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Clinics and Research in Hepatology and Gastroenterology (2015) xxx, xxx—xxx

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MINI REVIEW

MicroRNAs as biomarkers for hepatocellular carcinoma diagnosis and prognosis Song He a,∗, De-Chun Zhang b, Cheng Wei a a

Maanshan Center for Clinical Laboratory, Maanshan Municipal Hospital Group, 45, Hubei Road, 243000 Maanshan, China b Molecular Medicine & Tumor Research Center, Chongqing Medical University, Chongqing, China

Summary Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world, and it is the second leading cause of cancer-related deaths. Despite improvements in HCC therapy, the overall survival rate is still very low because of the late detection of the tumors. Thus, early detection of HCC offers the best chance of survival for patients. MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs involved in the regulation of gene expression and protein translation. Many studies have shown that they played a very important role in cancer progresses and outcomes. The aberrant expression of miRNAs is common in various human malignancies and it modulates cancer-associated genomic regions or fragile sites. As for the relationship between miRNAs and HCC, several studies have demonstrated that the aberrant expression of specific miRNAs can be detected in HCC patients’ serum and plasma or HCC cells and tissues, and miRNAs have shown great promise as diagnostic and prognostic markers for HCC. In the present review, we discussed the applications of miRNAs as biomarkers for HCC diagnosis and prognosis, and the association between miRNAs polymorphisms and the risk of HCC as well. © 2015 Elsevier Masson SAS. All rights reserved.

Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world, and it is the second leading cause of cancer-related deaths [1,2]. Globally, there are

∗

Corresponding author. Maanshan Center for Clinical Laboratory, Maanshan Municipal Hospital Group, 45, Hubei Road, 243000 Maanshan, China. E-mail address: [email protected] (S. He).

approximately 750,000 new cases of liver cancer reported per year [3]. Despite improvements in HCC therapy, the treatment of patients with HCC is still challenging because of the array of patient-specific, tumor-specific, and liverspecific variables that impact our ability to treat patients safely and effectively [3]. The overall 5-year survival rate of HCC is 5—9% from the time of clinical diagnosis, and the dismal prognosis is largely caused by late detection of the tumors [4,5]. However, if the tumor is detected early, particularly when it is a single nodule and smaller than 2 cm, and the patients have undergo hepatectomy, the 5-year

http://dx.doi.org/10.1016/j.clinre.2015.01.006 2210-7401/© 2015 Elsevier Masson SAS. All rights reserved.

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survival rate can reach 69% [6,7]. Therefore, early detection of HCC at a surgically resectable stage offers the best chance of survival for patients, and it is need to find better biomarkers for detecting HCC and tracking its development. Current diagnostic and prognostic methods are insufficient for detecting and prognosing HCC at early stages. To develop better biomarkers for HCC, numerous researchers have devoted to the studies on HCC diagnosis and prognosis. Serum ␣-fetoprotein (AFP) and Des-gamma carboxyprothrombin (DCP) levels have long been used as tumor biomarkers [8,9]. However, one-third cases of early-stage HCC (tumors < 3 cm) are missed using AFP analysis. Serum AFP levels may also elevate in patients with benign liver diseases, such as hepatitis and cirrhosis. Elevated DCP activity is present in only 44—47% of HCC patients with tumors < 3 cm [10—12]. Shen et al. [13] have assessed serum Dickkopf-1 (DKK1) as a new protein biomarker for the diagnosis of HCC. The assay of DKK1 is a complement measurement of AFP in HCC diagnosis. It will improve identification of patients with AFP-negative HCC and distinguish HCC from non-malignant chronic liver diseases. MicroRNAs (miRNAs) are small endogenous non-coding single-stranded RNAs that are 21—30 nucleotides in length. They modulate the expression of various target genes at the post-transcriptional and translational levels [14]. Aberrant expression of miRNAs is common in various human malignancies and it modulates cancer-associated genomic regions or fragile sites. As for the relationship between miRNAs and HCC, several studies have demonstrated that the aberrant expression of specific miRNAs can be detected in serum and plasma of HCC patients or HCC cells and tissues, and miRNAs have shown great promise as diagnostic and prognostic markers for HCC [14,15]. In this review, we discussed the applications of miRNAs as biomarkers for HCC diagnosis and prognosis, and the association between miRNAs polymorphisms and the risk of HCC as well.

