Expression of mir-122 plasma as a biomarker for hepatocellular carcinoma – Dang Chieu Duong

Journal of military pharmaco-medicine n 0 9-2018 95 EXPRESSION OF miR-122 PLASMA AS A BIOMARKER FOR HEPATOCELLULAR CARCINOMA Dang Chieu Dương1; Phan Quoc Hoan2; Le Huu Song2 Nguyen Linh Toan3; Ngo Tat Trung2 SUMMARY Objectives: MicroRNAs participate in cell proliferation, apoptosis and transformation. However, the use of microRNAs-122 in combination with alpha-fetoprotein has not been evaluated. In this study, we aim to evaluate the expression of microRNAs-122 plasma, and combination of microRNAs-122 with alpha-fetoprotein to diagnosis of hepatocellular carcinoma. Subjects and methods: Real-time PCR was employed to measure microRNAs-122 expression levels on 250 plasma samples of 101 patients with hepatitis B virus-related hepatocellular carcinoma, 46 chronic hepatitis B patients and 103 healthy controls. Results: The relative expression levels of microRNAs-122 in hepatitis B virus-related hepatocellular carcinoma patients were higher than chronic hepatitis B and healthy controls (p < 0.001). The individual microRNAs-122 acquired high diagnostic accuracy for hepatocellular carcinoma surveillance (hepatocellular carcinoma vs. chronic hepatitis B, AUC = 0.910; hepatocellular carcinoma vs. healthy controls, AUC = 0.989). When alpha-fetoprotein levels were lower than 20 ng/mL, microRNAs-122 still preserves accuracy to distinguish hepatocellular carcinoma from other groups (chronic hepatitis B, AUC = 0.915; healthy controls, AUC = 0.991). When microRNAs- 122 were used in combination with alpha-fetoprotein levels, the diagnostic performance was significantly improved in discriminating hepatocellular carcinoma from group chronic hepatitis B (AUC = 0.914). Conclusions: MicroRNAs-122 is a potential biomarker to improve diagnostic in hepatitis B virus-related hepatocellular carcinoma patients, especially in patients with hepatocellular carcinoma with normal alpha-fetoprotein level. * Keywords: Hepatocellular carcinoma; MicroRNAs-122; Alpha-fetoprotein. INTRODUCTION Liver cancer is currently the second most common cause of cancer-related death worldwide, and hepatocellular carcinoma (HCC) accounts for more than 90% of liver cancers [1]. One of the reasons for the high mortality in HCC is that the tumors are frequently detected at a stage when curative resection is no longer feasible because of intrahepatic and extrahepatic metastases. Today, the diagnosis of HCC relies on the finding of a liver mass in radiology imaging studies including ultrasonography, computed tomography (CT), and/or magnetic resonance imaging (MRI) [2]. However, the diagnosis of small lesions is relatively inaccurate. One of the common approaches used 1. Viettiep Hospital 2. 108 Military Central Hospital 3. Vietnam Military Medical University Corresponding author: Dang Chieu Duong (dcduong@gmail.com) Date received: 01/10/2018 Date accepted: 14/11/2018 Journal of military pharmaco-medicine n 0 9-2018 96 for screening HCC in a high risk- population is serum tumor markers such as alpha-fetoprotein (AFP) and Des- gamma-carboxy prothrombin (DCP). The performance of the currently used serum protein biomarkers for routine surveillance of HCC is unsatisfied [3]. Therefore, new biomarkers with better accuracy or with capabilities to complement for hepatic imaging are in need to improve the surveillance of HCC. MicroRNA (miR) is a small noncoding RNA gene product known to post- transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of its target mRNAs. MiRs control a wide array of biological processes, such as cell differentiation, proliferation, and apoptosis [4]. Expressions of miRs have been widely reported in human cancers with both up and down-regulation detected in neoplastic cells compared with their normal counterparts. Several recent studies reported that miRs are stably detectable in plasma and serum. The finding also raised the possibility that assaying miRs in plasma or serum may serve as a novel approach for blood- based detection of human cancers [5]. In this study, we focused on miR-122, which is one of the first miRs detected abundantly in HCC. We aim to: Evaluate the expression of miR-122 plasma, and combination of miR-122 with AFP to diagnosis of HCC. SUBJECTS AND METHODS 1. Subjects. Patients and sample: Plasma samples from 101 patients with hepatitis B virus (HBV)-related HCC, 46 chronic hepatitis B (CHB), and 103 healthy control (HC) were collected for this