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Journal of Digestive Diseases 2014; 15; 614–621
doi: 10.1111/1751-2980.12185
Original article
Serum microRNA-29 levels correlate with disease progression in patients with chronic hepatitis B virus infection Chong HUANG, Jian Ming ZHENG, Qi CHENG, Kang Kang YU, Qing Xia LING, Ming Quan CHEN & Ning LI Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
METHODS: Using real-time quantitative polymerase chain reaction assay, serum miR-29a, miR-29b and miR-29c levels were measured in patients with chronic HBV infection, and the correlation between serum miR-29 levels and the participants’ liver biochemistry, fibrotic stage and necroinflammation grade were also evaluated.
between S0/1 and S2/3 patients was not significant. miR-29b levels were higher in S0/1 patients than in other patient groups, but did not differ between S2/3 and S4 patients. At fibrotic stages of S0/1 and S2/3, patients with no or mild liver inflammation (G0/1) tended to express higher miR-29 levels than those with advanced inflammation (G2–4) (P > 0.05). miR29a–c showed significant correlation with alanine transaminase levels (P < 0.05 for miR-29a, miR-29b and miR-29c) in S0–3 patients. The expression of miR-29 was highest in immune-tolerant patients (P < 0.001).
RESULTS: Altogether 91 patients with chronic HBV infection were divided by fibrotic stage into S0/1 (no or mild fibrosis), S2/3 (progressive fibrosis) and S4 (cirrhosis) subgroups, and 12 healthy individuals were also included in the study. Serum miR-29a and miR-29c in S0–3 were significantly higher than those in S4 patients (P < 0.001); however, the difference
CONCLUSIONS: Serum miR-29 levels are negatively correlated with liver fibrotic stages and necroinflammation grades in patients with chronic HBV infection. miR-29 appears to be a novel biomarkers for predicting disease progression in these patients.
OBJECTIVE: To investigate the role of serum microRNA-29 (miR-29) as a biomarker for the prediction of disease progression in patients with chronic hepatitis B virus (HBV) infection.
KEY WORDS:
biomarker, chronic hepatitis B, hepatitis B virus, liver fibrosis, microRNAs.
INTRODUCTION Approximately 350–400 million patients worldwide suffered from chronic hepatitis B virus (HBV) Correspondence to: Ning LI, Department of Infectious Diseases, Huashan Hospital, Fudan University, 12 Middle Urumqi Road, Shanghai 200040, China. Email: [email protected] Conflict of interest: None. © 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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infection.1 End-stage liver disease (ESLD), including liver cirrhosis and hepatocellular carcinoma (HCC) occurs in a significantly high proportion of patients with chronic hepatitis B (CHB).2 Antiviral therapies with nucleoside analogues (NAs) or interferon (IFN) are effective in halting disease progression in such patients. These antiviral therapies are indicated when the patient’s viral load is high together with elevated peripheral liver enzyme levels and histological evidence of advanced necroinflammation or fibrosis. Liver biopsy has been proven to be valuable in predicting ESLD, especially in patients
Journal of Digestive Diseases 2014; 15; 614–621 presenting with persistently normal liver function and low viral load.3 However, liver biopsy is an invasive procedure that is associated with possible complications such as bleeding and sampling error. MicroRNAs (miRNAs) represent a family of short (18–25 nucleotides), noncoding RNAs that regulate gene expression at the level of transcription or translation.4 miRNAs are involved in the pathophysiological processes such as cellular differentiation, proliferation and apoptosis. Several studies5–8 have investigated the role of miRNAs in the pathogenesis of liver-related diseases including CHB. A fraction of cell-free miRNAs are detectable within the peripheral blood circulation as the result of their active secretion in the form of exosomes.9 The detection of cell-free miRNAs in the peripheral blood or body fluid provides a prospective, non-invasive tool to evaluate the disease progression in cancer and hematological diseases.10,11 Recently, serum miR-122, which was correlated with viral load, has been found to be capable of discriminating between patients with chronic HBV infection from healthy controls.12 The miR-29 family is composed of miR-29a, miR29b and miR-29c. The dysregulation of miR-29 contributes to the tumor pathogenesis, autoimmune disorders and fibrostic diseases including liver cirrhosis.13,14 The activation of hepatic stellate cells (HSC) plays a central role in the development of liver fibrosis.15 Quiescent HSC express higher miR-29 levels than activated HSC, and miR-29 family members have been found to downregulate in human and murine liver fibrosis.16 In contrast, the overexpression of miR-29b in HSC significantly downregulated the expressions of Col1a1, Col4a5 and Col5a3.16 During the activation of HSC in primary culture, miR-29b overexpression markedly attenuated Col1a1 and Col1a2 mRNA expression and additionally inhibited the increased expression of α-smooth muscle actin (α-SMA), discoidin domain receptor 2 (DDR2), fibronectin 1 (FN1), integrin β1 (ITGB1) and platelet-derived growth factor receptor-β (PDGFR-β), which are the key genes involved in the activation of HSC.17 Furthermore, miR-29atransgenic mice were protected from bile duct ligation-induced liver injury.18 As low circulating serum levels of miR-29a are characteristic of advanced liver fibrosis,16 this study aimed to investigate the potential role of serum miR-29 as a biomarker for the prediction of disease progression in CHB patients.
