Abstract and Introduction
Hepatocellular carcinoma (HCC) is currently the fifth most common cancer worldwide and the fourth leading cause of cancer-related death. The number of new cases is estimated to be more than 500 000 per year, accounting for 4% of all newly diagnosed cancers. Aetiological studies have shown that infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) and ingestion of aflatoxin B1-contaminated food may be important risk factors for the development of HCC. In addition, cirrhosis resulting from heavy alcohol intake represents a major risk factor in some Western countries.
The pathogenesis of HCC has been studied extensively, and molecular changes during malignant transformation have been identified. Hepatocarcinogenesis is considered as a multistep process originating from hepatic stem cells or mature hepatocytes, and evolving from chronic liver disease driven by chronic inflammation, oxidative stress, cell death followed by unrestricted proliferation/regeneration, and permanent liver remodelling. Finally, the accumulation of genetic and epigenetic alterations leads to an activation of oncogenes and inhibition of tumour suppressor genes accompanied by an escalation of genetic instability and the disruption of signalling pathways related to the main promoters of hepatocarcinogenesis, namely cell proliferation and angiogenesis.
Genomic studies have demonstrated that many portions of the human genome do not encode conventional protein-coding genes but encode biologically active non-coding RNA species. One class of such small non-coding RNAs is microRNAs (miRNAs), a group of regulatory RNAs of 19–22 nucleotides involved in control of gene expression at the post-transcriptional level. They accomplish this by binding to the 3′ untranslated region (3′UTR) of target messenger RNA (mRNA), resulting in either their degradation or inhibition of translation. Up to 30% of human protein-coding genes may be regulated by miRNAs, making them one of the largest classes of regulatory molecules in humans. The miRNAs are also involved in numerous cellular processes such as development, differentiation, apoptosis, cell proliferation, metabolism and immunity. Each mRNA can be regulated by several miRNAs, and one miRNA can recognize several targets. Intriguingly, miRNAs may also lead to an upregulation of gene expression. Recent studies provide clear evidence that miRNAs are abundant in the liver and modulate a diverse spectrum of liver functions. Many miRNAs, such as miR-122, miR-1, miR-16, miR-27b, miR-30d, miR-126, miR-133, miR-143 and the let-7 family, are abundantly expressed in adult liver tissue. Recent research has identified targets and functions of miRNAs, illustrating that some are oncogenic in nature whereas others show tumour suppressor activity. Oncogenic miRNAs are up-regulated in cancer and contribute to its pathology through various mechanisms such as targeting tumour suppressor genes. Several studies have shown that specific miRNAs are aberrantly expressed in malignant HCC cells or tissues compared to non-malignant hepatocytes or tissue. Selected miRNAs such as miR-21, miR-224, miR-34a, miR-221/222, miR-106a and miR-203 are upregulated in HCC. Certain miRNAs have been noted to be decreased in HCC compared to non-tumour tissue, such as miR-122a, miR-422b, miR-145 and miR-199a.
Every type of tumour classifies by globally abnormal miRNA expression patterns. The miRNA expression profiles are characterized by tumour classification, prognosis, and response to therapy. The miRNA expression profiles may prove useful as diagnostic and prognostic markers in cancer, and various miRNA-based therapies have shown promise as well. For example, some specific miRNAs have been linked with clinicopathological parameters of HCC that include metastasis, recurrence and prognosis. Down-regulation of miR-145 and miR-199b, and up-regulation of miR-224 are often detected in pre-malignant dysplastic nodules. These changes continue throughout HCC development. In addition, down-regulation of miR-145 has been associated with early stages of HBV-related hepatocarcinogenesis, which may be valuable as an early diagnostic HCC marker. Furthermore, miR-26 is a tumour suppressor and miR-26 silencing in hepatocytes contributes to the development of a more aggressive form of HCC. The latter study also revealed that miR-26 expression is an independent predictor of survival. However, only patients whose tumours were reduced in the expression of miR-26 had a favourable response to interferon therapy. These results indicate that miR-26 status in tumours may be a useful tool in estimating prognosis in patients with HCC and in assisting in the selection of patients who are likely to benefit from adjuvant therapy with interferon to prevent relapse. Moreover, miRNAs in serum and plasma sample from cancer patients have been considered as promising novel biomarkers for cancer diagnosis and prognosis. Elevated levels of serum miR-21, miR-122 and miR-223 are present in patients with HCC but not chronic hepatitis. Thus, these results suggest that serum miRNAs may serve as potential markers of HCC. In addition, in combination with conventional serum markers such as α-fetoprotein (AFP), lens culinaris agglutinin-reactive AFP (AFP-L3%), and des-γ-carboxyprothrombin (DCP), analysis of miR-16 levels in serum from 105 HCC patients improved sensitivity and specificity for HCC detection. Recently, a miR-196a2 polymorphism was reported in blood samples of 532 Chinese patients, and was significantly associated with cirrhosis-related HCC susceptibility.
