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Circulating MicroRNA-122 for the Diagnosis of Hepatocellular Carcinoma: A Meta-Analysis

Background Circulating microRNA-122 (miR-122) has been recognized as a marker of hepatocellular carcinoma (HCC). The current meta-analysis was performed to quantitatively evaluate the diagnostic performance of circulating miR-122 for HCC. Methods Related studies that evaluated the diagnostic performance of circulating miR-122 determined from pathophysiological examination for HCC were obtained by systematic searches of the PubMed and Embase databases. A randomized fixed effects model was applied according to the heterogeneity among studies. The pooled sensitivity, specificity, and area under the summary receiver operating characteristic curve (AUC) were calculated to evaluate the diagnostic accuracy. Publication bias was detected by Deeks' funnel plot asymmetry test. Results Thirteen studies providing data for 920 HCC patients and 1217 controls were included in the meta-analysis. The pooled sensitivities, specificities, and AUCs of serum miR-122 were 0.76, 0.75, and 0.82, respectively, for discriminating HCC patients from overall controls; 0.85, 0.83, and 0.91, respectively, for discriminating HCC patients from healthy controls; 0.79, 0.82, and 0.87, respectively, for discriminating HCC from HBV or HCV infection; and 0.65, 0.75, and 0.74, respectively, for discriminating HCC from liver cirrhosis or dysplastic nodule formation. No significant publication bias was detected. Conclusions Serum miR-122 confers moderate efficacy for discriminating HCC patients from healthy controls or patients with HBV or HCV infection, but not for discriminating HCC patients from those with liver cirrhosis or dysplastic nodule formation.


Introduction
Hepatocellular carcinoma (HCC) accounts for more than 80% of cases of primary liver cancer [1], and the prognosis of patients with HCC depends on the staging of the disease. The 5-year survival of patients with early-stage HCC was reported to be as high as 75%, whereas the 1-year survival of patients with widespread HCC was reported to be less than 10%, which highlights the importance of early diagnosis [1,2].
Currently, clinical screening strategies for HCC mainly involve ultrasonography [3,4]. However, the diagnostic performance of ultrasonography for early HCC is poor due to its low sensitivity [5]. Other imaging modalities such as computer tomography (CT) and magnetic resonance imaging (MRI) have limited value for HCC screening because of the risk of radiation exposure or high expense [3,4]. Serum biomarkers may be convenient and effective for early diagnosis of HCC. However, increased circulating alpha fetoprotein (AFP), the most commonly used circulating biomarker for HCC, has been observed in patients without HCC, such as those with chronic hepatitis B or C, suggesting the poor specificity of AFP for HCC [3,6].
Recent advances in biomedical research have demonstrated that microRNAs (miRNAs), an endogenous group of small noncoding RNAs that negatively regulate gene expression at the posttranscriptional level, are involved in many cellular processes, including carcinogenesis [7]. miR-NAs are highly stable and measurable from various tissues, including the peripheral circulation, which makes them potential biomarkers for cancer screening in clinics [8,9]. MicroRNA-122 (miR-122) has been demonstrated to be involved in the process of hepatocarcinogenesis, and differences in the circulating level of miR-122 have been observed in HCC patients compared with control individuals [8,10]. Although accumulating studies have evaluated the potential diagnostic performance of miR-122 for HCC, quantitative meta-analyses that summarize the diagnostic efficacy of miR-122 for HCC are still needed. In a previous metaanalysis that included four studies [11][12][13][14], Huang et al. concluded that serum miR-122 confers promising diagnostic efficacy for HCC [15]. However, due to the small number of studies included, they were unable to evaluate the efficacy of circulating miR-122 for discriminating HCC patients from various control patient populations [15]. Since the publication of their meta-analysis, several additional studies regarding the diagnostic efficacy of miR-122 for HCC have been published [16][17][18][19][20][21][22][23][24]. Therefore, we aimed to quantitatively evaluate the potential diagnostic performance of circulating miR-122 for HCC in an updated meta-analysis. Moreover, we explored whether circulating miR-122 confers similar efficacy for discriminating HCC patients from healthy controls, HCC patients from those with HBV or HCV infection, and HCC patients from those with liver cirrhosis.

