Hou J, Lin L, Zhou W, Wang Z, Ding G, Dong Q, et al. Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular cancer. Cancer Cell 2011;19:232-243.
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The full scale of human miRNome in specific cell or tissue, especially in cancers, remains to be determined. An in-depth analysis of miRNomes in human normal liver, hepatitis liver, and hepatocellular carcinoma (HCC) was carried out in this study. We found nine miRNAs accounted for 88.2% of the miRNome in human liver. The third most highly expressed miR-199a/b-3p is consistently decreased in HCC, and its decrement significantly correlates with poor survival of HCC patients. Moreover, miR-199a/b-3p can target tumorpromoting PAK4 to suppress HCC growth through inhibiting PAK4/Raf/MEK/ERK pathway both in vitro and in vivo. Our study provides miRNomes of human liver and HCC and contributes to better understanding of the important deregulated miRNAs in HCC and liver diseases.
MicroRNAs (miRNAs) are small RNAs of 22 nucleotides length that do not hold the sequential information to transcribe proteins, but function as critical regulators of gene expression in multicellular and some unicellular eukaryotes.1 Pre-miRNAs undergo sequential processing by the ribonuclease III endonucleases Drosha and Dicer, leading to the mature 20- to 23-nucleotide species.2 These in turn are integrated into the RNA-induced silencing complex (RISC) and recognize their target genes by interacting with complementary sequences in the 3′ untranslated region of their messenger RNAs (mRNAs). In the case of perfect or nearly perfect pairing, the target mRNA becomes degraded, whereas imperfect pairing leads to translational repression of the latter.3 Importantly, an individual miRNA can regulate hundreds of transcripts and thus can coordinate complex networks of gene expression and subsequently induce global changes in cellular physiology and pathophysiology. Indeed, in addition to genetic and epigenetic abnormalities modifying oncogenes and tumor suppressor genes, deregulation of miRNAs has been shown to contribute to carcinogenesis of both solid and hematological malignancy.4, 5
In recent years, significant efforts were taken to identify miRNAs that regulate hepatocarcinogenesis. Altered expression patterns of miRNAs have been described in both rodent and human hepatocellular carcinoma (HCC) in studies using microarray technology or quantitative polymerase chain reaction (qPCR). In 2006, Murakami et al. reported on a panel of eight miRNAs that were significantly altered in HCC, comprising the miR-199 family, which was also down-regulated in their collective.6 In the following years, a whole kaleidoscope of other deregulated miRNAs were reported by different groups in the context of HCC.7 Furthermore, specific targets were linked to miRNAs deregulated in HCC, including genes involved in tumor metastasis such as focal adhesion kinase (targeted by miR-151),8, 9 cell-cycle–modulating proteins such as cyclin G1 (targeted by miR-122),10 or the cyclin-dependent kinase inhibitors CDKN1B/p27 and CDKN1C/p57 (targeted by miR-221).11 However, as the authors of the present article point out, the results obtained from these previous microarray- or qPCR-based studies had certain limitations. They were at least partially controversial and demonstrated a large interstudy variance. In addition, they mainly focused on the alterations of individual miRNAs, but were unable to determine the abundance of each miRNA in the background of the entire miRNome. Based on the hypothesis that a minimum threshold amount must be reached for miRNAs to exert their function,12 it seemed likely that the abundantly expressed miRNAs might be more important than those expressed at relatively low levels.
In the present study by Hou and coworkers from the Second Military Medical University of Shanghai, China, the authors applied the innovative massively parallel signature sequencing (MPSS) technology to carry out a comparative in-depth analysis of the miRNomes in normal liver tissues and HCC.13 This technique provides the unique possibility to identify the individual miRNome in-depth and thereby to reveal miRNA expression differences in relation to the individual miRNA abundances. Using this technique, the authors identified specific miRNAs that were most abundant in their collectives of normal liver, hepatitis-infected livers, and HCCs. Within this panel of miRNAs, miR-199a/b-3p was markedly decreased in HCC samples as compared with matched non-neoplastic liver tissues. The authors next validated these MPSS-based expression data in livers from different large and well-defined cohorts of patients with HCC by qPCR and correlated these data with clinical features. Strikingly, within the panel of miRNAs tested, only the expression of miR-199 was found to correlate with patient outcome, and low miR-199a/b-3p levels were identified as an independent predictor for both poor overall and tumor-free survival. Together with the well-known down-regulation of miR-199a/b-3p in nonasiatic HCC collectives,14 these data highlight the importance of miR-199a/b-3p expression in liver diseases and HCC.
