 IU/mL) and elevated ALT (N40 U/L); HBe antigen positive: patients with HBe antigen-positive hepatitis.
level in patients with high levels of HBs antigen, who are at higher risk for viral flare-up, was significantly higher than that in patients with low levels of HBs antigen (P b 0.05, Fig. 4). These data suggest that serum miR-210 could serve as a potential marker to discriminate inactive carriers with lower or higher risk for disease progression. 3.5. Serum miR-210 and markers of liver injury As shown in Fig. 1A and Table 2, serum miR-210 levels were higher in the low replicative HBe antigen negative elevated ALT group than in the inactive carrier group (P b 0.05). In addition, serum miR-210 levels were significantly increased in the high replicative HBe antigen negative elevated ALT compared with high replicative HBe antigen negative normal ALT (P b 0.05, Fig. 1A and Table 2). We next correlated serum miR-210 level with parameters of liver inflammation and live damage. We compared miR-210 amounts with ALT values, HAI scores and fibrosis stages. Our results showed that there was a significant correlation between serum miR-210 level and ALT values (r = 0.557, P b 0.001, Fig. 5A). Notably, miR-210 level was significantly increased with HAI score, with the highest level observed in HAI score N11 (P b 0.001, Fig. 5B). However, there was no significant difference in patients with different fibrosis stages (P N 0.05, Fig. 5C). 3.6. Serum miR-210 and markers of liver function To investigate whether serum miR-210 is related to liver function, we correlated serum miR-210 level with markers of liver function including serum albumin concentration, bilirubin and international normalized ratio (INR). However, there was no significant correlation between serum miR-210 level and albumin concentration (r = −0.500, P N 0.05, Fig. 6A), as well as bilirubin concentration (r = − 0.094, P N 0.05, Fig. 6B). Similarly, INR also did not correlate with serum miR210 level (r = 0.152, P N 0.05, Fig. 6C), indicating that altered miR-210 level was not associated with liver function in CHB patients.
4. Discussion Due to the huge number of CHB patients and the increase trend of liver cirrhosis and HCC caused by chronic hepatitis B, numerous medical expenses have been spent on the treatment every year and will be further increased for the increasing demand, resulting in a serious health burden in China. Thus, it might be a good therapeutic strategy for early diagnosis and treatment for liver fibrosis to avoid disease progression. Liver biopsy is the gold standard for the assessment of HBV-induced liver injury severity and monitoring of CHB progression [23]. However, patients are not willing to accept it due to the invasive procedure and possible complications, such as bleeding and sampling error. With the development of the modern bio-technology, increasing evidence has shown that serum miRNAs are detectable in peripheral blood and might be used as non-invasive disease markers [24–26]. In support of this, differences of serum miRNA levels have been found between patients with a number of diseases and healthy controls [14]. Therefore, the detection of serum miRNA is a new approach for noninvasive clinical diagnosis. The development of liver fibrosis is promoted by a variety of factors including viral replication and immune status of patients. On the one hand, viral replication is very important for disease progression in CHB patients. It has been demonstrated that once patients are infected with HBV, the double-stranded HBV DNA could be converted into covalently closed circular DNA (cccDNA), which plays a vital role in the replication of viral DNA [1]. In addition, the cccDNA serves as a pool for the production of mRNA for the translation of viral proteins including HBs antigen [1]. HBs antigen level, secreted by infected cells or infectious viruses, represents the viral persistence in hepatocytes and could reflect the viral activity [20]. Therefore, HBs antigen is also considered as a surrogate marker for viral translation. On the other hand, CHB patients often undergo immune-tolerant with the normal ALT values. For the better understanding of disease progression in CHB patients, such patients, especially inactive HBV carrier, should also be included in the study. With the low levels of HBV DNA and normal
Fig. 2. Relation between serum miR-210 levels and HBV DNA levels in all CHB patients (A), HBe antigen-positive (B) and HBe antigen-negative (C).
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Fig. 3. Relation between serum miR-210 levels and HBs antigen levels in all CHB patients (A), HBe antigen-positive (B) and HBe antigen-negative (C).