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PATIENTS AND METHODS Patients Treatment-naive patients with chronic HBV infection who were admitted to the Department of Infectious Diseases, Huashan Hospital, Fudan University (Shanghai, China) from January 2009 to December 2013 were recruited in the study. All the patients were hepatitis B surface antigen (HBsAg)-positive for at least 6 months. Exclusion criteria were: (i) patients aged less than 16 years; (ii) patients having co-infected with human immunodeficiency virus (HIV); (iii) those with the coexistence of liver injury caused by any other etiologies including hepatitis C virus (HCV) infection, drug intake, alcohol consumption and autoimmune hepatitis, and so on; (iv) those with severe systematic diseases; (v) pregnancy and lactation. In all, 91 patients who underwent liver biopsy and 12 healthy individuals were finally enrolled in the study. Written informed consent was obtained from all the adult patients who participated in the study. Participants aged under 18 years provided their verbal assent and written informed consent was obtained from their parents. The study was performed in accordance with the Helsinki Declaration and was approved by the Ethical Committee of Huashan Hospital, Fudan University. Clinical characteristics, laboratory examinations and liver histology Peripheral blood samples were collected at the day of liver biopsy, and were centrifuged at 1500 ×g for 10 min at room temperature. The serum was immediately stored at −70°C until analysis. Liver function tests were determined by standard methods in a clinical setting. HBsAg titers were determined on stored frozen serum samples with an HBsAg quantitative assay (Abbott Laboratories, Abbott Park, IL, USA) based on the automated chemiluminescent microparticle immunoassay (Abbott Architect i2000SR analyzer; Abbott Laboratories). Samples with HBsAg >250 IU/mL were diluted to the calibration range. A domestic HBV DNA quantification assay (Shanghai Kehua Bio-engineering Co., Ltd., Shanghai, China) was used to quantify serum HBV DNA titers. Samples with HBV DNA > 4 × 107 IU/mL were further diluted and retested. Liver specimens obtained by liver biopsy in CHB patients were assessed by an experienced pathologist. Two components, grading (G) and staging (S), given
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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in a numerical value ranged from 0 to 4, were used to describe disease progression of CHB. Grading reflects the intensity of necroinflammatory activity, and staging is a measure of fibrosis and alteration to the lobular architecture in chronic hepatitis. G0 and S0 represent the absence of necroinflammation and fibrosis, while G4 and S4 represent bridging necrosis involving multiple lobules and cirrhosis. Detection of miR-29 by real-time polymerase chain reaction (PCR) Total miRNA was extracted and purified from 100 μL serum using the miRNeasy Serum/Plasma Kit (QIAGEN GmbH, Hilden, Germany). Before extraction, 3.5 μL cel-miR-39 prediluted at 1.6 × 108 copies/μL was added to each tube as a spike-in control for normalization. The purified miRNA was immediately reverse-transcribed with the miScript II Reverse Transcription (RT) Kit (QIAGEN GmbH). The expressions of serum miR-29a, miR-29b and miR-29c were quantified using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). A real-time reaction was performed using miScript SYBR Green PCR Kit with primers specific for miR-29a, miR-29b and miR-29c (all from QIAGEN GmbH) according to the manufacturer’s instructions. A comparative ΔCT method was used to compare each target with cel-miR39, and relative values were expressed as 2−ΔΔC T. Statistical analysis Statistical analyses were performed with the Graphpad 5.0 (Graphpad Software, San Diego, CA, USA). Table 1.
Variables were expressed as median and interquartile range (IQR) or numbers and percentages unless otherwise specified. Differences in the parameters were compared using non-parametric Mann–Whitney U–test, and the relationship between two variables was assessed by Spearman’s rank correlation. A twotailed P value of ≤0.05 was considered statistically significant. RESULTS Serum miR-29 levels was correlated with liver fibrosis stage in CHB patients In total, 91 treatment-naive patients with chronic HBV infection and 12 healthy individuals were recruited in the study. The patients’ characteristics are summarized in Table 1. Irrespective of the patients’ HBeAg status and viral load, histological evidence of progressive liver necroinflammation (G ≥ 2) and fibrosis (S ≥ 2) were strongly associated with ESLD. According to the Chinese Guideline of Prevention and Treatment for Chronic Hepatitis B (2010 version) patients with liver histology of G ≥ 2 and/or S ≥ 2 are strongly recommended for antiviral therapy,19 especially for those with persistently normal or nearnormal alanine transaminase (ALT) levels. But persistently elevated ALT level predicts disease progression in CHB patients, especially when ALT level is over twice the upper limit of normal (ULN).20,21 To investigate the role of miR-29 in predicting disease progression, patients were classified into three groups: S0/1 (no or mild fibrosis), S2/3 (progressive fibrosis) and S4 (cirrhosis) (Table 1). The S0/1 group included 13 immune-tolerant patients (HBeAg-positive patients
Characteristics of the enrolled patients with chronic hepatitis B virus infection and healthy individuals Fibrosis stage
Gender, n (M/F) Age, years (median [range]) HBsAg, log10IU/mL (median [range]) HBeAg, n (+/−) HBV DNA, log10IU/mL (median [range]) ALT, U/L (median [range]) Necroinflammation grade, n (%) G0–1 G2–4 WBC, ×109/L (median [range]) PLT, ×109/L (median [range])
S0/1 (n = 36)
S2/3 (n = 30)
S4 (n = 25)
28/8 32 (19–55) 4.38 (1.95–5.10) 27/9 7.54 (3.5–9.30) 75 (16–748)
23/7 27 (16–61) 4.02 (2.64–5.0) 25/5 7.63 (3.92–9.70) 179 (24–1721)
18/7 44 (21–64) 3.27 (0.35–3.70) 17/8 4.94 (0–7.29) 37 (15–138)
26 (72.2) 10 (27.8) 5.1 (3.2–12.7) 204 (109–289)
5 (16.7) 25 (83.3) 5.4 (3.5–9.9) 180 (92–291)
12 (48.0) 13 (52.0) 2.9 (1.0–11.5) 61 (23–227)
Healthy controls 8/4 33 (24–51) NA NA NA NA NA
NA NA
ALT, alanine transaminase; F, female; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; M, male; NA, not available; PLT, platelet; WBC, white blood cells.