MiRNAs used for HCC diagnosis A large number of reports have shown dysregulation of miRNAs in human HCC, and these miRNAs may be used as novel potential biomarkers for HCC diagnosis and prognosis. A Venn diagram has shown the role of miRNAs in HCC diagnosis, prognosis, or overlapping (Fig. 1A). Much efforts has been made to develop non-invasive circulation biomarkers for the diagnosis of HCC. We have got 13 individual studies [16—28] including 21 sets of data, from MEDLINE database, which have assessed the diagnostic accuracy of circulation miRNAs for HCC with sensitivity, specificity, and area under the curve (AUC) of receiver operating characteristic (ROC) (Table 1). The average sensitivity, specificity, and AUC of ROC of the 21 sets of data were 83.73% (95% confidence interval [95%CI], 78.59%—88.91%), 84.77% (95%CI, 77.89%—90.45%) and 0.8849 (95%CI, 0.8464—0.9191), respectively, as indicates good accuracy of miRNAs in the diagnosis for HCC. Nevertheless, the diagnostic accuracy of test performances has varied depending on different specimens. The average AUC of ROC of serum miRNAs for HCC was 0.8941 (95%CI, 0.8359—0.9433), indicating an overall very high diagnostic accuracy, while the AUC of plasma levels of miRNAs was lower (0.8664, 95%CI 0.8137—0.9170). When it comes to HCC

tissues, the average sensitivity, specificity, and AUC of two studies [29,30] is just 67%, 80%, and 0.758 respectively. By the use of urine as a sample source, the average sensitivity and specificity are even lower (64% and 67% respectively) [12]. It seems that miRNAs assayed from aerum have the highest diagnostic accuracy for HCC. It shows a greater differences between different miRNAs used for HCC diagnosis. There is a study assessing the diagnostic accuracy of miR-18a as a potential marker for screening hepatitis B virus-related hepatocellular carcinoma. The results have displayed an AUC of ROC of 0.881 with 86.1% sensitivity and 75.0% specificity in discriminating HBV-related HCC from healthy controls, and an AUC of ROC of 0.775 with 77.2% sensitivity and 70.0% specificity in discriminating HBV-related HCC from chronic hepatitis or cirrhosis [18]. But there is much higher diagnostic accuracy for miR-101-5p [16], miR-375 [21] and miR-885-5p [22] with a AUC of ROC of 0.976, 0.960, and 0.904 respectively. The published studies have shown that combined miRNAs have higher diagnostic value for HCC diagnosis. Liu et al.’s study [19] showed that combined miR-15b and miR-130b yielding a AUC of ROC of 0.981 (98.2% sensitivity and 91.5% specificity). Another study [21] had assessed different combinations of miRNAs used for HCC diagnosis, and the results revealed that the combined miR-23b, miR-423, miR-375, miR-23a and miR342-3p had an ROC of 0.999 (specificity, 96.9%; sensitivity, 99.4%) in HCC prediction, the combination of miR-10a and miR-125b had an ROC of 0.992 (specificity, 98.5%; sensitivity, 98.5%), and the combination of miR-375, miR-25 and let-7f had an ROC of 0.997 (specificity, 97.1%; sensitivity, 97.1%). Tan et al. [28] constructed a panel of 8 miRNAs which could differentiate HCC patients from healthy (AUC = 0.893) and cirrhosis (AUC = 0.892) patients. In addition, there are more researches have reported miRNAs used for HCC diagnosis as showed in Fig. 1A, although there have not assessed the diagnostic accuracy with sensitivity, specificity, and AUC of ROC, these miRNAs still may be the biomarkers with the highest potential. Of course, the discrepancy of a certain miRNA dysregulation also exists between different studies. For example, a study reported that the expression of serum miR-101 in patients with HBV-HCC was significantly higher than that in the healthy controls [31], while another study indicated that the serum miR-101 levels were found to be significantly downregulated in the HBV-HCC patients compared with the HBV-LC patients, CHB patients and healthy controls [16]. This discrepancy may be caused by the choice of control group populations or small sample sizes, and this need more and large-scale studies to prove and define it.