ALT values, many inactive HBV carriers could develop viral flares with consecutive disease progression, suggesting that these patients are also at the risk of developing liver cirrhosis and HCC. Recent studies have shown that inactive carriers with low HBs antigen levels (b1000–3500 IU/mL) and low serum HBV DNA levels (b2000 IU/mL) have a relative low risk of viral flare-up, suggesting that HBs antigen level could serve as a potential marker of risk stratification for inactive carriers [21,22]. Currently, the anti-viral therapy is applied to inhibit the disease progression of liver fibrosis. There are at least three criteria: high HBV DNA levels (N 2000 IU/mL), elevated liver enzymes and advanced fibrosis [27]. For instance, nucleotide and nucleoside analogues or pegylated interferon could be taken into account for treatment if needed [27]. It has been reported that cellular and viral miRNAs have been identified as a new class of regulators in viral pathogenesis [28]. miRNAs such as miR-125a-5p [29], miR-199-3p and miR-210 [9] can suppress HBV expression and replication through direct binding to viral transcripts. Further study demonstrated that liver miR-125-5p level correlated with HBV replication and disease progression in CHB patients [10]. Our previous study found that serum miR-125a-5p level also correlates with disease progression in CHB patients [30]. Murakami et al. reported that the progression of liver fibrosis is related with overexpression of the miR-199-3p [31]. However, it is still not clear whether serum miR-210 levels correlate with surrogate markers for viral replication and translation to serve as disease parameters in different groups of patients with CHB. In the present study, our results showed that serum miR-210 levels were increased in all CHB patients, with the highest level in HBe antigen positive group. Moreover, serum miR-210 levels were significantly increased in the high replicative group compared with the low replicative group. Serum miR-210 levels could discriminate patients with HBe antigen positive from HBe antigen negative. Further study showed that there was significant correlation between serum
miR-210 levels and the levels of HBV DNA (P b 0.001) and HBs antigen (P b 0.001). It has been demonstrated that miR-210 negatively regulates HBV replication by targeting the HBV pre-S1 region [9]. Of note, it was found that inactive carrier patients with high (N3500 IU/mL) or low (b3500 IU/mL) levels of HBs antigen could be differentiated by serum
Fig. 4. Serum miR-210 levels in HBV carriers were analyzed according to HBs antigen levels (HBs antigen b3500 IU/mL, n = 13 or N3500 IU/mL, n = 7). *P b 0.05.
Fig. 5. Relation between serum miR-210 levels and ALT (A), necroinflammation HAI score (B) and fibrosis stage (C). *P b 0.05 compared with HAI score 2.
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replication under hypoxic condition in HepG2.2.15 cells. It is also reported that serum miR-210 is associated with disease progression and hepatitis severity [36]. However, they showed that serum miR-210 correlated with liver function other than HBV DNA level in CHB patients, which is not consistent with our current result. It is speculated that the groups of their study were almost different from that of this study. Their groups including mild CHB, severe CHB and acute-on-chronic liver failure, focused on the severity of hepatitis, whereas both of the levels of HBV DNA and ALT values were taken into account in this study. The reason for different groups in this study is that the significance of serum miR-210 not only could be assessed in healthy controls, HBe antigen negative and HBe antigen positive patients, but also in the sub-groups of HBe antigen negative. Moreover, the association between serum miR-210 and patients' immune states was also analyzed. Therefore, it might be the reason for the different findings. Combined with these, the clinical significance of serum miR-210 in different groups of CHB might be different. This is a first report of serum miR-210 in CHB patients with immunotolerant or hepatitis. There are some limitations in this study. Firstly, how serum miR-210 levels were increased during liver fibrosis is not clear. It is speculated according to our results that increased miR-210 may be released from the injury hepatocytes during disease progression. Serum miR-210 levels were also not analyzed in immunotolerant with HBe antigen positive. Secondly, there were significant overlaps between the different groups of chronic HBV and the corresponding microRNA-210 levels. It may lead to some limitation for the usefulness of serum miR-210 in the clinical. It is known that receiver operating characteristic curve (ROC) could be used to evaluate the diagnostic value of serum miR-210 in the different groups of CHB patients. However, we considered that the relative number of each group is too small to provide a more accurate analysis. Therefore, ROC analysis was not analyzed in this study and studies with larger sample sizes are warranted to validate the efficacy of this marker. In conclusion, serum miR-210 levels are elevated in CHB patients and correlated with HBV replication and translation, and necroinflammation markers, highlighting the potential utility of this microRNA as a marker of CHB. Conflict of interest The authors declare that they have no conflict of interest. Fig. 6. Relation between serum miR-210 levels and serum albumin concentration (A), INR (B) and serum bilirubin concentration (C).
