Serum mir-182 and mir-331-3p as diagnostic and prognostic markers in patients with hepatocellular carcinoma

Tumor Biol. (2015) 36:7439 7447 DOI 10.1007/s13277-015-3430-2 RESEARCH ARTICLE Serum mir-182 and mir-331-3p as diagnostic and prognostic markers in patients with hepatocellular carcinoma Lin Chen 1 & Feihu Chu 2 & Yali Cao 1 & Jianguo Shao 1 & Feng Wang 3 Received: 29 October 2014 /Accepted: 7 April 2015 /Published online: 24 April 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015 Abstract Previous studies have identified a number of micrornas (mirnas) that were aberrantly expressed in hepatocellular carcinoma (HCC) tissues. Nevertheless, their diagnostic and prognostic value in serum has not been fully evaluated. Herein, the levels of five serum mirnas, namely, mir-182, mir-331-3p, mir-197, mir-492, and mir-581, were detected in 103 HCC patients, 95 benign liver diseases, and 40 healthy controls using real-time PCR technique. The results showed that, compared with benign liver diseases and healthy controls, the levels of serum mir-182 and mir-331-3p were significantly increased in HCC patients, both P<0.001. Area under the receiver operating characteristic (ROC) curves for serum mir-182 and mir-331-3p were 0.911 (95 % CI, 0.863 0.947) and 0.890 (95 % CI, 0.838 0.930), the sensitivity were 78.6 and 79.61 %, and the specificity were 91.58 and 86.32 %, respectively. Moreover, the combination of serum mir-182, mir-331-3p, and alphafetoprotein (AFP) can markedly increase the differential diagnostic value of benign and malignant liver diseases, especially better than serum AFP alone, P<0.05. Serum mir-182 was positively correlated with serum AFP (P=0.001), tumor size (P=0.013), and TNM classification of malignant tumors * Jianguo Shao xiaobei227@sina.com * Feng Wang richardwangf@163.com 1 2 3 Department of Gastroenterology and Clinical Laboratory, The Third People s Hospital of Nantong City, Jiangsu 226006, China Department of General Surgery, The Sixth People s Hospitalof Nantong City, Jiangsu 226011, China Department of Clinical Laboratory Center, Affiliated Hospital of Nantong University, Jiangsu 226001, China (TNM) stage (P=0.003); however, only TNM stage was demonstrated a significant correlation with serum mir-331-3p (P=0.006). In addition, Kaplan Meier survival curve, together with univariate and multivariate Cox proportional hazard analyses, further disclosed that serum mir-182 and mir-331-3p were associated with postoperative survival of HCC patients, and both of them were regarded to be independent prognostic factors for patients with HCC. Taken together, our present study indicates that serum mir-182 and mir- 331-3p, upregulated in HCC, can provide positive diagnostic and prognostic values for HCC. Keywords Hepatocellular carcinoma. MicroRNA-182. MicroRNA-331-3p. Alpha-fetoprotein. Diagnosis. Prognosis Introduction Hepatocellular carcinoma (HCC) is one of the most common human malignancies and is currently the third leading cause of cancer-related mortality worldwide. Currently, due to a lack of biomarkers for the early detection of HCC, only 30 40 % of patients with HCC are found eligible for potentially curative intervention. Although surgical resection, liver transplantation, and chemoembolization may be used to treat this deadly disease, 5-year survival rates have not changed much during the past several decades [1]. Serum alpha-fetoprotein (AFP) has been regarded as a gold standard serum marker for HCC since the 1970s. It is an important serum protein mainly secreted by the yolk sac, liver, and gastrointestinal tract during fetal development. The concentration of serum AFP may decrease gradually after birth in 12 18 months and can hardly be detected in adults, while the expression of serum AFP in adults is frequently related to HCC [2, 3].TheelevatedAFP

