MicroRNAs (miRNAs) are (mostly) endogenously developed fragments of single stranded non-coding RNA (19-25 nucleotides) that regulate more than 50% of all cell specific protein translation. The deregulation of miRNAs is linked to cancer because they play a role in modulating target genes responsible for cell proliferation, apoptosis, DNA repair, invasion and metastasis [
1]. The sensitivity of miRNA expression (transcription) alteration in cancer incidence is underlined by the location of their parent genes, often found in fragile chromosomal regions that exhibit DNA amplification, deletions and translocations which deregulate miRNA expression [
2]. Circulating miRNAs have been proposed as promising biomarkers for cancer pathologies because of their abundance in sera, as well as their stability under extreme conditions [
3,
4]. Serum miRNAs are resistant to ribonuclease digestion because they are protected in protein complexes or in membranous micro-vesicles that transport them in the circulatory system [
1,
5]. The stability of cell free, circulating miRNAs is underlined by the fact that successful quantification has been observed in samples stored up to 10 years at −80 degrees C [
1,
6]. Despite considerable investigation of extracellular miRNAs, the use of miRNAs as biomarkers of cancer is still regarded as a ‘work in progress’ and mostly restricted to research programs [
5,
7]. Continuing technological developments, however, like 2nd generation sequencing, as well as a better understanding of the pathobiological role of miRNAs, underline their future promise as clinical biomarkers [
7,
8].
A wide range of studies has investigated the diagnostic proficiency of circulating miRNAs in liver diseases, including hepatocellular carcinoma (HCC), chronic hepatitis (CH), non-alcoholic fatty liver disease (NAFLD), liver toxicity, cirrhosis and non-alcoholic steatohepatitis [
9‐
12]. The strong causal association between HCC and CH continues to influence HCC incidence [
3,
13] while emerging studies explain the biological role of viral miRNAs [
14]. Sub-Saharan Africa, and South Africa in particular, is an endemic region for both HBV and HIV infection, as well as rapid urbanization and lifestyle changes [
15]. The aim of this study is to investigate circulating miRNAs as biomarkers for HCC in South Africa against a background of both HIV/HBV mono and co-infection.
Empirical evidence of circulating miRNAs in HCC
In hepatocellular carcinoma (HCC), a range of miRNAs is deregulated in response to cancer cells that promote aberrant expressions in their target genes [
16]. HCC deregulates the expression of circulating miRNAs (upwards or downwards) to inversely influence the expression of target mRNAs/ specific genes involved in cell cycle regulation, apoptosis, DNA repair, invasion and metastasis [
2]. In HCC development, the miRNA mediated expression of mRNA can have either oncogenic effects or promote a loss of tumor suppressor function [
2,
14,
16].
Emerging evidence indicates multiple miRNAs are deregulated in HCC. A recent reviewed collated a wide range of studies to collectively indicate 55 miRNAs that are down-regulated and 41 miRNAs that are up-regulated in HCC [
16]. The presence and proficiency of circulating miRNA as biomarkers for HCC, have been tested both individually, as well as in selected groups. Examples of deregulated circulating miRNAs, identified in numerous studies, include miR-10a, miR-21, miR-23a/b, miR-25, miR-26a/b, miR-122, miR-125b, miR-192, miR-222, miR-223, miR-342-3p, miR-375, miR-423, miR-801, miR-885-5p, and miR-Let-7f [
3]. It has been suggested that miR-122a is the most abundant miRNA in hepatocytes [
9], that it is a reliable marker of viral infection [
17] and it is down-regulated in ~70% of HCC [
18]. MiR-500 is also abundantly expressed in liver cancer cell lines and deregulation of miRNA occurs in ~45% of HCC cases [
19].