miR-210 levels. It is known that high HBV DNA levels are often associated with a more severe liver disease [32]. These data suggest that serum miR-210 not only correlates with HBV replication and translation, but also is a marker for risk stratification. Recently, it has been reported that serum miR-210 level was increased in many diseases including cancers and inflammationassociated diseases, and could serve as a useful biomarker [33–35]. Due to immune responses to HBV infection, different degrees of liver injury and inflammation are often caused by CHB infection. We next determine the relationship between serum miR-210 levels and the severity of liver inflammation. Our results showed that serum miR-210 levels were associated with CHB patients' immune states. It was found that serum miR-210 levels were higher in hepatitis group (high replicative HBe antigen negative elevated ALT) than in immunotolerant group (high replicative HBe antigen negative normal ALT) (Fig. 1A). In addition, serum miR-210 levels correlated with liver inflammatory activity markers including ALT values and HAI scores, whereas it did not affect parameters of liver function as well as liver fibrosis stages. Therefore, serum miR-210 may be a useful biomarker for hepatitis severity. Interestingly, Song et al. reported that miR-210 negatively regulated HBV
Acknowledgments The project was supported by the National Natural Science Foundation of China (Nos. 81000176/H0317, 81100292/H0317 and 81500458/ H0317), Zhejiang Provincial Natural Science Foundation of China (Nos. Y2090326 and Y2110634), Wenzhou Municipal Science and Technology Bureau (Nos. Y20110033 and Y20120127), the Wang Bao-En Liver Fibrosis Foundation (Nos. 20100002 and 20120127), the incubator program of the First Affiliated Hospital of Wenzhou Medical University (HFY2014045) and the key disciplines in Colleges and Universities of Zhejiang Province. References [1] J.L. Dienstag, Hepatitis B virus infection, N. Engl. J. Med. 359 (2008) 1486–1500. [2] B.J. McMahon, Epidemiology and natural history of hepatitis B, Semin. Liver Dis. 25 (Suppl. 1) (2005) 3–8. [3] X. Liu, X. Wan, Z. Li, C. Lin, Y. Zhan, X. Lu, Golgi protein 73(GP73), a useful serum marker in liver diseases, Clin. Chem. Lab. Med. 49 (2011) 1311–1316. [4] C.J. Chen, H.I. Yang, J. Su, et al., Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level, JAMA, J. Am. Med. Assoc. 295 (2006) 65–73. [5] C.M. Croce, G.A. Calin, miRNAs, cancer, and stem cell division, Cell 122 (2005) 6–7. [6] A. Esquela-Kerscher, F.J. Slack, Oncomirs — microRNAs with a role in cancer, Nat. Rev. Cancer 6 (2006) 259–269. [7] W.P. Kloosterman, R.H. Plasterk, The diverse functions of microRNAs in animal development and disease, Dev. Cell 11 (2006) 441–450.
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Serum microRNA-210 levels in different groups of chronic hepatitis B patients Fujun Yu a, Jianhuan Yang b, Jinsheng Ouyang c, Yihu Zheng d, Bicheng Chen e, Guojun Li f, Zhongqiu Lu g, Peihong Dong a,⁎,1, Jianjian Zheng e,⁎⁎,1 a
Department of Infectious Diseases, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China Department of Children's internal medicine, The Second Affiliated Hospital & Yuying Children's hospital of Wenzhou Medical University, Wenzhou 325000, PR China Department of Respiratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China d Department of General Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China e Key Laboratory of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China f Department of Hepatology, Ningbo Yinzhou Second Hospital, Ningbo 315000, PR China g Emergency Department, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China b c
a r t i c l e
i n f o
Article history: Received 24 June 2015 Received in revised form 31 July 2015 Accepted 25 August 2015 Available online 28 August 2015 Keywords: MicroRNA-210 HBV carrier HBV DNA HBs antigen Chronic hepatitis B
a b s t r a c t Background: It has been reported that hepatitis B virus (HBV) replication can be suppressed by microRNA-210 (miR-210). However, whether serum miR-210 levels can serve as disease parameters in patients with chronic hepatitis B (CHB) remains unclear. Methods: Serum miR-210 levels were quantified in 115 CHB patients and 20 healthy controls by real-time PCR. Results: We found that serum miR-210 levels can discriminate the different groups of CHB patients from healthy control (P b 0.05), as well as patients with HBe antigen positive from those with HBe antigen negative (P b 0.05). Serum miR-210 levels correlated with HBV DNA and HBs antigen (r = 0.525, P b 0.001 and r = 0.348, P b 0.001). Notably, inactive carrier patients with high (N3500 IU/mL) or low (b 3500 IU/mL) levels of HBs antigen were differentiated by serum miR-210 levels (P b 0.05). Moreover, serum miR-210 levels correlated with liver inflammatory activity markers including alanine aminotransferase (ALT) and HAI score. However, there was no correlation of serum miR-210 levels with parameters of liver function including serum albumin, international normalized ratio and bilirubin, as well as the stages of liver fibrosis. Conclusions: Serum miR-210 can be used as an indicator of HBV replication and translation, and a potential marker of necroinflammation in patients with CHB. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chronic infection with the hepatitis B virus (HBV), accounting for 350–400 millions of patients worldwide, is a predominant etiological factor for liver disease in China [1,2]. In particular, about 7.8% of China population are HBV carriers (93 million, two-thirds of the world's total number of carriers) [3]. Once the infection becomes chronic, chronic hepatitis B (CHB) patients are at the risk of developing liver
Abbreviations: HBV, hepatitis B virus,; CHB, chronic hepatitis B; HCC, hepatocellular carcinoma; miRNA, microRNA; HAI, histological activity index; ALT, alanine aminotransferase; INR, international normalized ratio; cccDNA, covalently closed circular DNA; HIV, human immunodeficiency virus; HCV, hepatitis C virus; Ct, cycle threshold. ⁎ Correspondence to: P. Dong, Department of Infectious Diseases, The First Affiliated Hospital of Wenzhou Medical University, No. 2 FuXue lane, Wenzhou, Zhejiang, PR China. ⁎⁎ Correspondence to: J. Zheng, Key Laboratory of Surgery, The First Affiliated Hospital of Wenzhou Medical University, No. 2 FuXue lane, Wenzhou, Zhejiang, PR China. E-mail addresses: [email protected] (P. Dong), [email protected] (J. Zheng). 1 These authors share co-corresponding author.