7440 Tumor Biol. (2015) 36:7439 7447 serum level has been reported to be closely associated with the clinical pathologic characteristics of HCC including tumor size, TNM classification of malignant tumors (TNM) stage and invasive depth, as well as a prognostic marker for HCC [4, 5]. To date, although serum AFP has mainly been used in clinic for diagnosis and monitoring of HCC, its sensitivity and specificity are not still satisfying. The false-negative rate with AFP level alone may be as high as 40 % for early stage of HCC. In addition, the AFP levels may remain normal even in 15 30 % of the patients with advanced HCC [6, 7]. Therefore, novel diagnostic biomarkers are urgently needed in order to early detection and improve the prognosis of this deadly disease. MicroRNAs (mirnas) are a class of small (22 25 nucleotides in size), endogenous, noncoding RNAs that can regulate expression of their target genes posttranscriptionally contributing to diverse cellular processes such as proliferation, development, apoptosis, and differentiation [8]. Mounting evidence has been shown that mirnas are consistently deregulated and associated with the development and progression of various types of human cancer. Moreover, a part of these mirnas may be promising as potential biomarkers for diagnosis, prognosis, and personalized therapy of human cancers including HCC [9]. For example, the downregulation of mir-130a and mir-100 and the upregulation of mir-25 and mir-372 were associated with tumor progression and prognosis in HCC [10 13], showing great potential as tissue-based markers for cancer definition. On the other hand, mirnas in serum/plasma are emerging as a new class of potential markers for noninvasive diagnosis and monitoring of cancer. Owing to their resistance to endogenous RNase activity and high stability after prolonged incubation at room temperature and multiple freezing thawing processes, circulating mirnas are not only abundant in blood but also very stable. Furthermore, blood specimens can be easily obtained and have the advantages of minimally invasive continuous in vitro testing and high reproducibility. As a result, more and more attention has been paid to determine disease-specific circulating mirnas to predict and diagnose cancer [14]. Studies have showed that serum mir-24-3p, mir- 215, mir-143, mir-1, mir-122, etc. have the positive clinical application value in the diagnosis and prognosis of HCC [15 17]. Therefore, investigation of aberrant circulating mirnas as biomarkers for HCC will be more attractive and more promising. Our previous experiments have screened a number of mirnas that were aberrantly expressed in HCC tissues; however, their diagnostic and prognostic value in serum has not been fully evaluated. Therefore, in this study, the levels of five serum mirnas, namely, mir-182, mir-331-3p, mir-197, mir-492, and mir-581, were examined by real-time quantitative reverse transcription polymerase chain reaction (qrt- PCR), in order to evaluate whether these serum mirnas were abnormally expressed in HCC and whether they had potential diagnosis and prognosis value in patients with HCC. Our present study will shed new light on the identification of new serum diagnostic and prognostic markers for the deadly HCC. Materials and methods Ethical approval The project protocol was approved by the Ethics Committee of Jiangsu Province Medical Association. All patients and healthy controls have signed informed consent forms. Patients and samples Our study was performed in the Third People s Hospital of Nantong City, and a total of 238 individuals were enrolled in this study. Serum samples from 103 HCC patients in the preoperative state, 95 patients with benign liver diseases, and 40 healthy controls were collected. The diagnosis of all HCC patients was histologically confirmed. The tumor type and the grade of cell differentiation were evaluated according to the WHO classification system, whereas the pathological stage was determined by the International Union Against Cancer (UICC) TNM classification. Of the 103 HCC patients, 67 were followed up after they suffered from surgical resection in the subsequent 3-year period. The benign group was comprised of 39 patients with liver cirrhosis, 47 patients with chronic hepatitis, and 9 patients with nonalcoholic fatty liver diseases. The clinical characteristics of the subjects are listed in Table 1. Peripheral blood was obtained in the early morning from all individuals. Sera were separated by centrifuging the blood samples at 3000g for 15 min and stored at 80 C for further assay. MiRNAs quantitation by real-time PCR analysis