Abstract

INTRODUCTION

Hepatocellular carcinoma (HCC) is the seventh most common malignancy and the second leading cause of cancer-related deaths worldwide. Globally, there are approximately 750000 new cases and 700000 deaths of HCC reported per annum[1-3]. Two well-known risk factors for HCC are chronic viral hepatitis B (HBV) and C (HCV), which account for 80%-90% of all HCC cases worldwide. Other risks for HCC include obesity, diabetes, vitamin D deficiency, aflatoxin B1 exposure, alcoholic and non-alcoholic liver cirrhosis[4,5]. However, the underlying mechanism of HCC has not been entirely elucidated. Surgical resection and orthotopic liver transplantation are the best curative tools for the long-term survival of HCC patients. However, surgical resection is not feasible in more than 80% of HCC patients because of tumor location, tumor size or severity of the underlying liver disease. Only 5%-15% of HCC patients are potentially resectable[6,7]. The overall five-year survival rate in patients with HCC is very low, ranging from 5% to 9%. The cumulative five-year recurrence rate is approximately 70% to 80% even after curative surgical resection. Recurrence after resection generally results in a high rate of mortality[8]. Therefore, the most urgent needs are the identification of sensitive and specific markers for early diagnosis, the monitoring of recurrence and the prediction of prognosis for HCC.

Current methods for HCC diagnosis are classified into the following main categories: imaging [abdominal ultrasonography, magnetic resonance imaging (MRI), and contrast-enhanced computed tomography (CT)] and laboratory biomarker analysis [serum alpha-fetoprotein (AFP) levels][9,10]. However, the diagnostic performance of imaging technologies is unsatisfactory, particularly for the diagnosis of small lesions and early-stage HCC[11,12]. AFP is the most commonly used tumor marker for HCC diagnosis and prognosis prediction, but the false negative rate using AFP level alone is as high as 40% for patients with early-stage HCC. AFP levels remain normal in 15%-30% of all the patients, even patients with advanced HCC[6,13]. Therefore, the American Association for the Study of Liver Disease Practice Guidelines discarded AFP for surveillance and diagnosis because of its low sensitivity (39%-64%) and specificity (76%-94%)[14]. Therefore, the identification of novel and ideal biomarkers with high specificity and sensitivity for HCC diagnosis is desperately needed. Circulating microRNAs (miRNAs) have received remarkable attention because they offer great advantages and good performance as novel biomarkers for HCC diagnosis and prognosis prediction[15].

Emerging evidence demonstrated that miRNAs are an important class of non-coding RNAs as tumor oncogenes or suppressors that are involved in the HCC development[16]. miRNAs are endogenous nucleotides that are found in intra- and extracellular spaces, such as blood, urine and saliva[15,17]. Cellular miRNAs are also released, detected and quantified in the blood[18]. Circulating miRNAs exhibit several obvious advantages as potentially novel and ideal diagnostic biomarkers, including significant stability in various types of body fluids, resistance to endogenous RNase digestion, low cost, ease of analysis, and sensitive detection methods, such as real-time quantitative polymerase chain reaction (qRT-PCR)[19-21]. The above characteristics of circulating miRNAs and the limitations of currently available non-invasive methods to monitor HCC indicate that circulating miRNAs are a rapidly growing field of research that hold great promise for the development of reliable biomarkers for the diagnosis and monitoring of HCC[6,20,22].

This review highlights miRNA biogenesis and the types and origins of circulating miRNAs, summarizes recent findings of circulating miRNAs as prognostic and predictive biomarkers for HCC, and addresses challenges and perspectives for the clinical utility of circulating miRNAs in HCC.