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2014; 15; 614–621
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Figure 1. Serum microRNA-29 (miR-29) levels in patients at different stages of chronic hepatitis B-induced liver fibrosis and healthy controls (HC). (a–c) miR-29a, miR-29b and miR-29c levels; (d–f) those subdivided by grading (G0/1 and G2–4). □, G0/1; ■, G2–4. Values are expressed as median and interquartile range.
with persistently normal ALT level for at least one year and normal liver histology) and 23 CHB patients. No inactive HBsAg carriers (positive HBsAg for over 6 months with persistently normal liver function, negative HBeAg, positive HBeAb, HBV DNA 0.05) (Fig. 1). miR-29b levels were significantly higher in S0/1 patients than the other two patient groups (S0/1 vs S2/3, P < 0.05; S0/1 vs S4, P < 0.01), but was much lower than that of the healthy controls (P < 0.05) (Fig. 1). And the miR-29b levels between the S2/3 and S4 groups were not significantly different (P > 0.05). To discover whether inflammation grading affects serum miR-29 expression in the individual fibrotic stage, we divided each group into two subgroups based on grading. In S0/1 and S2/3 groups, all miR-29 tended to be higher in patients with no or mild liver necroinflammation (G0/1) than those with advanced inflammation (G ≥ 2),
although the difference was not statistically significant. In contrast, liver necroinflammation seemed to be irrelevant to miR-29 expression in the S4 group (Fig. 1). miR-29 levels differed between immune-tolerant and CHB patients In patients at liver fibrosis S0–3 all miR-29 levels were significantly correlated with ALT levels (P = 0.0258 for miR-29a, P < 0.0001 for miR-29b, P = 0.0044 for miR-29c) (Fig. 2a–c). We further divided patients into immune-tolerant, S0/1 CHB and S2/3 CHB subgroups. The expressions of miR29a, miR-29b and miR-29c were all highest in the immune-tolerant group than in the other CHB groups (P < 0.001) (Fig. 2d–f); miR-29a and miR-29c levels in the immune-tolerant group were even higher than those in the healthy controls (P < 0.01, data not shown). But no differences in miR-29 levels were observed between S0/1 and S2/3 CHB patients (Fig. 2d–f). The relationship between ALT levels and miR-29 expressions was not examined in S4 patients because only 2 of the 25 patients had an ALT level of at least twice ULN.
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Figure 2. (a–c) Correlation of serum microRNA-29 (miR-29) with alanine transaminase (ALT) levels in patients at liver fibrosis S0–3 and (d–f) miR-29 levels in immune-tolerant (IT) patients, S0/1, S2/3 chronic hepatitis B (CHB) patients and healthy controls (HC). Values in (d–f) are expressed as median and interquartile range.
The relationship between miR-29 and other biomarkers We then investigated whether peripheral blood white blood cell (WBC) or platelet (PLT) counts and the biomarkers reflecting the extent of liver fibrosis were correlated with miR-29 expressions. WBC count was observed to be correlated with miR-29a levels (P < 0.05), but not with miR-29b or miR-29c levels (Fig. 3a–c). In contrast, all miR-29 showed significant correlation with PLT count (Fig. 3d–f). miR-29 levels also showed a positive correlation with HBV DNA titer (log10IU/mL) (P < 0.05, data not shown). The correlation between miR-29a and miR-29c level and HBsAg titer (log10IU/mL) was also significant (P < 0.05, data not shown). However, low viral load in patients with advanced liver disease might have contributed to the statistical difference. DISCUSSION Chronic hepatitis B is a severe liver disease associated with significant morbidity and mortality.1 Predicting
disease progression in patients with chronic HBV infection is important because of the appropriate time for antiviral therapy. Liver biopsy has long been considered the gold standard for the staging of CHBinduced fibrosis, but it is an invasive procedure associated with occasional severe outcomes and has several limitations. In contrast, miRNAs derived from blood or body fluid are easily accessible potential biomarkers for the evaluation of disease severity. miR-29 is intimately involved in the pathogenesis of fibrotic diseases including liver cirrhosis, and an early study found that, compared with healthy individuals, in patients with advanced liver cirrhosis due to various etiologies serum miR-29a levels were significantly lower.16 In this study we investigated the correlation between serum miR-29 levels and disease progression in patients with chronic HBV infection, and found that serum miR-29 levels were negatively correlated with patient’s fibrotic stage. miR-29 expressions also readily differed between immune-tolerant patients and HBeAg-negative or HBeAg-positive CHB patients with mild fibrosis. Furthermore, a significant negative correlation between miR-29 expression and ALT levels
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2014; 15; 614–621 (a)
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Figure 3. Correlation between serum microRNA-29 (miR-29) levels and (a–c) white blood cell (WBC) count and (d–f) platelet (PLT) count. P values are calculated using the Spearman rank correlation.