Total RNAs from sera were extracted using the mirvana PARI S Kit (Ambion, Austin, TX, USA) according to the manufacturer s instructions. The RNA concentration was measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Reverse transcription was performed using the PrimeScript First Strand cdna synthesis kit (Takara Bio, Inc., Dalian, Japan) according to the manufacturer s instructions. Real-time PCR was performed using the All-in-One mirna quantitation by realtime PCR (qrt-pcr) detection kit (GeneCopoeia, Rockville, MD, USA) on the Applied Biosystems 7500 Real-time PCR System (ABI, Abilene, TX, USA). The PCR amplification protocol was as follows: 95 C for 10 min, 40 cycles of

Tumor Biol. (2015) 36:7439 7447 7441 Table 1 Demographic and clinical characterization of study population Characteristics HCC (n=103) Benign liver diseases (n=95) Healthy controls (n=40) Age (years) Median (range) 52 (39 80) 50 (37 76) 49 (30 78) Gender Male 89 80 31 Female 14 15 9 HBV infection Positive 72 63 0 Negative 31 32 40 Liver cirrhosis Presence 61 39 0 Absence 42 56 40 Serum AFP (ng/ml) 20 26 82 40 >20 77 13 0 Tumor size (cm) 5 36 >5 67 Tumor differentiation Well 23 Moderate 49 Poor 31 TNM stage I 18 II 34 III 30 IV 21 denaturation at 95 C for 15 s, then 1 min of annealing/ extension at 60 C. The comparative cycle threshold (CT) method was applied to quantify the expression levels of mirnas. The small nuclear U6 RNA was used as internal control. The relative amount of mirnas was calculated using the equation 2 ΔΔCT. All the reactions were run in triplicate, and the primers for mirnas and U6 were purchased from RiboBio (Guangzhou, China). Serum AFP detection The concentrations of AFP in the serum samples from HCC patients were measured in the Clinical Pathology Laboratory of the third Affiliated Hospital of Nanjing Medical University by ARCHITECT i2000 SR (Abbott, Chicago, IL, USA). A content of AFP >20 ng/ml was considered abnormal. Statistical analysis Values were expressed as median, range, or interquartile range (IQR, 25 75). Serum mirna levels were compared using appropriate nonparametric Mann Whitney U test or Kruskal Wallis H test, and the distribution of the patients was measured by the χ 2 test. Receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to assess the diagnostic value of mir-182 and mir-331-3p for HCC. Survival curve was constructed with the Kaplan Meier method and compared by the log-rank test. Cox proportional hazard regression model was then applied to evaluate survival data. Statistical analyses were performed using SPSS Statistics 17.0 software (SPSS, Chicago, IL, USA) and P<0.05 was considered to be statistically significant. Results Patient characteristics Demographic and clinical characteristics of study population are summarized in Table 1. A total of 238 participants including 103 patients with HCC, 95 patients with benign liver diseases, and 40 healthy controls were recruited in this study. No significant differences of age (Kruskal Wallis H test, P=

7442 Tumor Biol. (2015) 36:7439 7447 0.368) or gender (χ 2 test, P=0.426) were found among three groups. Increased levels of serum mir-182 and mir-331-3p in patients with HCC The levels of five serum mirnas were analyzed in HCC group, benign liver diseases group, and healthy controls group. No significant difference was observed in the levels of serum mir-197, mir-492, and mir-581 among three groups, all P>0.05 (Table 2). Therefore, these three mirnas were not included in further analyses. On the other hand, the levels of serum mir-182 in HCC group were 14.86 (10.44 18.24) [median (IQR 25 75)], which were drastically higher than those in benign group [4.56 (3.00 7.71)] and control group [4.24 (2.75 5.32)], both P<0.001. Nevertheless, there was no statistical difference of serum mir-182 level between the benign and control group (P=0.111; Table 2, Fig. 1a). Meanwhile, the levels of serum mir-331-3p in HCC group were 9.79 (6.28 12.88), which were obviously higher than those in benign group [3.87 (2.76 4.79)] and control group [3.63 (2.61 4.18)], both P<0.001, whereas there was no significantly difference of serum mir-331-3p level between the benign group and control group (P= 0.085; Table 2, Fig. 1b). Our results