study between 2014 and 2017. Biopsies were taken in all patients with HCC and CHB. HCC and CHB were confirmed histologically in the groups. Blood samples were obtained from all patients and healthy controls, plasma samples were immediately separated from blood cells and were stored at -800C until use. MiRNA extraction and cDNA synthesis: Total RNA, including miRNA fractions was isolated from 200 µL serum with TRIzol reagent and reconstituted in 50 µL water treated with diethylpyrocarbonate (DEPC). The quality of total RNA preparations was assessed by NanoDrop spectrometer at 260 and 280 nm (A260/280). Approximately 300 ng of total RNA were used for reverse transcription (RT) by RevertAid First Strand cDNA Synthesis Kit following the manufacturer's instruction. Primers used for cDNA synthesis were designed according to stem-loop. Quantification of miR-122 by real-time PCR: After reverse transcription, cDNA was reconstituted in 100 µL 25 mM-Tris-HCl pH 8.0. The real- time PCR (qPCR) reaction mixtures consisted of 10 µL of 2x Sybr-Green I master mix, 5 µL of cDNA preparation, 5 pmoL of miR-122 universal reverse primer GTGCAGGGTCCGAGGT and 5 pmoL of forward primer specific for miR-122. The qPCR reaction was performed using the Stratagene M3000p device with a pre-incubation step at 500C for 15 minutes, initial denaturation at 950C for 5 minutes, followed by 45 cycles of 950C for 15 sec and 600C for 60 seconds. The RT-PCR reactions were finalized by Journal of military pharmaco-medicine n 0 9-2018 97 amplicon melting dissociation. The cycle of threshold (Ct) values were recorded and analyzed according to the comparative Ct method, in which the Ct value of miR-16 was used as normalization factor [6]. Statistical analysis: The HCC staging was performed according to the Barcelona clinic liver cancer (BCLC) staging system. Data ware expressed as mean ± SD. Differences between groups were assessed by the t-test, or the Mann- Whitney U test, p value < 0.05 denoted the presence of a statistically significant difference. The diagnostic value for differentiating between HCC patients and the control was assessed by calculating the area under the receiver operator characteristic (ROC) curve (AUC). All statistical analyses were performed using SPSS (version 21.0). RESULTS The main clinical and demographic characteristics such as age, gender, viral loads and the tumour marker AFP for HCC patients, CHB patients, and 103 healthy controls are summarized in table 1. Table 1: Clinical characteristics of the studied patients. Characteristics HCC (n = 101) CHB (n = 46) HC (n = 103) Age (years) 55.6 ± 12.34 41.43 ± 11.54 24 ± 6.42 Gender (M: %) 93.1 95.7 76.7 AFP (ng/mL)* 339.29 ± 574.02 43.81 ± 142.97 NA HBV-DNA (log)* 5.01 ± 1.52 6.63 ± 1.97 NA Tumor number (solitary/multiple) 65/36 NA NA Maximum tumor size (cm) 7.78 ± 3.41 NA NA Vascular invasion (yes/no) 23/78 NA NA Tumor differentiation (high/intermediate/low) 25/46/10 NA NA BCLC staging (A/B/C/D) 16/50/34/1 NA NA (*: p < 0.05 for comparison HCC vs. CHB; NA: Not applicable) Levels of HBV loads were significantly higher in CHB compared to HCC group (p < 0.05). As expected, the levels of the tumour marker AFP were observed significantly higher in HCC patients compared to CHB patients (p < 0.05). Journal of military pharmaco-medicine n 0 9-2018 98 Figure 1: Differential expression miR-122 levels in different groups. The plasma miR-122 expression in patients with HCC was significantly higher than in patients with CHB and HC (p < 0.001, p < 0.001, respectively). Figure 2: Diagnostic performance of miR-122 and miR-122 in combination with AFP in differentiating HCC from other groups. (A: HCC vs. CHB; B: HCC [AFP ≤ 20 ng/mL] vs. CHB; C: HCC vs. HC and D: HCC [AFP ≤ 20 ng/mL] vs. HC) Journal of military pharmaco-medicine n 0 9-2018 99 Table 2: Diagnostic performance of miR-122 and miR-122 in combination with AFP in differentiating HCC against control subjects. AUC Variable(s) HCC vs. CHB HCC vs. HC HCC (AFP ≤ 20 ng/mL) vs. CHB HCC (AFP ≤ 20ng/mL) vs. HC AFP* 0.758 NA NA NA miR-122* 0.910 0.989 0.915 0.991 AFP + miR-122** 0.914 NA NA NA (*: p < 0.05 for comparison AFP vs. miR-122; **: p < 0.05 for comparison AFP vs. miR-122+AFP; A: Not applicable) Diagnostic performance of miR-122 was significantly higher than AFP levels in differentiating HCC from CHB (p < 0.05). When miR-122 is combined with AFP levels to form the so-called (miR-122 + AFP) panel under the logistic regression model, the diagnostic