was found. Therefore, serum miR-29 might be a potential biomarker to assess disease progression in patients with CHB. Studies have investigated the potential of various peripheral circulation miRNAs in the diagnosis of liver diseases, including miR-122, miR-22, miR-20a, miR-192 and miR-210,5,22–25 among which miR-122 has been the most studied. To our knowledge, miR-29 is the only validated miRNA that is downregulated in the progress of liver injury, while the other miRNAs are reported to be upregulated. Serum miR-122 levels are increased with the severity of liver injury, as shown by its good correlation with aminotransferase level and necroinflammation.12,22 However, miR-122 levels are downregulated in patients with decompensated cirrhosis compared with the compensated patients.25 Low serum miR-122 level has been found to be an independent marker for poor survival in cirrhotic patients.25 Serum miR29a has been reported to be negatively correlated with the model for end-stage liver disease (MELD) score,16 and in our cohorts it was negatively correlated with the extent of cytopenia, which is a biomarker for advanced liver cirrhosis. Therefore, unlike miR-122, which manifests a biphasic course in
a continuum of disease severity, miR-29 is consistently downregulated along with the severity of liver injury. The combined detection of serum miR-122 and miR-29 may further improve their diagnostic value in predicting disease progression. Researchers have found that at least some circulating miRNAs are selectively and actively secreted by parent cells to function as signaling molecules for intercellular communication.26 Circulating miRNAs are transported on various kinds of carriers (e.g. exosomes, lipoproteins and argonaute 2), which protect them from being degraded by the ubiquitous ribonuclease.26 Some authors have also suggested that circulating miRNAs might result from their passive release as by-products of dead cells;27 however, that hypothesis has yet to be validated. Till now, the exact forms and origins of circulating miR-29s are unknown. miR-29 is abundant in the liver compartment. Although hepatocytes markedly outnumber HSCs, these two cell types contribute the same amount of liver miR-29.16 Immunocytes also express a high level of miR-29 to maintain immune homeostasis by inhibiting interleukin (IL)-23 and IL-17 signaling.28 Profibrotic and inflammatory chemokines induce the downregulation of miR-29.16
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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In CHB patients, HBV infection-induced liver disease and systemic inflammation may overwhelm the miR-29 signaling pathway. However, massive liver necroinflammation will inevitably result in the release of cell components, including miRNAs. This counteracts the phenomenon observed in our study in which serum miR-29 levels were downregulated, but not upregulated with ongoing necroinflammation. A possible explanation is that the decreased secretion of miR-29 far exceeds the miR-29 passively released from hepatocytes. Another explanation might be that passively released miR-29 does not possess a protective carrier and is readily degraded upon release. To verify this hypothesis, we need to detect both circulating and liver miR-29 in animal models of fulminant liver failure, in which massive liver destruction occurs in a very short period of time. In contrast, with hepatitis C virus,29 HBV infection is unlikely to disrupt the biosynthesis of miR-29 in hepatocytes, as demonstrated by high miR-29 levels in immune-tolerant patients. In this study, the expressions of miR-29a, miR-29b and miR-29c showed similar trends in different patient groups but did not always fit each other exactly. This might be due to distinct regulating mechanisms possessed by each miR-29 member. miR-29 levels show overlap between different fibrotic stages and the relevant subgroups, and it is likely that miR-29 expression is susceptible to regulation by other comorbidities besides CHB. Among the miR-29 members, serum miR-29b levels seemed to be more sensitive that reflects progressive liver fibrosis, in which serum miR-29b levels not miR-29a or miR-29c levels could differentiate no or mild fibrosis (S0/1) from significant fibrosis (S2/3). miR-29b levels were also proven to have a better correlation with ALT levels than miR-29a or miR-29c, indicating that miR-29b levels were more susceptible to systemic or liver inflammation induced by HBV infection. In the future, more comprehensive studies covering all natural stages of chronic HBV infection are needed to establish the role of serum miR-29. In conclusion, serum miR-29 levels were negatively correlated with liver necroinflammation and fibrosis in patients with chronic HBV infection. Serum miR-29 levels helped to differentiate immune-tolerant patients from CHB patients with no or mild fibrosis. miR-29 may thus serve as a novel non-invasive biomarker to evaluate disease progression in CHB patients in the future.
Journal of Digestive Diseases 2014; 15; 614–621 ACKNOWLEDGMENTS We thank Professor Chang Sheng DU and Dr Zhi Heng HE for their valuable advice and excellent technical assistance. The work was supported by the National Natural Science Foundation of China (No. 81101240).