show that serum mir-182 and mir-331-3p are abnormally increased in patients with HCC, indicating their potential diagnostic value for HCC. Diagnosis value of serum mir-182 and mir-331-3p for HCC ROC curve was drawn for distinguishing benign liver diseases from malignant conditions (Fig. 2). The AUC of serum mir- 182 was 0.911 (95 % CI, 0.863 0.947). Youden index was maximal when the level of serum mir-182 was 9.56, and therefore, it was defined as the cutoff value. At this cutoff value, the sensitivity was 78.64 % (95 % CI, 69.5 86.1) at a specificity of 91.58 % (95 % CI, 84.1 96.3), and the positive predictive value, the negative predictive value, and the total efficacy were 91.01, 79.82, and 84.85 %, respectively. The AUC of serum mir-331-3p was 0.890 (95 % CI, 0.838 0.930). When the cutoff value was defined at 5.55, the sensitivity was 79.61 % (95 % CI, 70.5 86.9) at a specificity of 89.47 % (95 % CI, 81.5 94.8), and the positive predictive value, negative predictive value, and total efficacy were 89.13, 80.19, and 84.34 %, respectively. The AUC of serum AFP was 0.840 (95 % CI, 0.781 0.888). When 20 ng/ml was defined as the cutoff value, the sensitivity was 74.76 % (95 % CI, 65.2 82.8) at a specificity of 86.32 % (95 % CI, 78.1 92.3), and the positive predictive value, negative predictive value, and total efficacy were 85.56, 75.93, and 80.30 %, respectively (Table 3). These results suggest that serum mir- 182 and mir-331-3p, as potential biomarkers, may have positive diagnostic value for HCC. We further evaluated the diagnosis of malignant and benign liver diseases by a combination of serum mir- 182, mir-331-3p, and AFP. Compared with serum mir- 182, mir-331-3p, or AFP alone, the sensitivity, negative predictive value, and total efficacy were significantly improved by a combination of serum mir-331-3p and AFP, P<0.05, whereas there was some extent improvement in diagnostic efficiency by a combination of serum mir-182 and AFP, P>0.05. Moreover, the combination of serum mir-182, mir-331-3p, and AFP can markedly increase the diagnostic efficiency of HCC and differential diagnostic value of benign and malignant liver diseases, especially better than serum AFP alone, P<0.05 (Table 3). Our data show that serum AFP, combined with mir-182 and mir-331-3p, can be used in efficient diagnosis of benign and malignant liver diseases. Correlates of serum mir-182 and mir-331-3p with clinicopathological features of HCC To better understand the potential roles of serum mir- 182 and mir-331-3p in HCC development and progression, we further analyzed the potential association of serum mir-182 and mir-331-3p levels with various clinicopathological features of HCC. In the present study, the levels of serum mir-182 and mir-331-3p were classified as low or high on the basis of the median value. The statistical analysis revealed that high Table 2 The levels of serum mirnas in patients with HCC or benign liver diseases and healthy controls [median (IQR, 25 75)] Serum marker HCC group (n=103) Benign group (n=95) Control group (n=40) mir-182 14.86 (10.44 18.24) a 4.56 (3.00 7.71) 4.24 (2.75 5.32) mir-331-3p 9.79 (6.28 12.88) a 3.87 (2.76 4.79) 3.63 (2.61 4.18) mir-197 4.73 (2.25 7.09) 4.45 (2.52 6.75) 4.38 (2.36 6.13) mir-492 3.37 (2.12 4.91) 3.51 (2.31 5.14) 3.19 (2.05 4.36) mir-581 5.21 (3.45 8.17) 4.85 (3.02 7.66) 5.03 (3.37 6.94) a Compared with benign group and control group, P<0.001

Tumor Biol. (2015) 36:7439 7447 7443 Fig. 1 Scatter plots of serum levels of mir-182 (a) and mir-331-3p (b) in healthy controls (control group, n=40), patients with benign liver diseases (benign group, n=95), and patients with HCC (HCC group, level of serum mir-182 was positively correlated with serum AFP (P=0.001), tumor size (P=0.013), and TNM stage (P=0.003); however, there was no significant correlation of serum mir-182 with other clinical features, such as age, gender, hepatitis B virus (HBV) infection, liver cirrhosis, and tumor differentiation, P>0.05. On the other hand, only TNM stage, rather than other clinical features, demonstrated a significant correlation with the level of serum mir-331-3p (P=0.006) (Table 4). The associations of serum mir-182 and mir-331-3p with TNM stages were further demonstrated