http://dx.doi.org/10.1016/j.cca.2015.08.022 0009-8981/© 2015 Elsevier B.V. All rights reserved.
cirrhosis and hepatocellular carcinoma (HCC). The increased risks of liver cirrhosis and HCC associated with HBV DNA level and the persistence of HBe antigen have been well documented [4]. MicroRNAs (miRNAs) are evolutionarily highly conserved, small (18–24 nucleotides long) non-coding RNAs and play a vital role in many physiological processes, including cellular development, differentiation, metabolism, apoptosis and proliferation [5]. They are additionally implicated in pathogenesis of chronic inflammation, cardiovascular disease and cancer [6–8]. Notably, it has been demonstrated that miRNAs have entered the stage of virology. For example, Zhang et al. found that miR-210 negatively regulates HBV replication by targeting the HBV pre-S1 region in HepG2 2.2.15 cells under normoxic condition [9]. Coppola et al. reported that liver hsa-miR-125a-5p correlates with HBV replication and disease progression [10]. Recently, it has been demonstrated that cell-free miRNAs could circulate in the blood and serve as stable biomarkers for diseases [11–14]. Here, we aimed to study the significance of serum miR-210 levels in treatment-naive patients with different stages of CHB and compared them with surrogate markers of viral replication and translation.
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2. Methods
2.2. Liver histology
2.1. Patient selection
Liver biopsy was performed using a 16-gauge Menghini needle. Each liver biopsy case was advised by physicians in care, and liver specimens at least 2.0 cm in length were obtained. Samples were fixed in formalin, embedded in paraffin, and stained with hematoxylin–eosin. Results were reviewed by experienced hepatopathologists [16]. In addition, at least 8–10 portal tracts in samples are required to admit patients. Histological activity index (HAI) and fibrosis stages (F0 = no fibrosis–F6 = cirrhosis) were assessed according to the Ishak scoring system [17].
Serum samples were obtained from patients attending the First Affiliated Hospital of Wenzhou Medical University in Wenzhou and Ningbo Yinzhou Second Hospital in Ningbo from 2007.1 to 2012.8 (Table 1). 115 treatment-naive patients with CHB were included in this study and underwent liver biopsy. Inclusion criteria were CHB defined by detectable serum HBs antigen and serum HBV DNA for more than six months. Exclusion criteria were: (i) patients aged less than 16 years, (ii) coinfection with human immunodeficiency virus (HIV), (iii) coexistence of liver injury caused by other etiologies, including hepatitis C virus (HCV) infection, drug intake, alcohol consumption and auto-immune hepatitis, (iv) severe systematic diseases, and (v) pregnancy and lactation [15]. Demographic and clinical information was obtained, and blood samples collected from all patients on the day of diagnostic liver biopsy. 20 healthy controls (with normal liver biochemistry, no history of liver disease or alcohol abuse and no viral hepatitis) served as control patients. Written informed consent for the use of blood samples was obtained from all participants. The project was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). All procedures were performed in accordance with the current international guidelines, standards on human experimentation of the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China), and the Helsinki Declaration of 1975, revised in 1983.
Table 1 Patient characteristics. Parameter
CHB patients
Healthy controls
Epidemiology Gender, m/f (%) Age, years, median (range)
69/46 (60.0/40.0) 53 (41.7, 61.2)
11/9 (55.0/45.0) 50 (40.3, 59.6)
Virology HBs antigen (log10 IU/mL), median (range) HBV DNA (log10 IU/mL), median (range) HBe antigen positive, n (%) HBe antigen negative Inactive carriers, n (%) Low replicative, elevated ALT n (%) High replicative, normal ALT n (%) High replicative, elevated ALT n (%)
3.60 (1.50, 5.00) 3.88 (1.69, 8.91) 16 (13.9) 20 (17.4) 9 (7.8) 31 (26.9) 39 (33.9)
HAI 2, n (%) 3, n (%) 4, n (%) 5, n (%) 6, n (%) 7, n (%) 8, n (%) 9, n (%) ≥11, n (%)
13 (11.3%) 25 (21.7%) 8 (7.0%) 10 (8.7%) 12 (10.4%) 16 (13.9%) 12 (10.4%) 9 (7.8%) 10 (8.7%)
Fibrosis F0, n (%) F1, n (%) F2, n (%) F3, n (%) F4, n (%) F5, n (%) F6, n (%)
15 (13.0%) 19 (16.5%) 24 (20.9%) 20 (17.4%) 21 (18.3%) 6 (5.2%) 10 (8.7%)
Inactive carriers: patients with low replicative (HBV DNA b 2000 IU/mL) and normal ALT (b40 U/L); low replicative HBe antigen negative elevated ALT: patients with low replicative (HBV DNA b2000 IU/mL) and elevated ALT (N40 U/L); high replicative HBe antigen negative normal ALT: patients with high replicative (HBV DNA N2000 IU/mL) and normal ALT (b40 U/L); high replicative HBe antigen negative elevated ALT: patients with high replicative (HBV DNA N2000 IU/mL) and elevated ALT (N40 U/L); HBe antigen positive: patients with HBe antigen-positive hepatitis.