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Journal of Digestive Diseases 2014; 15; 614–621
doi: 10.1111/1751-2980.12185
Original article
Serum microRNA-29 levels correlate with disease progression in patients with chronic hepatitis B virus infection Chong HUANG, Jian Ming ZHENG, Qi CHENG, Kang Kang YU, Qing Xia LING, Ming Quan CHEN & Ning LI Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
METHODS: Using real-time quantitative polymerase chain reaction assay, serum miR-29a, miR-29b and miR-29c levels were measured in patients with chronic HBV infection, and the correlation between serum miR-29 levels and the participants’ liver biochemistry, fibrotic stage and necroinflammation grade were also evaluated.
between S0/1 and S2/3 patients was not significant. miR-29b levels were higher in S0/1 patients than in other patient groups, but did not differ between S2/3 and S4 patients. At fibrotic stages of S0/1 and S2/3, patients with no or mild liver inflammation (G0/1) tended to express higher miR-29 levels than those with advanced inflammation (G2–4) (P > 0.05). miR29a–c showed significant correlation with alanine transaminase levels (P < 0.05 for miR-29a, miR-29b and miR-29c) in S0–3 patients. The expression of miR-29 was highest in immune-tolerant patients (P < 0.001).
RESULTS: Altogether 91 patients with chronic HBV infection were divided by fibrotic stage into S0/1 (no or mild fibrosis), S2/3 (progressive fibrosis) and S4 (cirrhosis) subgroups, and 12 healthy individuals were also included in the study. Serum miR-29a and miR-29c in S0–3 were significantly higher than those in S4 patients (P < 0.001); however, the difference
CONCLUSIONS: Serum miR-29 levels are negatively correlated with liver fibrotic stages and necroinflammation grades in patients with chronic HBV infection. miR-29 appears to be a novel biomarkers for predicting disease progression in these patients.
OBJECTIVE: To investigate the role of serum microRNA-29 (miR-29) as a biomarker for the prediction of disease progression in patients with chronic hepatitis B virus (HBV) infection.
KEY WORDS:
biomarker, chronic hepatitis B, hepatitis B virus, liver fibrosis, microRNAs.
INTRODUCTION Approximately 350–400 million patients worldwide suffered from chronic hepatitis B virus (HBV) Correspondence to: Ning LI, Department of Infectious Diseases, Huashan Hospital, Fudan University, 12 Middle Urumqi Road, Shanghai 200040, China. Email: [email protected] Conflict of interest: None. © 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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infection.1 End-stage liver disease (ESLD), including liver cirrhosis and hepatocellular carcinoma (HCC) occurs in a significantly high proportion of patients with chronic hepatitis B (CHB).2 Antiviral therapies with nucleoside analogues (NAs) or interferon (IFN) are effective in halting disease progression in such patients. These antiviral therapies are indicated when the patient’s viral load is high together with elevated peripheral liver enzyme levels and histological evidence of advanced necroinflammation or fibrosis. Liver biopsy has been proven to be valuable in predicting ESLD, especially in patients
Journal of Digestive Diseases 2014; 15; 614–621 presenting with persistently normal liver function and low viral load.3 However, liver biopsy is an invasive procedure that is associated with possible complications such as bleeding and sampling error. MicroRNAs (miRNAs) represent a family of short (18–25 nucleotides), noncoding RNAs that regulate gene expression at the level of transcription or translation.4 miRNAs are involved in the pathophysiological processes such as cellular differentiation, proliferation and apoptosis. Several studies5–8 have investigated the role of miRNAs in the pathogenesis of liver-related diseases including CHB. A fraction of cell-free miRNAs are detectable within the peripheral blood circulation as the result of their active secretion in the form of exosomes.9 The detection of cell-free miRNAs in the peripheral blood or body fluid provides a prospective, non-invasive tool to evaluate the disease progression in cancer and hematological diseases.10,11 Recently, serum miR-122, which was correlated with viral load, has been found to be capable of discriminating between patients with chronic HBV infection from healthy controls.12 The miR-29 family is composed of miR-29a, miR29b and miR-29c. The dysregulation of miR-29 contributes to the tumor pathogenesis, autoimmune disorders and fibrostic diseases including liver cirrhosis.13,14 The activation of hepatic stellate cells (HSC) plays a central role in the development of liver fibrosis.15 Quiescent HSC express higher miR-29 levels than activated HSC, and miR-29 family members have been found to downregulate in human and murine liver fibrosis.16 In contrast, the overexpression of miR-29b in HSC significantly downregulated the expressions of Col1a1, Col4a5 and Col5a3.16 During the activation of HSC in primary culture, miR-29b overexpression markedly attenuated Col1a1 and Col1a2 mRNA expression and additionally inhibited the increased expression of α-smooth muscle actin (α-SMA), discoidin domain receptor 2 (DDR2), fibronectin 1 (FN1), integrin β1 (ITGB1) and platelet-derived growth factor receptor-β (PDGFR-β), which are the key genes involved in the activation of HSC.17 Furthermore, miR-29atransgenic mice were protected from bile duct ligation-induced liver injury.18 As low circulating serum levels of miR-29a are characteristic of advanced liver fibrosis,16 this study aimed to investigate the potential role of serum miR-29 as a biomarker for the prediction of disease progression in CHB patients.