in Fig. 3. Our present results suggest that serum mir-182 and mir-331-3p, especially serum mir-182, could help us predict the tumor stage and judge the progression of HCC. Serum mir-182 and mir-331-3p dynamics in posttreatment HCC patients Serum mir-182 and mir-331-3p was measured in 18 HCC patients who received trans-arterial chemoembolization Fig. 2 ROC curve for distinguishing benign from liver malignant conditions. The AUC of serum mir-182, mir-331-3p, and AFP was 0.911 (95 % CI, 0.863 0.947), 0.890 (95 % CI, 0.838 0.930), and 0.840 (95 % CI, 0.781 0.888), respectively n=103). The levels of mirnas were analyzed by qrt-pcr and normalized to U6 snrna. The line is the median value. P values were determined using the Mann Whitney U test (TACE) treatment and were followed up at four time periods, namely, pretherapy, 3 15 days, 16 50 days, and 51 120 days after treatment. As shown in Fig. 4, there was a common trend that serum mir-182 and mir-331-3p were significantly lower in HCC patients with TACE treatment than those in pretherapy, and decreased progressively with time-dependent manner in the follow-up period after treatment. Therefore, detection the changes of serum mir-182 and mir-331-3p may prove useful for dynamic monitoring of the treatment outcome of HCC patients who underwent TACE intervention. Serum mir-182 and mir-331-3p correlate with prognosis in patients with HCC To investigate the prognostic value of serum mir-182 and mir-331-3p in patients with HCC, survival analysis was performed in 67 cases according to follow-up data. During the 3- year follow-up period, 31 patients died. As shown in Fig. 5, patients with low levels of serum mir-182 and mir-331-3p were significantly associated with prolonged postoperative survival of HCC patients, P=0.001 and 0.013, respectively. Cox proportional hazards analyses were applied to further evaluate the potential of serum mir-182 and mir-331-3p as prognostic biomarkers. Univariate survival analyses indicated that serum AFP (P=0.034), tumor size (P=0.019),TNM stage (P<0.001), mir-182 (P=0.001), and mir-331-3p (P=0.014) were significantly associated with prognosis in HCC patients, while gender, age, HBV infection, liver cirrhosis, and tumor differentiation were not associated with prognosis, all P>0.05 (Table 5). In the multivariate Cox proportional hazards analyses, which included high level of serum mir-182 (hazard ratio, 2.321; 95 % CI, 1.136 4.741; P=0.021) and mir-331-3p (hazard ratio, 2.010; 95 % CI, 1.043 3.871; P=0.037), as well as TNM stage (III+IV) (hazard ratio, 2.756; 95 % CI, 1.318 5.761; P=0.007), were strongly associated with poor survival in HCC patients (Table 5). Our data suggest that serum mir-182 and mir-331-3p are identified to be independent prognostic factors for survival in HCC patients.

7444 Tumor Biol. (2015) 36:7439 7447 Table 3 Diagnosis of malignant and benign liver diseases by serum mir-182, mir-331-3p, and AFP Biomarker Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Efficiency (%) mir-182 78.64 (81/103)* # 91.58 (87/95) 91.01 (81/89) 79.82 (87/109)* # 84.85 (168/198)* # mir-331-3p 79.61 (82/103)* # 89.47 (85/95) 89.13 (82/92) 80.19 (85/106)* # 84.34 (167/198)* # AFP 74.76 (77/103)* # 86.32 (82/95)* 85.56 (77/90)* 75.93 (82/108)* # 80.30 (159/198)* # mir-182+afp 82.52 (85/103)* 94.74 (90/95) 94.44 (85/90) 83.33 (90/108) 88.38 (175/198)* mir-331-3p+afp 91.26 (94/103) 92.63 (88/95) 93.07 (94/101) 90.72 (88/97) 91.92 (182/198) mir-182+mir-331-3p+afp 93.20 (96/103) 95.79 (91/95) 96.00 (96/100) 92.86 (91/98) 94.44 (187/198) *P<0.05, compared with mir-182+mir-331-3p+afp; # P<0.05, compared with mir-331-3p+afp Discussion Owing to HCC cells be characterized by self-sufficient growth signals, insensitivity to antigrowth signals, sustained angiogenesis, tissue invasion and metastasis, limitless replicative potential, and evasion of apoptosis, HCC represents one of the most common cancers worldwide with high mortality rate. The early diagnosis of HCC is of great clinical desirable, and the improved prognosis of HCC if the patients could get surgical treatment early. Table 4 Correlations of serum mir-182 and mir-331-3p with clinicopathologicol characteristics in patients with HCC Characteristics n Serum