2.3. Blood sample processing Blood samples were centrifuged at 3400 g for 7 min at room temperature within 4 h of acquisition. Sera were transferred into Eppendorf tubes and additionally centrifuged at 12,000 g for 10 min at 4 °C to remove the remaining cells. Serum samples were stored at − 80 °C pending RNA extraction.
2.4. Detection of miR-210 with real-time quantitative RT-PCR According to the manufacturer's instructions for liquid samples, total RNA was extracted with the miRNeasy Mini Kit (Qiagen, Carlsbad, California, USA). Then DNase treatment (Qiagen, Carlsbad, California, USA) was performed to remove DNA contaminants in total RNA and final elution volume was 20 μL. In addition, serum RNA preparations were quantified using NanoDrop 1000 (Nanodrop, Wilmingtion, Delaware, USA) before the reverse transcription reaction. Reverse transcription was performed in a 20 μL reaction volume using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). For cDNA synthesis, reaction mixtures were sequentially incubated at 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. miR-210 was quantified in triplicate via qRT-PCR using the TaqMan MicroRNA Assay Kit (Applied Biosystems, Foster City, CA). According to the standard TaqMan MicroRNA assay protocol, real-time PCR was performed on an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA) under the following conditions: 95 °C for 10 min, followed by 35 cycles of 95 °C for 15 s and 60 °C for 60 s. Each PCR mixture (20 μL) included the reverse transcription products, TaqMan 2X Universal PCR Master Mix without UNG Amperase, miRNA-specific TaqMan probes, and primers supplied by Applied Biosystems. The cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to cross the threshold in qPCR. Ct values were calculated with SDS 2.0.1 software (Applied Biosystems, Foster City, CA). The formula 2−ΔCt was used to calculate the miRNA levels in serum, where ΔCt = mean (Ct of internal references) − Ct of target miRNA. The relative expression levels of miR210 were calculated and normalized to miR-16 (Applied Biosystems, Foster City, CA) using the comparativeΔCt method and the equation 2−ΔCt, as described previously [18]. 2.5. Serum HBV DNA and serum hepatitis marker analysis For all patients, serum HBV DNA was quantified using an Artus HBV QS-RGQ Kit (Qiagen, Hilden, Germany), with a lower detection limit of 10.2 IU/mL. HBsAg, HBeAg, and antibodies against HBsAg (anti-HBs), HBeAg (anti-HBe) and hepatitis B core antigen (anti-HBc) were determined using the Roche Modular E170 Immunoassay Analyzer (Roche, Basel, Switzerland). HBsAg titres were additionally determined with the HBsAg quantitative assay (Abbott Laboratories, Abbott Park, IL, USA) based on an automated chemiluminescent microparticle immunoassay (Abbott Architect i2000SR analyzer; Abbott Laboratories), according to the manufacturer's instructions. Architect HBsAg QT measures a range of HBsAg from 0.05 to 250 IU/mL. Therefore, the dilution for samples with higher HBsAg titre is required.
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2.6. Statistical analysis
3.2. The levels of serum miR-210 and HBV DNA in CHB patients
Data were analyzed using SPSS 13.0 (IBM, Armonk, NY). Statistical significance was determined using nonparametric Kruskal–Wallis or nonparametric Wilcoxon–Mann–Whitney test. Correlation coefficients (r) were calculated using Spearman correlation. In the box plots, vertical lines indicate the range, and horizontal boundaries of the boxes represent the first and third quartiles. Data were considered significant at P b 0.05.
To assess whether serum miR-210 level was associated with viral replication, the correlation between serum miR-210 concentrations and the viral load in CHB patients was analyzed. Serum miR-210 was significantly correlated with HBV DNA in all sera from CHB patients (r = 0.525, P b 0.001, Fig. 2A). According to HBe antigen status, CHB patients were divided into two subgroups: HBe antigen-positive and HBe antigen-negative. Then, the correlation between serum miR-210 and HBV DNA was further investigated in HBe antigen-positive and HBe antigen-negative patients. Accordingly, there was a significant correlation between serum miR-210 and HBV DNA in HBe antigen-positive patients (r = 0.365, P b 0.001, Fig. 2B). Similar result was also observed in HBe antigen-negative patients (r = 0.459, P b 0.001, Fig. 2C). These data suggest that serum miR-210 correlates with the viral load in CHB patients.