Database
Searches. This systematic review and metaanalysis was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [25] and Cochrane Handbook [26]. The literature reports were obtained via electronic searches of the PubMed (MEDLINE) and Embase databases using the following terms: "microRNA-122", "miRNA-122", or "miR-122", combined with "hepatocellular cancer", "hepatocellular tumor", "hepatocellular carcinoma", "hepatocellular neoplasm", "liver cancer", "liver tumor", "liver carcinoma", "liver neoplasm", or "HCC", with a limitation of studies in humans. The date of the final database search was March 12, 2019. The reference lists of relevant original and review articles were manually searched for potential studies.

Inclusion and Exclusion
Criteria. The following inclusion criteria were applied: (1) full-length article published in English; (2) histopathological examination used as the referenced standard for HCC diagnosis; (3) studies aimed at evaluating the diagnostic performance of circulating miR-122 for HCC diagnosis; and (4) reported data being adequate for extraction or calculation of the true-and false-positive values as well as true-and false-negative values for HCC diagnosis with miR-122 as determined by histopathological examination. Review articles, repeated reports, and preclinical studies were excluded.

Data Extraction and Quality
Assessment. The literature search, data extraction, and quality assessment were independently performed by two authors, with discrepancies resolved by discussion with the corresponding author. The following data were extracted: name of the first author; year of publication; study location; number, age, and gender of HCC patients and controls; characteristics of controls; and miR-122 sampling and measurement methods. True-and false-positive data and true-and false-negative data for the diagnosis of HCC based on miR-122 were extracted or calculated for meta-analysis. The quality evaluation was performed with the QUADAS (quality assessment tool for diagnostic accuracy studies) scale [27]. The QUADAS scale is a validated tool for quality evaluation of the diagnostic accuracy studies, with the highest score of 14 indicating optimal study quality.

Statistical
Analysis. The summary sensitivity and specificity were calculated from 2 × 2 forms with corresponding 95% confidence intervals (CIs). The area under the receiver operating characteristic (AUC) curve derived from the data was taken to reflect the overall effectiveness of each quantitative method. Interstudy heterogeneity was formally tested using Cochran's Q test, and significant heterogeneity was defined as P < 0:10. We also examined the I 2 statistic to reflect the heterogeneity of the included studies, which describes the percentage of total variation across studies that is due to heterogeneity rather than chance. An I 2 > 50% was considered indicative of significant heterogeneity [28]. In cases of significant heterogeneity as reflected by I 2 > 50%, a random effects model was used to estimate the overall effect; otherwise, a fixed effects model was applied. Because the characteristics of controls may affect the diagnostic efficacy of circulating miR-122 for HCC, we subsequently analyzed the performance of miR-122 for discriminating HCC patients from healthy controls, from patients with HBV or HCV infection, and from patients with liver cirrhosis or dysplastic nodule formation. Deeks' funnel plot asymmetry test was used to evaluate publication bias. Statistical analyses were performed using Stata 12.0. All statistical tests were two-sided, with P < 0:05 indicating statistical significance.

Study Characteristics and Quality
Evaluation. The characteristics of the included studies are summarized in Table 1. Overall, 13 studies reporting data for 920 patients with histopathologically confirmed HCC and 1217 control individuals were included. Seven of the studies were performed in China 2 BioMed Research International [11-14, 17, 21, 23], three in Egypt [16,18,19], one in Vietnam [22], one in Italy [20], and one in Australia [24]. The characteristics of the control populations were mixed, with inclusion of healthy controls, participants with HBV or HCV infection, and patients with liver cirrhosis or dysplastic nodules. All of the included studies measured serum miR-122 expression by quantitative real-time PCR. The QUADAS scale scores were between 6 and 12 for the included studies, indicating moderate study quality.