mir-182 level Serum mir-331-3p level High Low P value High Low P value Age 0.327 0.845 <52 51 23 28 25 26 52 52 29 23 27 25 Gender 0.775 0.246 Male 89 44 45 47 42 Female 14 8 6 5 9 HBV infection 0.136 0.288 Positive 72 40 32 39 33 Negative 31 12 19 13 18 Liver cirrhosis 0.232 0.111 Presence 61 34 27 35 26 Absence 42 18 24 17 25 Serum AFP (ng/ml) 0.001 0.497 20 26 6 20 15 11 >20 77 46 31 37 40 Tumor size (cm) 0.013 0.101 5 36 12 24 14 22 >5 67 40 27 38 29 Tumor differentiation 0.232 0.072 Well 23 8 15 7 16 Moderate 49 27 22 26 23 Poor 31 17 14 19 12 TNM stage 0.003 0.006 I 18 5 13 3 15 II 34 13 21 16 18 III 30 17 13 19 11 IV 21 17 4 14 7

Tumor Biol. (2015) 36:7439 7447 7445 Fig. 3 Scatter plots of serum levels of mir-182 (a) and mir- 331-3p (b) in TNM I+II stages (n=52) vs. III+IV stages (n=51), P=0.001 and 0.002, respectively Currently, serum AFP has mainly been used in clinic for diagnosis of HCC; however, its sensitivity and specificity are not satisfying [1, 6, 7]. Discovery of new and more sensitive biomarkers for early detection of HCC would play a pivotal role in improving the prognosis of this deadly disease. Ideal biomarkers should be easy to measure and have a strong association with clinical outcome. mirnas could match these proposed criteria. Studies showed that mirnas, which are involved in tumorigenesis and in the development of various types of cancer, are detectable in the plasma/serum [14, 18, 19]. These circulating mirnas originate from cancer tissues and can act as potential biomarkers for diagnosis and prognosis of various types of cancer, including HCC. MiR-182 belongs to the mir-183 family that is located at human chromosome 7q32.2. Studies have showed that mir-182 acts as an important regulator of various physiological processes, such as mammalian circadian rhythm, T cell development and DNA repair, etc. Several studies have also illustrated that mir-182 is abnormally expressed in various tumors and directly involves in human cancer processes, such as tumorigenesis, migration, metastasis, etc. [20 22]. In addition, mir-182 contributes to metastasis of HCC by downregulation metastasis suppressor 1 and increases drug resistance in cisplatin-treated HCC cell by regulating tumor protein 53-induced nuclear protein 1 (TP53INP1) [23, 24]. On the other hand, the human mir-331 gene is located at 12q22. Although mir-331-3p is downregulated in many other cancers [25 27], it promotes proliferation and metastasis of HCC by targeting PH domain and leucine-rich repeat protein phosphatase (PHLPP) [28]. These studies indicate that both mir- 182 and mir-331-3p may play oncogenic roles in HCC. Subsequently, we further hypothesized that mir-182 and mir-331-3p were dysregulated in sera and might have potential diagnosis and prognosis values for HCC. In the present study, we found that serum levels of mir-182 and mir-331-3p were significantly elevated in patients with HCC when compared to those in healthy controls and benign liver diseases, P<0.001. Moreover, our ROC curve analysis showed that serum mir-182 yielded an AUC of 0.911 (95 % CI, 0.863 0.947), with the sensitivity of 78.64 % and the specificity of 91.58 %. Serum mir-331-3p yielded an AUC of 0.890 (95 % CI, 0.838 0.930), with the sensitivity of 79.61 % and the specificity of 89.47 %. The results indicate that serum mir-182 and mir-331-3p may be new potential diagnostic biomarkers for HCC. The capability for differential diagnosis of benign and malignant liver diseases was further analyzed by serum mir-182, mir- 331-3p, and AFP. Our results showed that the combina- Fig. 4 Line charts of serum levels of mir-182 (a) and mir- 331-3p (b) in 18 TACE treatment HCC patients. The follow-up time was from 0 to 120 days after posttreatment. The levels of mirnas were analyzed by qrt- PCR and normalized to U6 snrna