3. Results 3.1. Serum miR-210 levels in different groups of patients with CHB To investigate whether serum miR-210 levels were abnormally altered in CHB patients, serum miR-210 levels were detected in sera from 115 patients with CHB as well as 20 healthy controls. According to HBV DNA levels and HBe antigen status of EASL guidelines, CHB patients were classified to three groups including inactive carriers, HBe antigen-negative hepatitis and HBe antigen-positive hepatitis. Moreover, we added two additional groups of patients with HBe antigennegative: patients with low replicative (HBV DNA b2000 IU/mL) and elevated alanine aminotransferase (ALT), and patients with high replicative (HBV DNA N2000 IU/mL) and normal ALT. qRT-PCR analysis revealed that compared with healthy controls, serum miR-210 levels were increased in all subgroups of CHB patients, with the highest levels in HBe antigen positive hepatitis group (P b 0.05, Fig. 1A and Table 2). In the patients with HBe antigen negative, serum miR-210 levels were significantly higher in the high HBV replicative group than in the low HBV replicative group (P b 0.05, Fig. 1A and Table 2). We also observed that compared with HBe antigen positive hepatitis group, serum miR-210 levels were reduced in healthy controls, inactive carriers, low-replicative HBe antigen-negative patients with elevated ALT, high-replicative HBe antigen-negative patients with normal ALT and high-replicative HBe antigen-negative patients with elevated ALT, with the lowest levels in healthy controls (P b 0.05, Fig. 1A and Table 2). Our data suggest that serum miR-210 levels were elevated in CHB patients and might be associated with viral replication. In contrast, the Ct value of miR-16, an ubiquitous miRNA which is not specific for the liver, did not differ between healthy controls and all the patients (P N 0.05, Fig. 1B). Serum miR-16 level could be served as an internal control for normalization, which is consistent with the previous study [19].
3.3. The levels of serum miR-210 and HBs antigen in CHB patients To further confirm the correlation between viral replication and serum miR-210 in CHB patients, we correlated serum miR-210 levels with the viral activity. HBs antigen levels, correlating with the amount of HBV-infected cells and hepatic HBs antigen, were reported as markers of viral activity [20]. Next, the correlation between serum miR-210 and HBs antigen levels was analyzed in CHB patients. Our results showed that there was a significant correlation between serum miR-210 and HBs antigen levels (r = 0.348, P b 0.001, Fig. 3A). Notably, it was also found that serum miR-210 correlated with HBs antigen levels in HBe antigen-positive patients (r = 0.294, P b 0.001, Fig. 3B) and HBe antigen-negative patients (r = 0.284, P b 0.001, Fig. 3C). Combining the above data, our results suggest that serum miR-210 correlates with viral replication in CHB patients. 3.4. Serum miR-210 serves as a potential marker to discriminate inactive carriers with lower or higher risk for disease progression It is known that many inactive carriers are at risk for viral flare-up and HBs antigen level could serve as a useful marker for the identification of carriers with low and high risk of viral flare-up [21,22]. Next, to determine the correlation between serum miR-210 and HBs antigen level in patients with hepatitis B carrier status, serum miR-210 levels were analyzed in HBV carrier with high and low levels of HBs antigen (higher and lower than 3500 IU/mL). We found that serum miR-210
Fig. 1. Increased serum miR-210 levels in CHB patients. (A) Serum miR-210 levels in different groups of CHB patients and healthy controls. Vertical lines indicate the range, and horizontal boundaries of the boxes represent the first and third quartiles. *P b 0.05 compared with healthy controls, **P b 0.05 compared with inactive carrier, ***P b 0.05 compared with low replicative HBe antigen negative elevated ALT, #P b 0.05 compared with high replicative HBe antigen negative normal ALT and ##P b 0.05 compared with high replicative HBe antigen negative elevated ALT. (B) Serum miR-16 levels in all CHB patients and healthy controls. There is no significant difference between CHB patients and healthy controls (P N 0.05).
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Table 2 Serum miR-210 levels in different groups. Group
Number
Median
Data range (miR-210/miR-16) 25%
75%
Healthy controls Inactive carriers Low replicative HBe antigen negative elevated ALT High replicative HBe antigen negative normal ALT High replicative HBe antigen negative elevated ALT HBe antigen positive
20 20 9 31 39 16
0.115 0.490 0.690 0.780 1.120 2.215
0.072 0.382 0.590 0.650 0.880 1.673
0.177 0.687 0.770 1.002 1.420 2.733
Inactive carriers: patients with low replicative (HBV DNA b2000 IU/mL) and normal ALT (b40 U/L); low replicative HBe antigen negative elevated ALT: patients with low replicative (HBV DNA b2000 IU/mL) and elevated ALT (N40 U/L); high replicative HBe antigen negative normal ALT: patients with high replicative (HBV DNA N2000 IU/mL) and normal ALT (b40 U/L); high replicative HBe antigen negative elevated ALT: patients with high replicative (HBV DNA N2000 IU/mL) and elevated ALT (N40 U/L); HBe antigen positive: patients with HBe antigen-positive hepatitis.