Discussion
In this meta-analysis of 13 studies reporting data for 920 HCC patients and 1217 control individuals, we found that serum miR-122 conferred moderate diagnostic accuracy for HCC overall, with a summary AUC of 0.82. Subsequent analyses indicated that the serum miR-122 level confers acceptable efficacy for discriminating HCC patients from healthy controls (AUC: 0.91) or patients with HBV or HCV infection (AUC: 0.87), but less optimal efficacy for discriminating HCC patients from those with liver cirrhosis or dysplastic nodule formation (AUC: 0.74). These results suggest that measurement of serum miR-122 may be of significance for HCC surveillance in apparently healthy people or carriers of HBV or HCV, while for those with liver cirrhosis, imaging exanimations are essential. Previous findings from experimental studies indicated that miR-122 regulates various physiological and pathological processes within hepatic cells, such as lipid metabolism, the response to drug or alcoholic hepatic injury, the response to viral infection, and hepatic fibrosis formation [10]. Accumulating evidence indicates that miR-122 expression is downregulated in the HCC tissue compared with the normal hepatic cells [29] and thus may predict poor prognosis in these patients [30]. Further studies have confirmed that miR-122 may function as a tumor suppressor during the process of hepatocarcinogenesis [31][32][33]. Interestingly, a higher level of circulating miR-122 has been observed in HCC patients versus those without HCC, suggesting serum miR-122 as a potential biomarker of HCC [8]. The results of our present meta-analysis further demonstrated that measurement of circulating miR-122 may confer moderate diagnostic efficacy for HCC according to histopathological examination, particularly for the discrimination of HCC patients from healthy controls or patients with HBV or HCV infection. These results are of clinical significance because they support the inclusion of serum miR-122 measurement in HCC surveillance in apparently healthy people or carriers of HBV or        Figure 2: Forest plots for the diagnostic performance of circulating miR-122 for HCC: (a) summarized sensitivity and specificity of serum miR-122 for discriminating HCC patients from overall controls; (b) summarized sensitivity and specificity of serum miR-122 for discriminating HCC patients from healthy controls; (c) summarized sensitivity and specificity of serum miR-122 for discriminating HCC patients from patients with HBV or HCV infection; (d) summarized sensitivity and specificity of serum miR-122 for discriminating HCC patients from patients with liver cirrhosis or dysplastic nodule formation. 6 BioMed Research International  To the best of our knowledge, only one previous metaanalysis evaluated the diagnostic role of miR-122 for HCC. This meta-analysis only included four available studies published before 2015 and concluded that miR-122 may be used to distinguish HCC patients from healthy controls. Our meta-analysis included a total of 13 studies with 920 HCC patients and 1217 controls. The larger datasets enable our study to confirm the discriminating role of circulating miR-122 for HCC from healthy controls, with comprehensively summarized diagnostic parameters including sensitivities, specificities, and AUCs. Compared to healthy controls, discriminating patients with HCC from high-risk population, such as those with HBV/HCV infection or liver cirrhosis or dysplastic nodule formation, is of more clinical importance [35,36]. Although previous meta-analysis suggested a discriminating role of circulating miR-122 for HCC from healthy controls, its ability for the discriminating of HCC from high-risk population remains undetermined. Our meta-analysis showed that miR-122 may be useful to discriminate HCC from patients with HBV or HCV infection (AUC: 0.87), but with less optimal efficacy for discriminating HCC patients from those with liver cirrhosis or dysplastic nodule formation (AUC: 0.74). These results demonstrated that serum miR-122 may be of significance for HCC surveillance in apparently healthy people, but for patients with liver cirrhosis or dysplastic nodule formation, additional strategies are required.
Our study also has a few limitations. First, the heterogeneity among the included studies was considerable based on the I 2 statistics and results of Cochran's Q tests. Differences in study characteristics, such as comorbidities of the patients, miR-122 cutoff values, and internal controls used during quantitative PCR, may contribute to the heterogeneity. Unfortunately, we were unable to explore the influences of the above factors on the results of the meta-analysis, because these factors were rarely reported in the included studies. Secondly, the diagnostic performance of circulating miR-122 for HCC was only moderate based on our results. Strategies to improve this efficacy deserve investigation. For example, recent studies indicated that the measurement of miR-122 from circulating exosomes may provide better diagnostic efficacy for HCC than the measurement of miR-122 in serum [37,38]. Other strategies such as using miRNA panels [39] or combining conventional diagnostic strategies with circulating miR-122 measurement [40] may also improve the diagnostic efficacy for HCC. Finally, because the incidence of HCC is high in Chinese patients, a large proportion of studies regarding HCC diagnosis and prevention have been performed in China, as reflected in our meta-analysis. The potential diagnostic efficacy of circulating miR-122 for HCC in patients from other countries deserves further evaluation.
In conclusion, the results of our study indicate that serum miR-122 confers moderate efficacy for discriminating HCC patients from healthy controls or patients with HBV or HCV infection, but not for discriminating HCC patients from patients with liver cirrhosis or dysplastic nodule formation. Measurement of serum miR-122 may be of significance for HCC surveillance in apparently healthy people.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declared that they have no conflict of interest.