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PATIENTS AND METHODS Patients Treatment-naive patients with chronic HBV infection who were admitted to the Department of Infectious Diseases, Huashan Hospital, Fudan University (Shanghai, China) from January 2009 to December 2013 were recruited in the study. All the patients were hepatitis B surface antigen (HBsAg)-positive for at least 6 months. Exclusion criteria were: (i) patients aged less than 16 years; (ii) patients having co-infected with human immunodeficiency virus (HIV); (iii) those with the coexistence of liver injury caused by any other etiologies including hepatitis C virus (HCV) infection, drug intake, alcohol consumption and autoimmune hepatitis, and so on; (iv) those with severe systematic diseases; (v) pregnancy and lactation. In all, 91 patients who underwent liver biopsy and 12 healthy individuals were finally enrolled in the study. Written informed consent was obtained from all the adult patients who participated in the study. Participants aged under 18 years provided their verbal assent and written informed consent was obtained from their parents. The study was performed in accordance with the Helsinki Declaration and was approved by the Ethical Committee of Huashan Hospital, Fudan University. Clinical characteristics, laboratory examinations and liver histology Peripheral blood samples were collected at the day of liver biopsy, and were centrifuged at 1500 ×g for 10 min at room temperature. The serum was immediately stored at −70°C until analysis. Liver function tests were determined by standard methods in a clinical setting. HBsAg titers were determined on stored frozen serum samples with an HBsAg quantitative assay (Abbott Laboratories, Abbott Park, IL, USA) based on the automated chemiluminescent microparticle immunoassay (Abbott Architect i2000SR analyzer; Abbott Laboratories). Samples with HBsAg >250 IU/mL were diluted to the calibration range. A domestic HBV DNA quantification assay (Shanghai Kehua Bio-engineering Co., Ltd., Shanghai, China) was used to quantify serum HBV DNA titers. Samples with HBV DNA > 4 × 107 IU/mL were further diluted and retested. Liver specimens obtained by liver biopsy in CHB patients were assessed by an experienced pathologist. Two components, grading (G) and staging (S), given
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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in a numerical value ranged from 0 to 4, were used to describe disease progression of CHB. Grading reflects the intensity of necroinflammatory activity, and staging is a measure of fibrosis and alteration to the lobular architecture in chronic hepatitis. G0 and S0 represent the absence of necroinflammation and fibrosis, while G4 and S4 represent bridging necrosis involving multiple lobules and cirrhosis. Detection of miR-29 by real-time polymerase chain reaction (PCR) Total miRNA was extracted and purified from 100 μL serum using the miRNeasy Serum/Plasma Kit (QIAGEN GmbH, Hilden, Germany). Before extraction, 3.5 μL cel-miR-39 prediluted at 1.6 × 108 copies/μL was added to each tube as a spike-in control for normalization. The purified miRNA was immediately reverse-transcribed with the miScript II Reverse Transcription (RT) Kit (QIAGEN GmbH). The expressions of serum miR-29a, miR-29b and miR-29c were quantified using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). A real-time reaction was performed using miScript SYBR Green PCR Kit with primers specific for miR-29a, miR-29b and miR-29c (all from QIAGEN GmbH) according to the manufacturer’s instructions. A comparative ΔCT method was used to compare each target with cel-miR39, and relative values were expressed as 2−ΔΔC T. Statistical analysis Statistical analyses were performed with the Graphpad 5.0 (Graphpad Software, San Diego, CA, USA). Table 1.
Variables were expressed as median and interquartile range (IQR) or numbers and percentages unless otherwise specified. Differences in the parameters were compared using non-parametric Mann–Whitney U–test, and the relationship between two variables was assessed by Spearman’s rank correlation. A twotailed P value of ≤0.05 was considered statistically significant. RESULTS Serum miR-29 levels was correlated with liver fibrosis stage in CHB patients In total, 91 treatment-naive patients with chronic HBV infection and 12 healthy individuals were recruited in the study. The patients’ characteristics are summarized in Table 1. Irrespective of the patients’ HBeAg status and viral load, histological evidence of progressive liver necroinflammation (G ≥ 2) and fibrosis (S ≥ 2) were strongly associated with ESLD. According to the Chinese Guideline of Prevention and Treatment for Chronic Hepatitis B (2010 version) patients with liver histology of G ≥ 2 and/or S ≥ 2 are strongly recommended for antiviral therapy,19 especially for those with persistently normal or nearnormal alanine transaminase (ALT) levels. But persistently elevated ALT level predicts disease progression in CHB patients, especially when ALT level is over twice the upper limit of normal (ULN).20,21 To investigate the role of miR-29 in predicting disease progression, patients were classified into three groups: S0/1 (no or mild fibrosis), S2/3 (progressive fibrosis) and S4 (cirrhosis) (Table 1). The S0/1 group included 13 immune-tolerant patients (HBeAg-positive patients
Characteristics of the enrolled patients with chronic hepatitis B virus infection and healthy individuals Fibrosis stage
Gender, n (M/F) Age, years (median [range]) HBsAg, log10IU/mL (median [range]) HBeAg, n (+/−) HBV DNA, log10IU/mL (median [range]) ALT, U/L (median [range]) Necroinflammation grade, n (%) G0–1 G2–4 WBC, ×109/L (median [range]) PLT, ×109/L (median [range])
S0/1 (n = 36)
S2/3 (n = 30)
S4 (n = 25)
28/8 32 (19–55) 4.38 (1.95–5.10) 27/9 7.54 (3.5–9.30) 75 (16–748)
23/7 27 (16–61) 4.02 (2.64–5.0) 25/5 7.63 (3.92–9.70) 179 (24–1721)
18/7 44 (21–64) 3.27 (0.35–3.70) 17/8 4.94 (0–7.29) 37 (15–138)
26 (72.2) 10 (27.8) 5.1 (3.2–12.7) 204 (109–289)
5 (16.7) 25 (83.3) 5.4 (3.5–9.9) 180 (92–291)
12 (48.0) 13 (52.0) 2.9 (1.0–11.5) 61 (23–227)
Healthy controls 8/4 33 (24–51) NA NA NA NA NA
NA NA
ALT, alanine transaminase; F, female; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; M, male; NA, not available; PLT, platelet; WBC, white blood cells.