In further experiments, the authors explored the functional significance of miR-199a/b-3p down-regulation in HCC. First, restoration of miR-199a/b-3p expression in HCC cell lines inhibited cell growth, induced apoptosis, and inhibited cell cycle progression, indicating that miR-199a/b-3p may function as a tumor suppressor in vitro. Moreover, infection of a human HCC-bearing nude mouse model (SMMC-LTNM model) with an adeno-associated virus 8 (AAV-8) vector system to overexpress miR-199a/b-3p led to inhibition of tumor growth and reduction of serum alpha-fetoprotein, even after a single tail vein injection.
As pointed out before, miRNAs can suppress transcription and translation of hundreds of target mRNAs. To identify the relevant targets of miR-199a/b-3p in the context of hepatocarcinogenesis, the authors followed different approaches, including in silico analysis of databases, gene enrichment, and ontology analysis. By combining these respective approaches, the authors could identify the mitogen-activated protein kinase (MAPK) signaling pathway as a potential target of miR-199a/b-3p. Of this pathway, only PAK4 (p21 protein [Cdc42/Rac]-activated kinase 4) contains putative miR-199a/b-3p target sites. Strikingly, expression of PAK4 could not only be down-regulated by miR-199a/b-3p transfection and overexpression, but high PAK4 protein levels also correlated with low miR-199a/b-3p expression in HCC samples. The pathophysiological significance of this finding was highlighted by the fact that intratumoral injection of cholesterol-conjugated small, interfering RNA against PAK4 led to inhibited tumor growth and reduced serum alpha-fetoprotein levels in the SMMC-LTNM model. On a molecular level, inhibition of miR-199a/b-3p or overexpression of PAK4 inhibited the activation of the Raf/MEK/ERK (extracellular signal-regulated kinase) cascade, which is believed to promote hepatocarcinogenesis in humans.14 Interestingly, besides PAK4, miR-199a/b-3p also targets signaling of c-met and mammalian target of rapamycin, and it seems likely that a deregulation in the whole network of these “pro-oncogenic” pathways rather than a down-regulation of a single gene contribute to the procarcinogenic effects of miR-199a/b-3p silencing in HCC (Fig. 1).
HCC represents the fifth most common cause for cancer-related death worldwide, and in some African or Asian countries, HCC is even the leading cause of cancer-related morbidity. Despite the clinical relevance and enormous efforts in both basic and clinical research spent on the development of novel systemic therapeutic options for patients with advanced HCC, results are still disappointing, considering that even the gold standard in treatment of those patients, Sorafenib (Nexavar), provides only a marginal survival benefit of less than 3 months.15 In this context, Hou and colleages identified the network of low miR-199a/b-3p expression, high PAK expression and consecutive activation of the Raf/MEK/ERK cascade as a new key player in development and progression of HCC. Notably, most of the patients analyzed in their study suffered from hepatitis B virus–associated HCC, and further studies are needed to clarify whether these data can be transferred one-to-one to HCC caused by other etiologies. Interestingly, the authors further verified an increase of miR-199a/b-3p in nontumorous but fibrotic tissues, which is in line with previous array-based data from mouse models of liver fibrosis and from patients with liver cirrhosis6, 16, 17 and suggests that this specific miRNA might functionally be involved in the processes driving the transition from fibrosis to HCC.
Finally, in this elegant article, the authors provided evidence that genetic delivery of miR-199a/b-3p might represent a promising option for pharmacological manipulation of tumor progression in patients with HCC. However, major obstacles have to be overcome on the way to a successful miRNA-based therapy. First, the optimal way for an effective and safe delivery of miRNA into cancer cells or the tumor microenvironment still remains unclear. They can be delivered by retroviruses, adenoviruses, or adeno-associated viruses or injected in cholesterol-modified form to the tissue of interest.18 The authors and other groups demonstrated the feasibility of both approaches in rodents and nonhuman primates. Second, the high number of potential targets for each individual miRNA may give rise to unexpected and fatal toxicity. If these concerns can be addressed, the promise of an miR-based anticancer therapy might become reality.