MiRNAs used for HCC prognosis Increasing evidence suggests that dysregulation of miRNAs is correlated with malignant transformation and tumor development. Many miRNAs are dysregulated in HCC, and the miRNA levels are also affected by HCC progression. That’s to say, these miRNAs can be used for HCC prognosis and monitoring the tumor progress, such as TNM stage, tumor invasion, metastasis, migration, recurrence, size, grade, and so on. The miRNAs network used for HCC prognosis has been shown in Fig. 1B.

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MicroRNAs for hepatocellular carcinoma

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Figure 1 The role of miRNAs in HCC diagnosis and prognosis. A. Venn diagram showing the role of miRNAs in HCC diagnosis, prognosis, or overlapping. B. The relation network of miRNAs used for HCC prognosis. Abbreviations: MiRNAs, MicroRNAs; HCC, Hepatocellular carcinoma; TNM, Tumor node metastasis; HBsAg, Hepatitis B surface antigen; HBV, Hepatitis B virus.

MiRNAs prognosing current situations of HCC TNM staging system is one of the most common tumor staging system in the world, which becomes the malignant tumor staging standard method for doctors and medical scientific workers now. The TNM stage of HCC could be indicated by detecting the level of tissue or serum miRNAs. Su et al.’s study [32] showed that the expression of miR-25 was significantly upregulated in HCC tissues, and miR-25 expression was significantly correlated with TNM stage. Besides, the expression of miR-183 [29], miR-191 and miR-221 [33], miR372 [34] from tissue samples, expression of miR-17-5p [35] and miR-222 [36] from serum samples were overexpressed and significantly correlated with TNM stage of HCC. The level of miR-29b [17], miR-34a [37], miR-100 [38], miR-146a [39], miR-206 [40] and miR-338 [41] correlated with TNM stage

were downregulated in HCC tissues and serum from HCC patients. Low miR-100 expression was confirmed to be correlated with not only TNM stage, but also significantly correlated with higher tumor grade, higher incidence of lymph node metastasis, and higher incidence of tumor recurrence in HCC patients [38], and low level of miR-206 was also associated with poor tumor differentiation, multiple tumor nodes, lymph node metastasis [40]. The expression of serum miR-16 is down-regulated, and let-7f is up-regulated in the patients with a tumor more than 5 cm in diameter [42]. The level of miR-221 [33,43] and miR-338 [41] expression also reflects the tumor size of HCC. MiR-146a level in HCC tissues was downregulated and related to portal vein tumor embolus and number of tumor nodes [39]. High level of miR-222 expression was also correlated with tumor number and portal vein

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S. He et al. Table 1

MiRNAs used for HCC diagnosis.

MiRNAs

Sample

Alteration

SEN (%)

SPE (%)

AUC

References

miR-101 miR-29b miR-18a

Serum Serum Serum

Downregulated Downregulated Upregulated

Serum Serum Serum Serum

Upregulated Upregulated Upregulated Upregulated

90.2 89.5 75 70 91.5 95.2 100 99.4

0.976 0.855 0.881c 0.775d 0.981 0.758 0.96 0.999

[16] [17] [18]

Combination of miR-15b and miR-130b miR-21 miR-375 Combination of miR-23b, miR-423, miR-375, miR-23a and miR-342-3p Combination of miR-10a and miR-125b Combination of miR-375, miR-25 and let-7f miR-885-5p Combination of miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a and miR-801