miRNA BIOGENESIS AND FUNCTION

miRNAs are single-stranded, non-coding RNA strands of 19-25 nucleotides that regulate more than 50% of all protein-coding genes in mammals[23,24]. There are 1881 human miRNAs sequences registered in the miRBase database. The synthesis of miRNAs primarily consists of two steps, including nuclear synthesis within and outside of the nucleus[25] (Figure ​(Figure1).1). First, RNA polymerase II transcribes miRNAs in the nucleus into primary miRNAs (pri-miRNAs) that are several hundred nucleotides in length[26,27]. Subsequently, the microprocessor complex, which consists of DiGeorge syndrome critical region gene (DGCR8) and the nuclease Drosha, cleaves the pri-miRNAs into miRNA precursors (pre-miRNAs) of approximately 60 to 70 nucleotides[3,28,29]. Exportin-5 transports the pre-miRNAs from the nucleus to the cytoplasm[25,30,31]. The pre-miRNAs are further processed in the cytoplasm by RNase Dicer to final miRNA duplexes (miRNA-miRNA^*) of 19-25 nucleotides[32,33]. Finally, the mature single-stranded miRNAs are generated and retained to form RISC (RNA-induced silencing complex), which is destined for mRNA repression/cleavage, but its passenger strand (miRNA^*) undergoes degradation[33-35]. Mature miRNAs recognize and bind to the 3′ untranslated region (UTR) of multiple mRNA targets with imperfect or perfect complementarity, which enables one specific miRNA to inhibit the translation of multiple genes[36]. Multiple miRNAs may also regulate one mRNA[17]. miRNAs participate in many key cellular processes, including proliferation, cell cycle, apoptosis, and metastasis[7,37]. The involvement of miRNAs in cancer is well established because miRNAs behave as tumor-suppressor genes or oncogenes, depending on the cellular function of their targets[38,39]. Growing evidence indicates that miRNAs may become a new tool for HCC diagnosis, prognosis, and therapy[38,40,41].

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Figure 1

MicroRNAs biogenesis and function. In the nucleus, pri-miRNAs are transcribed by RNA Polymerase II (Pol II) to pre-miRNAs after processing by the microprocessor complex, which consists of Drosha and DGCR8. Pre-miRNAs are exported from the nucleus to the cytoplasm by exportin-5 and processed into the miRNA-miRNA* duplex by Dicer. The duplex is separated into two strands: one strand is selected as the mature miRNA, and the other strand is degraded. Mature miRNAs are incorporated into RISC (RNA-induced silencing complex) to produce target mRNA repression (imperfect complementarity) or mRNA cleavage (perfect complementarity). pri-miRNAs: Primary miRNAs; Pol II: RNA Polymerase II; pre-miRNAs: MiRNAs precursor; DGCR8: DiGeorge syndrome critical region gene; miRNA: MicroRNA; RISC: RNA-induced silencing complex.

ORIGINS, TYPES, AND FUNCTION OF CIRCULATING MIRNAS

The mechanisms through which circulating miRNAs originate have been established recently. Figure ​Figure22 shows that circulating miRNAs originate from the following mechanisms: (1) active secretion via membrane vesicles; (2) active secretion in protein-miRNA complexes and lipoprotein complexes (such as high-density lipoproteins); (3) active secretion via exosomes when a multivesicular body fuses with the cell membrane; and (4) passive secretion originating from cell necrosis and apoptosis[42-46]. Some research has demonstrated that circulating miRNAs exist in two different types, circulating protein-bound miRNAs or circulating microparticle miRNAs[18,43,47,48]. Circulating microparticle miRNAs are circulating miRNAs that are packaged into microparticles. Three different types of microparticles were defined by their size, mechanisms of production, and protein and lipid content[49]: (1) exosomes (40 to 100 nm in diameter; released by exocytosis from multivesicular bodies); (2) microvesicles (100 to 1000 nm in diameter; released via plasma budding); and (3) apoptotic bodies (1000 to 4000 nm in diameter; released by cells executing apoptotic process)[20,43,50,51]. Circulating protein-bound miRNAs are circulating miRNAs that are associated with specific RNA-binding proteins and lipoproteins, such as Ago2[52], which is a component of the RNA-induced silencing complex, and high-density lipoproteins (HDL)[53]. One study demonstrated that the majority of circulating miRNAs were associated with protein complexes rather than vesicles[52], but other reports indicated that circulating miRNAs were selectively sorted to microparticles or protein complexes[54-57]. Notably, both types of circulating miRNAs vary in different types of liver disease. Bala and co-workers reported that circulating miR-122 and miR-155 are predominantly associated with the exosome-rich fraction in alcoholic liver disease and inflammatory liver injury, but these miRNAs are present in the protein-rich fraction in drug-induced liver injury. Therefore, these results suggest that the shift in different types of circulating miRNAs may provide further specificity of the mechanisms of liver pathology[22].

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Figure 2

Mechanisms of microRNAs secretion into blood vessels; origins and types of circulating microRNAs. miRNAs can be secreted from living cells into the extracellular environment (such as blood vessels) via the following mechanisms: (1) active secretion via exosomes; (2) active secretion via microvesicles; (3) active secretion in protein-miRNA complex (Ago2) and lipoprotein complex (such as HDL); and (4) passive secretion through apoptotic bodies. Circulating miRNAs may originate from immune cells, endothelial cells of other organs, and cancer-specific cells. In the blood, miRNAs can circulate as circulating protein-bound miRNAs or circulating microparticle miRNAs. Three types of microparticles are found in circulating blood: exosomes, microvesicles and apoptotic body. Circulating miRNAs are also present as protein-bound miRNAs. These miRNAs are primarily associated with specific RNA-bind proteins and lipoproteins, such as Ago2 and HDL. AGO: Argonaute; HDL: High-density lipoprotein; miRNAs: MicroRNAs; MVB: Multivesicular body.