performance in discriminating HCC from CHB obtained higher accuracy (AUC = 0.914, p < 0.05). When the AFP levels were lower than 20 ng/mL, miR-122 still preserved its diagnostic accuracy to distinguish HCC from other groups including CHB patients (AUC = 0.915), with either HC (AUC = 0.991). DISCUSSION Cancer-specific alterations of miRs expression are common in various cancers and act as critical roles in cancer progression. More and more evidence indicated that circulating plasma miRs served as promising biomarker for diagnosing and monitoring treatment response. Circulating miRs have been found to be stable in serum and plasma, suggesting its potential ability to be diagnostic biomarkers. Currently, AFP is often criticized for its high false positivity in distinguishing HCC with other liver diseases. In addition, it is estimated that 30 - 40% of all HCC patients are of AFP- negative status, making it difficult to diagnose and assess treatment response [7]. Thus, identifying a new biomarker that could complement AFP is of great importance. For now, several miRs have been identified as novel biomarkers for HCC [8]. In this study, we conducted analysis to evaluate whether plasma miR-122 had the potential ability to be a new biomarker for detecting HCC. In pilot group, we assessed the expression of miR-122 on 250 plasma samples of 101 patients with HBV-related HCC, 46 chronic hepatitis B patients and 103 HC. The present study demonstrated that plasma miR-122 expression in patients with HCC was significantly higher than in patients with CHB and HC (p < 0.001, p < 0.001, respectively). ROC analyses for the diagnostic power of plasma miR-122 yielded an AUC of 0.910 in differentiating patients with HCC from those with CHB, AUC of 0.989 in differentiating patients with HCC from HC. These results suggest that plasma miR-122 is a valuable biomarker for HCC. Furthermore, the superiority of the Journal of military pharmaco-medicine n 0 9-2018 100 differentiating power of a single measurement of plasma miR-122 compared with AFP was statistically confirmed, and the differentiating power of the combination of plasma miR-122 and AFP was significantly stronger than AFP alone, suggesting that measurement of both plasma miR-122 and AFP has a better differentiating power than plasma miR- 122 and AFP alone. Furthermore, plasma miR-122 level was significantly elevated even in patients with HCC who have AFP levels under 20 ng/mL. When the AFP levels were lower than 20 ng/mL, miR- 122 still preserved its diagnostic accuracy to distinguish HCC from other groups including CHB patients (AUC = 0.915), with either HC (AUC = 0.991). Whatever the reason, that differentiating power of plasma miR-122 was significantly superior to that of AFP, we suggested that plasma miR-122 is a useful diagnostic marker for HCC. At the same time, we must keep in mind that, as even patients with advanced HCC were included in the present study, the enrollment of the patients was not designed for the examination of diagnostic markers, suggesting the possibility that the aforementioned sensitivity and specificity might be over- estimated. In addition, we did not examine plasma miR-122 level in cirrhotic patients who also have possibility for developing HCC in the present study. We should investigate whether the miR-122 measurement is useful in differentiating HCC patients from cirrhotic patients in the future. Furthermore, large-scale cohorts and strict study design are needed to further confirm the diagnostic role of miR-122. CONCLUSIONS Our findings suggest that plasma miR- 122 levels may help enhance the diagnosis of HCC, especially for AFP- negative HCC. REFERENCES 1. Omata M, Cheng A.L, Kokudo N et al. Asia-Pacific clinical practice guidelines on the management of HCC: A 2017 update. Hepatol Int. 2017, 11 (4), pp.317-370. 2. Yu W.B, Rao A, Vu V et al. Management of centrally located HCC: Update 2016. World J Hepatol. 2017, 9 (13), pp.627-634. 3. Toyoda H, Kumada T, Tada T et al. Tumor markers for HCC: Simple and significant predictors of outcome in patients with HCC. Liver Cancer. 2015, 4 (2), pp.126-136. 4. Tsuchiya N, Sawada Y, Endo I et al. Biomarkers for the early diagnosis of HCC. World J Gastroenterol. 2015, 21 (37), pp.10573-10583. 5. Hayes C.N, Chayama K. Micro-RNAs as biomarkers for liver disease and HCC. J Mol Sci. 2016, 17 (3), p.280. 6. Livak K.J, Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. 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