95.5 75.9 86.1 77.2 98.2 53.3 96 96.9

Serum Serum Serum Plasma

Upregulated Upregulated Upregulated

miR-139 miR-483-5p miR-21

Plasma Plasma Plasma

Downregulated Upregulated Upregulated

miR-221 Combination of miR-206, miR-141-3p, miR-433-3p, miR-1228-5p, miR-199a-5p, miR-122-5p, miR-192-5p, and miR-26a-5p miR-183 miR-29a-5p

Serum Serum

Downregulated

98.5 97.1 90.53 83.2 79.1 75 80.6 75.5 61.1 87.3 87 82.8 81.6

98.5 99.1 79.17 93.9 76.4 91.1 58.1 89.9 83.3 92 40 83.3 84.6

0.992 0.997 0.904 0.941e 0.842f 0.88g 0.764 0.908 0.773 0.953 0.655 0.893e 0.89g

Tissues Tissues

Upregulated Upregulated

59.8 74.2

91.8 68.2

0.808 0.708

a

b

[19] [20] [21]

[22] [23]

[24] [25] [26] [27] [28]

[29] [30]

Abbreviations: miRNAs: microRNAs; HCC: hepatocellular carcinoma; SEN: sensitivity; SPE: specificity; AUC: area under the curve of receiver operating characteristic. a miR-192, miR-21, miR-801 upregulated, and others downregulated. b miR-206, miR-141-3p, miR-433-3p, miR-1228-5p upregulated, and others downregulated. c Discriminating HBV-related HCC from healthy controls. d Discriminating HBV-related HCC from chronic hepatitis or cirrhosis. e Discriminating the HCC group from the healthy group. f Discriminating the HCC group from the chronic hepatitis B group. g Discriminating the HCC group from the cirrhosis group.

tumor thrombosis [36]. A lower expression of miR-29b in serum and miR-22 in HCC tissues was also confirmed reflecting the lower differentiation of HCC tumour cells [17,44]. Most recently, miR-148a was observed downregulated significantly in HCC tissues, and also correlated to clinical TNM stage, metastasis, status of capsular infiltration and numbers of tumor nodes [45].

MiRNAs prognosing the tumor development trends Numerous recent studies on HCC have analyzed the association between various miRNAs and HCC progression and development. Patients with higher serum miR-1 and miR122 levels showed longer overall survival than individuals with lower miR-1 and miR-122 serum concentrations [46]. HCC patients highly expressing miR-224 had worse 5-year disease-free survival and 5-year overall survival [47]. MiR125b [48], miR-200a [49] and miR-210 [50] were also correlated with the overall survival of HCC patients. The expression level of miR-17-5p was not only correlated with overall survival, but also reflects the metastasis

status [35,51]. And also miR-155 reflected tumor recurrence, micro-vascular invasion and recurrence-free survival [52,53]. And miR-29a-5p, miR-185, miR-200a, miR-19a, miR886-5p, miR-126, miR-223, miR-24 and miR-147 levels were significantly associated with tumor early recurrence and overall survival of tumor [30,49,54,55]. The significantly low expression of miR-148a in the HCC tissues was confirmed correlated with capsular infiltration and lower time-torecurrence [45]. At present, the downregulation of miR-101 [56], miR148b [57] and miR-214 [58] in clinical HCC tissues were confirmed to be correlated with vein invasion of HCC. Chen et al.’s and Zheng et al.’s studies showed that in HCC with metastasis, there were higher miR-17-5p levels than that without metastasis [35,51], and some other studies indicated that the over-expression of miR-182 was correlated with intrahepatic metastasis [59], while miR-221 dysregulation in HCC tissues could be used for predicting local recurrence and distant metastasis after curative surgery [60]. Also, low miR-100 and miR-22 expression was observed to be significantly correlated with higher incidence of lymphatic metastasis [38,44]. A bead-based

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MicroRNAs for hepatocellular carcinoma Table 2

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Association between miRNAs polymorphisms and the risk of HCC.