7446 Tumor Biol. (2015) 36:7439 7447 Fig. 5 Kaplan Meier survival curve analyses were performed according to the levels of serum mir-182 (a) and mir-331-3p (b) in patients with HCC. Difference in survival between high- and low-level group was analyzed by the log-rank test tion assay of serum mir-182, mir-331-3p, and AFP can markedly increase differential diagnostic value of benign and malignant liver diseases, especially better than serum AFP alone, P<0.05. In addition, we also analyzed the potential association of serum mir-182 and mir-331-3p levels with various clinicopathological features of HCC. Our results showed that serum mir-182 was positively correlated with serum AFP (P=0.001), tumor size (P=0.013), and TNM stage (P=0.003); however, only TNM stage was demonstrated a significant correlation with the level of serum mir-331-3p (P=0.006). These data suggest that serum mir-182 and mir-331-3p, especially serum mir-182, could help us predict the tumor stage and judge the progression of HCC. The dynamics of serum mir-182 and mir-331-3p in posttreatment HCC patients were further evaluated following TACE treatment, which showed that dynamic monitoring these two mirnas could observe curative effect in HCC patients. On the other hand, the levels of serum mir-182 and mir-331-3p were correlated with postoperative survival in HCC patients, P=0.001 and 0.013, respectively, showing potential prognosis values for HCC. Furthermore, we also found serum mir-182 (hazard ratio, 2.321; 95 % CI, 1.136 4.741; P=0.021) and mir-331-3p (hazard ratio, 2.010; 95 % CI, 1.043 3.871; P=0.037) to be independent prognostic factors for survival in HCC patients. However, our study is a preliminary report on serum mir-182 and mir-331-3p in patients with HCC, further examination with long-term followup is necessary to evaluate predictability of these two biomarkers. Besides, the value of serum mir-182 and mir- 331-3p for early detection HCC and whether both of them could improve diagnosis of AFP-negative HCC should be further assessed. In summary, although mir-182 and mir-331-3p warrants further evaluation as both early detection markers and prognostic factors in HCC, this study demonstrates, for the first time, that serum mir-182 and mir-331-3p, elevated in HCC patients, may serve as novel noninvasive biomarkers to distinguish patients with benign and malignant liver diseases, as well as help us predict the tumor stage and judge the progression of HCC. Our findings facilitate further studies with a large cohort of patients determining a panel of serum mirna biomarkers, or combining mirna biomarkers with serum AFP and image examinations, which will improve the sensitivity and organ specificity for HCC screening and timely predict poor prognosis for this deadly liver disease. Table 5 Univariate and multivariate regression analyses of parameters associated with prognosis of HCC patients Parameters Subset Univariate analysis Multivariate analysis Hazard ratio (95 % CI) P value Hazard ratio (95 % CI) P value Age <52/ 52 1.249 (0.638 2.446) 0.516 Gender Male/female 1.098 (0.551 2.190) 0.790 HBV infection Positive/negative 1.510 (0.775 2.943) 0.226 Liver cirrhosis Presence/absence 1.567 (0.813 3.021) 0.180 Serum AFP (ng/ml) 20/>20 2.136 (1.060 4.307) 0.034 1.652(0.837 3.261) 0.148 Tumor size (cm) 5/>5 2.350 (1.149 4.807) 0.019 1.723(0.884 3.358) 0.110 Tumor differentiation Well/moderate/poor 1.921 (0.968 3.810) 0.062 TNM stage I/II/III/IV 3.584 (1.731 7.422) <0.001 2.756(1.318 5.761) 0.007 mir-182 High/low 3.264 (1.593 6.685) 0.001 2.321(1.136 4.741) 0.021 mir-331-3p High/low 2.486 (1.204 5.131) 0.014 2.010(1.043 3.871) 0.037

Tumor Biol. (2015) 36:7439 7447 7447 Acknowledgments This study was supported by National Natural Science Youth Foundation of China (No. 81201351), the project of Jiangsu Provincial Commission of Health and Family Planning (No. H201453), the project of Health Bureau of Nantong city (No. WQ2014065), and the science and technology development project of Nantong City, China (Nos. HS2014061, HS2013054, and HS2011058). Conflicts of interest References None 1. Bruix J, Gores GJ, Mazzaferro V. Hepatocellular carcinoma: clinical frontiers and perspectives. Gut. 2014;63:844 55. 2. Xu WJ, Guo BL, Han YG, Shi L, Ma WS. Diagnostic value of alpha- fetoprotein-l3 and Golgi protein 73 in hepatocellular carcinomas with low AFP levels. Tumour Biol. 2014;35(12):12069 74. 3. Debruyne EN, Delanghe JR. Diagnosing and monitoring hepatocellular carcinoma with alpha-fetoprotein: new aspects and applications. Clin Chim Acta. 2008;395(1 2):19 26. 4. Park H, Park JY. 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