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Figure 1. Serum microRNA-29 (miR-29) levels in patients at different stages of chronic hepatitis B-induced liver fibrosis and healthy controls (HC). (a–c) miR-29a, miR-29b and miR-29c levels; (d–f) those subdivided by grading (G0/1 and G2–4). □, G0/1; ■, G2–4. Values are expressed as median and interquartile range.
with persistently normal ALT level for at least one year and normal liver histology) and 23 CHB patients. No inactive HBsAg carriers (positive HBsAg for over 6 months with persistently normal liver function, negative HBeAg, positive HBeAb, HBV DNA 0.05) (Fig. 1). miR-29b levels were significantly higher in S0/1 patients than the other two patient groups (S0/1 vs S2/3, P < 0.05; S0/1 vs S4, P < 0.01), but was much lower than that of the healthy controls (P < 0.05) (Fig. 1). And the miR-29b levels between the S2/3 and S4 groups were not significantly different (P > 0.05). To discover whether inflammation grading affects serum miR-29 expression in the individual fibrotic stage, we divided each group into two subgroups based on grading. In S0/1 and S2/3 groups, all miR-29 tended to be higher in patients with no or mild liver necroinflammation (G0/1) than those with advanced inflammation (G ≥ 2),
although the difference was not statistically significant. In contrast, liver necroinflammation seemed to be irrelevant to miR-29 expression in the S4 group (Fig. 1). miR-29 levels differed between immune-tolerant and CHB patients In patients at liver fibrosis S0–3 all miR-29 levels were significantly correlated with ALT levels (P = 0.0258 for miR-29a, P < 0.0001 for miR-29b, P = 0.0044 for miR-29c) (Fig. 2a–c). We further divided patients into immune-tolerant, S0/1 CHB and S2/3 CHB subgroups. The expressions of miR29a, miR-29b and miR-29c were all highest in the immune-tolerant group than in the other CHB groups (P < 0.001) (Fig. 2d–f); miR-29a and miR-29c levels in the immune-tolerant group were even higher than those in the healthy controls (P < 0.01, data not shown). But no differences in miR-29 levels were observed between S0/1 and S2/3 CHB patients (Fig. 2d–f). The relationship between ALT levels and miR-29 expressions was not examined in S4 patients because only 2 of the 25 patients had an ALT level of at least twice ULN.
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Figure 2. (a–c) Correlation of serum microRNA-29 (miR-29) with alanine transaminase (ALT) levels in patients at liver fibrosis S0–3 and (d–f) miR-29 levels in immune-tolerant (IT) patients, S0/1, S2/3 chronic hepatitis B (CHB) patients and healthy controls (HC). Values in (d–f) are expressed as median and interquartile range.
The relationship between miR-29 and other biomarkers We then investigated whether peripheral blood white blood cell (WBC) or platelet (PLT) counts and the biomarkers reflecting the extent of liver fibrosis were correlated with miR-29 expressions. WBC count was observed to be correlated with miR-29a levels (P < 0.05), but not with miR-29b or miR-29c levels (Fig. 3a–c). In contrast, all miR-29 showed significant correlation with PLT count (Fig. 3d–f). miR-29 levels also showed a positive correlation with HBV DNA titer (log10IU/mL) (P < 0.05, data not shown). The correlation between miR-29a and miR-29c level and HBsAg titer (log10IU/mL) was also significant (P < 0.05, data not shown). However, low viral load in patients with advanced liver disease might have contributed to the statistical difference. DISCUSSION Chronic hepatitis B is a severe liver disease associated with significant morbidity and mortality.1 Predicting
disease progression in patients with chronic HBV infection is important because of the appropriate time for antiviral therapy. Liver biopsy has long been considered the gold standard for the staging of CHBinduced fibrosis, but it is an invasive procedure associated with occasional severe outcomes and has several limitations. In contrast, miRNAs derived from blood or body fluid are easily accessible potential biomarkers for the evaluation of disease severity. miR-29 is intimately involved in the pathogenesis of fibrotic diseases including liver cirrhosis, and an early study found that, compared with healthy individuals, in patients with advanced liver cirrhosis due to various etiologies serum miR-29a levels were significantly lower.16 In this study we investigated the correlation between serum miR-29 levels and disease progression in patients with chronic HBV infection, and found that serum miR-29 levels were negatively correlated with patient’s fibrotic stage. miR-29 expressions also readily differed between immune-tolerant patients and HBeAg-negative or HBeAg-positive CHB patients with mild fibrosis. Furthermore, a significant negative correlation between miR-29 expression and ALT levels
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Figure 3. Correlation between serum microRNA-29 (miR-29) levels and (a–c) white blood cell (WBC) count and (d–f) platelet (PLT) count. P values are calculated using the Spearman rank correlation.