HCC, viral infection and circulating miRNAs
Viruses encode their own sets of miRNA which are used to control the expression of their host’s genes [
20]. The ability of a virus to package its own miRNAs into exosomes and transport them to non-infected cells was first demonstrated by the EBV virus [
21]. Both viral transcripts and proteins can affect host miRNA expression, which can modulate viral and/or host protein expression [
22]. MiRNAs can bind to viral genomes or transcripts and regulate viral infection and, conversely, viral infection (e.g. HIV/HCV) can modulate host-cell microRNA machinery [
23]. The role of miRNAs in viral infection is being demonstrated in an increasing number of studies. MiR-122, for instance, down-regulates HBV replication by binding to the viral target sequence [
24] and, conversely, binds to the HCV genome to increase viral translation and replication [
25‐
27]. MiR-199a and miR-210 bind to different sites on mRNA coding of HBsAg, reducing HBsAg expression in HepG2 2.2.15 cells [
28]. MiR-15b has also been shown to modulate HBV replication by targeting the hepatocyte nuclear factor 1α (HNF1α) [
29], while miR-130a expression is increased in HCV infection.
Two review papers, summarizing a wide range of studies [
2,
3], identified a marker group of seven circulating miRNAs, including miR-122, miR-192, miR-223, miR-21, miR-26a, miR-27a, miR-801 that were able to distinguish between HCC, HBV, cirrhosis and healthy controls, as well as identify HCC tumor stages. Others have shown that serum levels of miR-10a and miR-125b were lower in HBV infected HCC patients than in chronic hepatitis B (CHB) patients and that a triplet of circulating miRNAs [namely miR-375, miR-25, miR-Let-7f] were able to diagnose HCC with ~98% accuracy [
3]. Circulating miR-21 was also higher in HCC than chronic hepatitis patients and healthy controls; furthermore, its levels correlated with miR-21 expressed in HCC tumor tissue and it had better diagnostic sensitivity than alpha fetoprotein (AFP) [
2,
3]. In another study, it was found that serum miR-21, −122, and −223 were higher in HCC and CH versus controls, whereas miR-122 and miR-21 were higher for CH than HCC but not miR-223 [
30].
Biological relevance of deregulated miRNAs in HCC
Various studies are increasingly beginning to explain the biology of specific circulating miRNAs and their potential role in HCC, with respect to cell proliferation, angiogenesis and metastasis (see Additional file
1: Table S1). Cell proliferation in HCC is promoted by the downregulation of miR-26a, which acts as a partner with miR-195 to overcome the G1/S cell cycle blockade through the repression of E2F expression. Cell proliferation in HCC is also influenced by upregulated miRNAs (e.g. miR-21, miR-216a) that promote tyrosine kinase by downregulating the PTEN tumour suppressor protein [
14]. MiR-122, for example, can inhibit angiogenesis and intrahepatic metastasis by suppressing the expression of the tumour necrosis factor-α- converting enzyme (TACE) [
14]. The metastasis of HCC is also influenced by miR-10a which regulates ephrin-type-A-receptor-4 mediated mesenchymal transition [
14,
16].
Controversial issues in circulating miRNA research
Recent research indicates that miRNAs are found in all cellular components, where they regulate transcription, translation, alternative splicing and DNA repair [
31]. A number of unsolved issues continues to delay the use of circulating miRNAs as viable cancer/ disease biomarkers. The mechanism of their generation and possible pathways is still being investigated [
31] and their biological role as messenger miRNAs in signaling, remains unclear [
32,
33]. A question also remains as to whether only certain types of extracellular vesicles (e.g. exosomes) transport messenger MiRNAs and others merely transport small RNA as debris [
7,
34]. In addition, the elevation of extracellular miRNAs in the blood sera of cancer patients has been attributed to general conditions like inflammation, rather than as a result of an early stage tumor [
7] Another issue is that recent reviews suggest that methodological problems in many earlier studies re non standardization of sample collection, sample quality control, RNA isolation, RT-qPCR and data normalization, have rendered their findings questionable [
7,
17,
35]. Finally, the ability of miRNAs to silence their target mRNAs, is also influenced by polymorphisms in their parent genes that cause small changes in the miRNA nucleotides, thus inducing a change in their ability to bind to mRNA targets [
16].