Role

SNP ID

miRNA (allele)

References

Decreased risk of HCC

rs1057035 rs1076064 rs10773771 rs11614913 rs12304647 rs16405 rs16917496 rs2292832 rs3746444 rs3783553 rs3859501 rs7963551 rs999885 rs1042522 rs11134527 rs11614913 rs17875871 rs2910164 rs3746444 rs3803012 rs4309483 rs4938723 rs6147150 rs999885 s10877887 rs11134527 rs4938723

DICER1(C>T) miR-378(A>G) PIWIL1(C>T) miR-196a2(C>T) miR-196a-2(A>C) miR-920 Ins/Del miR-502(T>C) miR-149(C>T) miR-499(A>G) miR-122 Ins/Del miRNAs-371-373(C>A) RAD52(G>T) miR-106b-25(A>G) TP53 Arg72Pro(G>C) miR-218(A>G) miR-196a2(C>T) IFNAR1 miR-146a(G>C) miR-499(A>G) RAN(A>G) miR-122(C>A) miR-34b/c(T>C) miR-let-7c Ins/Del miR-106b-25(A>G) let-7(T>C) miR-218(A>G) miR-34b/c(T>C)

[89] [71] [89] [74,87] [90] [91] [92] [78,84] [82—84] [93] [94] [95] [96] [73] [97] [85,86] [98] [74,75] [78—81] [89] [72] [73,99,100] [101] [76] [77] [102] [103,104]

Increased risk of HCC

Increased or decreased the risk of HCC based on different genotypes

Abbreviations: miRNAs: microRNAs; HCC: hepatocellular carcinoma; SNP: single nucleotide polymorphism; Ins/Del: insertion/deletion.

microarray analysis of microRNA expression in HCC revealed that the level of miR-338 expression could be associated with tumor-node-metastasis stage, vascular invasion and intrahepatic metastasis [41]. MiR-191 and miR-221 were also associated with HCC metastasis and capsular invasion [33]. Meanwhile, miR-122 was confirmed to play an important role in the control of cell migration and invasion [61]. Qi et al. [15] found that miRNAs also weighed heavy in HBV- or HCV-related HCC as novel potential biomarkers, as well as in HCC progression from cirrhosis to cancer. Salvi et al.’s study [62] revealed that miR-24 and miR-27a were significantly downregulated in HCCs from cirrhotic liver tissues in comparison to those from non-cirrhotic liver tissues. In cirrhotic HCC tissues the downregulation of miR-24 was correlated with poorer prognosis in patients with HBV and HCV infection. The expression of miR-101 was downregulated in HBV-related HCC tissues compared with adjacent noncancerous tissues, while the serum miR-101 in patients with HBV-related HCC was significantly higher than that in the healthy controls. And this increase was correlated with hepatitis B surface antigen positivity and HBV-DNA levels [31]. A work of Gao et al. [63] has shown that miR-145 and miR-199b were downregulated while miR-224 upregulated in HBV-related HCC patients. Pineau et al. [64] presented the miRNAs expression profile of a large number of paired HCC-nontumor samples as well as liver cancer cell lines with the methods of microarray and quantitative real-time

PCR (qPCR). They identified that the miRNAs were significantly dysregulated in liver tumorigenesis. Expression of miR-106b, miR-21, miR-210, miR-221, miR-222, miR-224, miR-34a, miR-425, miR-519a, miR-93, and miR-96 were all increased, and let-7c was lost during liver tumor progression. Their findings suggested that the expression levels of some miRNAs had changed gradually during the progression of liver disease [65].

Association between miRNAs polymorphisms and the risk of HCC Single nucleotide polymorphisms (SNPs) in miRNA genes are regarded to affect function through the transcription of the primary transcript, or through pri-miRNA and pre-miRNA processing. Additionally, they can act through the effects on miRNA-mRNA interactions. Recent studies have demonstrated that some SNPs are present in the miRNA genes, which can alter miRNA expression and/or maturation and are associated with the development and progression of cancer [66]. Numerous studies have reported the associations between miRNA SNPs and the risk of cancer, and many researchers performed meta-analyses to evaluate the association between them [67—69]. There are also many literatures reporting that miRNA SNPs were associated with the risk of HCC, including increase or decrease the risk of HCC (Table 2).