A recent study identified miRNAs in many body fluids, including serum and plasma[58]. Circulating miRNAs in body fluids are packaged into microparticles or bound to proteins, which provides stability and resistance to plasma RNase digestion and enables miRNA transfer from one cell to another during diverse biological processes[59]. Circulating miRNAs exhibit paracrine effects on tumor growth[60,61]. If tumors are defined as relatively homogeneous cancer cells that are formed in an independent microenvironment, circulating miRNAs may play a novel role as regulators of intercellular communications during tumor formation[62,63]. Aside from their functional roles in tumorigenesis, circulating miRNAs represent a less invasive alternative for diagnostic testing. Easily assessable serum-based miRNAs may provide novel biomarkers of diagnostic, prognostic and predictive values for HCC, specifically in the field of hepatology.

CIRCULATING MIRNAS AS NOVEL BIOMARKERS FOR HCC DIAGNOSIS AND PROGNOSIS

The first description of the presence of circulating miRNAs in serum and their potential as cancer markers was reported by Lawrie et al[64] in 2008. This study demonstrated the overexpression of miR-155, miR-21 and miR-210 in patients with diffuse large B-cell lymphoma compared to healthy controls. Notably, high miR-21 levels were associated with the relapse-free survival of these patients[60]. Multiple studies identified circulating miRNAs as diagnostic and prognostic markers for a wide range of malignancies, which primarily used blood plasma or serum for their analyses[48].

Evidence also suggests that miRNAs are widely involved in HCC carcinogenesis, differentiation and metastasis. Aberrant miRNA expression was reported in HCC patients and cell lines. More information on aberrant miRNA expression in HCC is available in comprehensive reviews[3,40]. Therefore, circulating miRNAs should be affected during HCC development and progression. Various reports demonstrated the potential clinical application of circulating miRNAs in HCC diagnosis and prognosis.

CONCLUSION

The use of circulating miRNAs as biomarkers for HCC has received attention since the first circulating miRNAs signatures were reported as potential diagnostic tools in oncology in 2008. This review highlights the process of miRNAs biogenesis and elaborates the origins and types of circulating miRNAs. Notably, we have summarized recent advances in circulating miRNAs signatures as non-invasive biomarkers in the diagnosis and prognosis of HCC. Overall, our review illustrates the potential application of circulating miRNAs, either as a single marker or as a panel of circulating miRNAs or in combination with conventional markers, as biomarkers for HCC diagnosis and prognosis predictions.

Encouraging progress in the use of circulating miRNAs in HCC diagnosis and prognosis has been made, and circulating miRNAs are extremely promising biomarkers. However, there are no clinically used miRNAs biomarkers for HCC, and further investigation is required to overcome some of the challenges in their application. First, the origin of circulating miRNAs has not been fully elucidated. Circulating miRNAs in serum/plasma have a heterogeneous origin, including from blood cells, endothelia cells, and other high blood-flow organs, which demonstrates that the expression of tumor-specific miRNA signatures may be masked by circulating miRNAs from other origins[19,81-83]. Another issue is that well-standardized protocols are not implemented for pre-analytical decisions of circulating miRNAs (e.g., sample storage, sample processing, the profiling method) to post-analytical processing (e.g., data normalization), which makes definitive comparisons between studies difficult[43,84]. Unfortunately, there is no widely accepted endogenous normalization control for circulating miRNAs, despite the use of qRT-PCR, which is the most commonly used technology for the detection of miRNAs in circulation. Furthermore, most miRNAs studies use small sample sizes, limited number of screened miRNAs, fail to differentiate HCC from hepatitis B and C, and lack large-scale prospective studies[9,20,74]. Moreover, much work is also required to compare values of circulating protein-bound miRNAs and microparticle miRNAs in different HCC patients. Finally, it is very important to use appropriate controls that are well matched in age, race, gender, etiology and severity of underlying liver disease when evaluating the sensitivity and specificity of circulating miRNAs for the diagnosis and monitoring of HCC[20,85,86].

Research on circulating miRNAs is in its infancy, and there are some challenges in the clinical application of circulating miRNAs. However, an improved understanding of the origins, stability, detection methods, and roles of circulating miRNAs in HCC will support the great potential of circulating miRNAs to become diagnostic and prognostic tools for HCC in the future.

Footnotes

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