level in patients with high levels of HBs antigen, who are at higher risk for viral flare-up, was significantly higher than that in patients with low levels of HBs antigen (P b 0.05, Fig. 4). These data suggest that serum miR-210 could serve as a potential marker to discriminate inactive carriers with lower or higher risk for disease progression. 3.5. Serum miR-210 and markers of liver injury As shown in Fig. 1A and Table 2, serum miR-210 levels were higher in the low replicative HBe antigen negative elevated ALT group than in the inactive carrier group (P b 0.05). In addition, serum miR-210 levels were significantly increased in the high replicative HBe antigen negative elevated ALT compared with high replicative HBe antigen negative normal ALT (P b 0.05, Fig. 1A and Table 2). We next correlated serum miR-210 level with parameters of liver inflammation and live damage. We compared miR-210 amounts with ALT values, HAI scores and fibrosis stages. Our results showed that there was a significant correlation between serum miR-210 level and ALT values (r = 0.557, P b 0.001, Fig. 5A). Notably, miR-210 level was significantly increased with HAI score, with the highest level observed in HAI score N11 (P b 0.001, Fig. 5B). However, there was no significant difference in patients with different fibrosis stages (P N 0.05, Fig. 5C). 3.6. Serum miR-210 and markers of liver function To investigate whether serum miR-210 is related to liver function, we correlated serum miR-210 level with markers of liver function including serum albumin concentration, bilirubin and international normalized ratio (INR). However, there was no significant correlation between serum miR-210 level and albumin concentration (r = −0.500, P N 0.05, Fig. 6A), as well as bilirubin concentration (r = − 0.094, P N 0.05, Fig. 6B). Similarly, INR also did not correlate with serum miR210 level (r = 0.152, P N 0.05, Fig. 6C), indicating that altered miR-210 level was not associated with liver function in CHB patients.
4. Discussion Due to the huge number of CHB patients and the increase trend of liver cirrhosis and HCC caused by chronic hepatitis B, numerous medical expenses have been spent on the treatment every year and will be further increased for the increasing demand, resulting in a serious health burden in China. Thus, it might be a good therapeutic strategy for early diagnosis and treatment for liver fibrosis to avoid disease progression. Liver biopsy is the gold standard for the assessment of HBV-induced liver injury severity and monitoring of CHB progression [23]. However, patients are not willing to accept it due to the invasive procedure and possible complications, such as bleeding and sampling error. With the development of the modern bio-technology, increasing evidence has shown that serum miRNAs are detectable in peripheral blood and might be used as non-invasive disease markers [24–26]. In support of this, differences of serum miRNA levels have been found between patients with a number of diseases and healthy controls [14]. Therefore, the detection of serum miRNA is a new approach for noninvasive clinical diagnosis. The development of liver fibrosis is promoted by a variety of factors including viral replication and immune status of patients. On the one hand, viral replication is very important for disease progression in CHB patients. It has been demonstrated that once patients are infected with HBV, the double-stranded HBV DNA could be converted into covalently closed circular DNA (cccDNA), which plays a vital role in the replication of viral DNA [1]. In addition, the cccDNA serves as a pool for the production of mRNA for the translation of viral proteins including HBs antigen [1]. HBs antigen level, secreted by infected cells or infectious viruses, represents the viral persistence in hepatocytes and could reflect the viral activity [20]. Therefore, HBs antigen is also considered as a surrogate marker for viral translation. On the other hand, CHB patients often undergo immune-tolerant with the normal ALT values. For the better understanding of disease progression in CHB patients, such patients, especially inactive HBV carrier, should also be included in the study. With the low levels of HBV DNA and normal
Fig. 2. Relation between serum miR-210 levels and HBV DNA levels in all CHB patients (A), HBe antigen-positive (B) and HBe antigen-negative (C).
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Fig. 3. Relation between serum miR-210 levels and HBs antigen levels in all CHB patients (A), HBe antigen-positive (B) and HBe antigen-negative (C).