was found. Therefore, serum miR-29 might be a potential biomarker to assess disease progression in patients with CHB. Studies have investigated the potential of various peripheral circulation miRNAs in the diagnosis of liver diseases, including miR-122, miR-22, miR-20a, miR-192 and miR-210,5,22–25 among which miR-122 has been the most studied. To our knowledge, miR-29 is the only validated miRNA that is downregulated in the progress of liver injury, while the other miRNAs are reported to be upregulated. Serum miR-122 levels are increased with the severity of liver injury, as shown by its good correlation with aminotransferase level and necroinflammation.12,22 However, miR-122 levels are downregulated in patients with decompensated cirrhosis compared with the compensated patients.25 Low serum miR-122 level has been found to be an independent marker for poor survival in cirrhotic patients.25 Serum miR29a has been reported to be negatively correlated with the model for end-stage liver disease (MELD) score,16 and in our cohorts it was negatively correlated with the extent of cytopenia, which is a biomarker for advanced liver cirrhosis. Therefore, unlike miR-122, which manifests a biphasic course in
a continuum of disease severity, miR-29 is consistently downregulated along with the severity of liver injury. The combined detection of serum miR-122 and miR-29 may further improve their diagnostic value in predicting disease progression. Researchers have found that at least some circulating miRNAs are selectively and actively secreted by parent cells to function as signaling molecules for intercellular communication.26 Circulating miRNAs are transported on various kinds of carriers (e.g. exosomes, lipoproteins and argonaute 2), which protect them from being degraded by the ubiquitous ribonuclease.26 Some authors have also suggested that circulating miRNAs might result from their passive release as by-products of dead cells;27 however, that hypothesis has yet to be validated. Till now, the exact forms and origins of circulating miR-29s are unknown. miR-29 is abundant in the liver compartment. Although hepatocytes markedly outnumber HSCs, these two cell types contribute the same amount of liver miR-29.16 Immunocytes also express a high level of miR-29 to maintain immune homeostasis by inhibiting interleukin (IL)-23 and IL-17 signaling.28 Profibrotic and inflammatory chemokines induce the downregulation of miR-29.16
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In CHB patients, HBV infection-induced liver disease and systemic inflammation may overwhelm the miR-29 signaling pathway. However, massive liver necroinflammation will inevitably result in the release of cell components, including miRNAs. This counteracts the phenomenon observed in our study in which serum miR-29 levels were downregulated, but not upregulated with ongoing necroinflammation. A possible explanation is that the decreased secretion of miR-29 far exceeds the miR-29 passively released from hepatocytes. Another explanation might be that passively released miR-29 does not possess a protective carrier and is readily degraded upon release. To verify this hypothesis, we need to detect both circulating and liver miR-29 in animal models of fulminant liver failure, in which massive liver destruction occurs in a very short period of time. In contrast, with hepatitis C virus,29 HBV infection is unlikely to disrupt the biosynthesis of miR-29 in hepatocytes, as demonstrated by high miR-29 levels in immune-tolerant patients. In this study, the expressions of miR-29a, miR-29b and miR-29c showed similar trends in different patient groups but did not always fit each other exactly. This might be due to distinct regulating mechanisms possessed by each miR-29 member. miR-29 levels show overlap between different fibrotic stages and the relevant subgroups, and it is likely that miR-29 expression is susceptible to regulation by other comorbidities besides CHB. Among the miR-29 members, serum miR-29b levels seemed to be more sensitive that reflects progressive liver fibrosis, in which serum miR-29b levels not miR-29a or miR-29c levels could differentiate no or mild fibrosis (S0/1) from significant fibrosis (S2/3). miR-29b levels were also proven to have a better correlation with ALT levels than miR-29a or miR-29c, indicating that miR-29b levels were more susceptible to systemic or liver inflammation induced by HBV infection. In the future, more comprehensive studies covering all natural stages of chronic HBV infection are needed to establish the role of serum miR-29. In conclusion, serum miR-29 levels were negatively correlated with liver necroinflammation and fibrosis in patients with chronic HBV infection. Serum miR-29 levels helped to differentiate immune-tolerant patients from CHB patients with no or mild fibrosis. miR-29 may thus serve as a novel non-invasive biomarker to evaluate disease progression in CHB patients in the future.
Journal of Digestive Diseases 2014; 15; 614–621 ACKNOWLEDGMENTS We thank Professor Chang Sheng DU and Dr Zhi Heng HE for their valuable advice and excellent technical assistance. The work was supported by the National Natural Science Foundation of China (No. 81101240).
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