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MiR-378 has been reported to be related to cell survival, tumor growth and angiogenesis [70]. Genetic variants in primary miR-378 (pri-miR-378) may impact miR-378 expression and contribute to the risk and survival of HCC. One study had assessed the associations between a genetic variant in primary miR-378 (rs1076064 A>G) and HCC susceptibility and prognosis. The results indicated that the variant genotypes of rs1076064 were associated with a decreased HCC risk in HBV carriers, and HCC patients with the variant genotypes were associated with a better survival [71]. Besides, a lot of miRNA SNPs were reported to decrease the risk of HCC, such as rs1057035 (DICER1 C>T), rs10773771 (PIWIL1 C>T), rs12304647 (miR-196a-2 A>C), rs16917496 (miR-502 T>C), rs2292832 (miR-149 C>T), and so on (showed in Table 2). On the other hand, many miRNA SNPs increased the risk of HCC. MiR-122 plays a vital role in the development of chronic HBV infection and HCC. The CA/AA genotypes of a miR-122 SNP (rs4309483 C>A) were significantly associated with the increasing risk of HCC compared with HBV carriers [72]. The miRNA SNPs of TP53 Arg72Pro (rs1042522 G>C) [73], miR146a (rs2910164 G>C) [74,75] and miR-106b-25 (rs999885 A>G) [76] also increased the risk of HCC (Table 2). In addition, HCC patients carrying the C allele of let-7 (rs10877887 T>C) had a significantly increasing risk for HCC compared to those with T allele [77]. There are some relevant studies reported miRNA SNPs such as miR-499 (rs3746444 A>G) and miR-196a2 (rs11614913 C>T) with inconsistent conclusions. Some studies indicated that the SNP of miR-499 (rs3746444 A>G) increased the risk of HCC [78—81], while other studies showed the effect of decreasing risk of HCC [82—84]. The SNP of miR-196a2 (rs11614913 C>T) has been reported to increase the risk of HCC [85,86]. Recently, there are two new researches concluded a new result which decreased the risk of HCC [74,87]. These controversial results perhaps because the choice of populations and different genotypes. In order to avoid these kinds of bias, several meta-analyses were conducted [68,69], but the results were still not perfect, and these need more and large-scale studies to define and prove it. The SNPs of miR-218 (rs11134527 A>G) and miR-34b/c (rs4938723 T>C) were reported to increase or decrease the risk of HCC based on different genotypes (showed in Table 2).

Conclusions Since the first miRNA was identified in 1993, researchers are aimed to better understand the miRNA function in humans. They have gradually realized that miRNA expression profiles are more informative than mRNA expression profiles in a number of diseases [88]. With the successful detection of stable miRNAs in bodily fluids of humans, the use of miRNAs as potential biomarkers became reality. MiRNAs represent critical regulators in cancer development. MiRNAs also play a key role in cancer progresses such as tumor cell differentiation, proliferation, cell cycle progression, invasion, and metastasis and outcomes. They could be used as potential biomarkers for cancer prognosis as well as in cancer diagnosis. Previous studies showed that it is optimistic and hopeful to adopt miRNA in HCC diagnosis and prognosis. However, the diagnostic value of miRNAs for HCC varies depending on

different specimens, study areas, and the choice of control groups. And the specimen types used for HCC developments and outcomes prognosing are mainly tissues, which means there are invasive to the human body. It requires comprehensive systematic reviews and meta-analyses, or large-scale randomized controlled multicenter trials to avoid all kinds of bias as much as possible, and select better non-invasive biomarkers for HCC diagnosing and prognosing.

Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.

Acknowledgment This work was supported by Maanshan Municipal Hospital Group and Chongqing Medical University, and partially funded by Maanshan Municipal Public Health Bureau. We thank our colleagues for useful discussion.

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