ALT values, many inactive HBV carriers could develop viral flares with consecutive disease progression, suggesting that these patients are also at the risk of developing liver cirrhosis and HCC. Recent studies have shown that inactive carriers with low HBs antigen levels (b1000–3500 IU/mL) and low serum HBV DNA levels (b2000 IU/mL) have a relative low risk of viral flare-up, suggesting that HBs antigen level could serve as a potential marker of risk stratification for inactive carriers [21,22]. Currently, the anti-viral therapy is applied to inhibit the disease progression of liver fibrosis. There are at least three criteria: high HBV DNA levels (N 2000 IU/mL), elevated liver enzymes and advanced fibrosis [27]. For instance, nucleotide and nucleoside analogues or pegylated interferon could be taken into account for treatment if needed [27]. It has been reported that cellular and viral miRNAs have been identified as a new class of regulators in viral pathogenesis [28]. miRNAs such as miR-125a-5p [29], miR-199-3p and miR-210 [9] can suppress HBV expression and replication through direct binding to viral transcripts. Further study demonstrated that liver miR-125-5p level correlated with HBV replication and disease progression in CHB patients [10]. Our previous study found that serum miR-125a-5p level also correlates with disease progression in CHB patients [30]. Murakami et al. reported that the progression of liver fibrosis is related with overexpression of the miR-199-3p [31]. However, it is still not clear whether serum miR-210 levels correlate with surrogate markers for viral replication and translation to serve as disease parameters in different groups of patients with CHB. In the present study, our results showed that serum miR-210 levels were increased in all CHB patients, with the highest level in HBe antigen positive group. Moreover, serum miR-210 levels were significantly increased in the high replicative group compared with the low replicative group. Serum miR-210 levels could discriminate patients with HBe antigen positive from HBe antigen negative. Further study showed that there was significant correlation between serum
miR-210 levels and the levels of HBV DNA (P b 0.001) and HBs antigen (P b 0.001). It has been demonstrated that miR-210 negatively regulates HBV replication by targeting the HBV pre-S1 region [9]. Of note, it was found that inactive carrier patients with high (N3500 IU/mL) or low (b3500 IU/mL) levels of HBs antigen could be differentiated by serum
Fig. 4. Serum miR-210 levels in HBV carriers were analyzed according to HBs antigen levels (HBs antigen b3500 IU/mL, n = 13 or N3500 IU/mL, n = 7). *P b 0.05.
Fig. 5. Relation between serum miR-210 levels and ALT (A), necroinflammation HAI score (B) and fibrosis stage (C). *P b 0.05 compared with HAI score 2.
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replication under hypoxic condition in HepG2.2.15 cells. It is also reported that serum miR-210 is associated with disease progression and hepatitis severity [36]. However, they showed that serum miR-210 correlated with liver function other than HBV DNA level in CHB patients, which is not consistent with our current result. It is speculated that the groups of their study were almost different from that of this study. Their groups including mild CHB, severe CHB and acute-on-chronic liver failure, focused on the severity of hepatitis, whereas both of the levels of HBV DNA and ALT values were taken into account in this study. The reason for different groups in this study is that the significance of serum miR-210 not only could be assessed in healthy controls, HBe antigen negative and HBe antigen positive patients, but also in the sub-groups of HBe antigen negative. Moreover, the association between serum miR-210 and patients' immune states was also analyzed. Therefore, it might be the reason for the different findings. Combined with these, the clinical significance of serum miR-210 in different groups of CHB might be different. This is a first report of serum miR-210 in CHB patients with immunotolerant or hepatitis. There are some limitations in this study. Firstly, how serum miR-210 levels were increased during liver fibrosis is not clear. It is speculated according to our results that increased miR-210 may be released from the injury hepatocytes during disease progression. Serum miR-210 levels were also not analyzed in immunotolerant with HBe antigen positive. Secondly, there were significant overlaps between the different groups of chronic HBV and the corresponding microRNA-210 levels. It may lead to some limitation for the usefulness of serum miR-210 in the clinical. It is known that receiver operating characteristic curve (ROC) could be used to evaluate the diagnostic value of serum miR-210 in the different groups of CHB patients. However, we considered that the relative number of each group is too small to provide a more accurate analysis. Therefore, ROC analysis was not analyzed in this study and studies with larger sample sizes are warranted to validate the efficacy of this marker. In conclusion, serum miR-210 levels are elevated in CHB patients and correlated with HBV replication and translation, and necroinflammation markers, highlighting the potential utility of this microRNA as a marker of CHB. Conflict of interest The authors declare that they have no conflict of interest. Fig. 6. Relation between serum miR-210 levels and serum albumin concentration (A), INR (B) and serum bilirubin concentration (C).
miR-210 levels. It is known that high HBV DNA levels are often associated with a more severe liver disease [32]. These data suggest that serum miR-210 not only correlates with HBV replication and translation, but also is a marker for risk stratification. Recently, it has been reported that serum miR-210 level was increased in many diseases including cancers and inflammationassociated diseases, and could serve as a useful biomarker [33–35]. Due to immune responses to HBV infection, different degrees of liver injury and inflammation are often caused by CHB infection. We next determine the relationship between serum miR-210 levels and the severity of liver inflammation. Our results showed that serum miR-210 levels were associated with CHB patients' immune states. It was found that serum miR-210 levels were higher in hepatitis group (high replicative HBe antigen negative elevated ALT) than in immunotolerant group (high replicative HBe antigen negative normal ALT) (Fig. 1A). In addition, serum miR-210 levels correlated with liver inflammatory activity markers including ALT values and HAI scores, whereas it did not affect parameters of liver function as well as liver fibrosis stages. Therefore, serum miR-210 may be a useful biomarker for hepatitis severity. Interestingly, Song et al. reported that miR-210 negatively regulated HBV
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