In this issue of Liver International, Augello et al. report important data consisting of 664 mature miRNA expression profiles in 60 HCV-positive liver lesions representative of six classes of liver disease (cirrhosis, cirrhosis associated HCC, low grade dysplastic nodules, high grade dysplastic nodules, early HCC and progressed HCC). They identified a miRNA gene cluster on the long arm of chromosome 19 (C19MC). The C19MC is the largest human miRNA gene cluster and is localized within chromosome 19q13.41. The C19MC accounts for an estimated 10% of all known human miRNA genes. Recent evidence has revealed a subset of HCCs that overexpress a family of miRNAs on C19MC. These miRNAs tend to promote malignant phenotype in HCC cells and in mouse model of HCC by miR-517a or miR-520c of C19MC family members. In addition, bisulphite-modified methylation analysis demonstrated that hypermethylation at the examined CpG islands of the C19MC is the mechanism of C19MC miRNAs overexpression. These results indicate that C19MC miRNA is an oncogenic miRNA that promotes tumour progression. Augello and colleagues also report that C19MC is progressively up-regulated in neoplastic lesions compared to cirrhotic liver parenchyma. High levels of miR-517c, 518a-3p, or 525-3p of C19MC miRNA members were significantly associated with micro-vascular invasion. The miR-515-3p, 525-3p, or miR-515-5p, 518a-3p and 520f were markedly increased in HCC with advanced tumour stage. These findings suggest that over-expression of the C19MC cluster is associated with clinical features of HCC. Interestingly, they were linked to high C19MC miRNA levels and post-operative recurrence, and overall survival. Survival analysis associated a high level of miR-517c with an increased risk of tumour recurrence. In addition, shorter overall survival was found in patients with high expression of miR-515-3p, miR-520h, miR-520f or miR-520g. The authors are able to conclude that the C19MC cluster is a novel molecular alteration characteristic of HCC and predictor of poor prognosis, and that C19MC is an attractive candidate for novel HCC therapies.
Utilization of miRNAs in therapy is an interesting idea and a major focus of current research activities. The miRNA-based therapeutic approaches for HCC would be promising data in HCC pathogenesis and clinical oncology. In a recent study, infection of locked nucleic acid oligonucleotides to miR-122 led to suppression of HCV viraemia with minimal toxicities, indicating the possibility of a therapeutic approach for targeting antiviral intervention in the liver. Furthermore, miR-26a delivered in an adeno-associated virus (AAV) vector can be used as general anti-cancer therapeutics. Expression of miR-26a in human liver cells induces G1 arrest that is linked to the targeting of cyclins D2 and E2. In a mouse model of HCC, AAV-mediated miR-26a suppressed tumour progression and activated tumour-specific apoptosis. These findings suggest the potential of targeting tumour-suppressing miRNAs or aberrantly expressed miRNA as a specific therapeutic modality for HCC. Moreover, miRNAs are often located in amplified or deleted genomic regions, so the analysis of miRNA expression together with single nucleotide polymorphism array analysis for genomic copy alterations will be helpful to detect new miRNAs with oncogenic or tumour suppressor properties. Also, gene expression profiles and the study of specific proteins in combination with miRNA expression analysis will provide valuable information about genomic alterations and regulatory function.
Although the results of Augello et al. are very important, additional in vitro or in vivo studies of liver cancer are needed to verify these initial observations and will certainly broaden our understanding of the pathogenesis of HCC. Genetic information of new genomic technologies and approaches is rapidly accumulating. Based on new technologies, including gene expression profiling and proteomics, it may be possible to seek molecular tumour markers and to identify molecules involved in hepatocarcinogenesis. However, our understanding of the molecular mechanisms of hepatocarcinogenesis is rudimentary. Considerable effort has now focused on unravelling the molecular pathogenesis of HCC to design better treatments or prevent the disease altogether.