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Keywords:

  • apoptosis;
  • c-met;
  • cell cycle;
  • hepatocellular carcinoma;
  • metastasis;
  • microRNA;
  • PI3K-Akt;
  • Ras-Raf-Mek-Erk;
  • receptor tyrosine kinase;
  • transforming growth factor-β

Abstract

  1. Top of page
  2. Abstract
  3. MicroRNA: A tiny molecule with enormous impacts
  4. Involvements of microRNAs in HCC etiologic factors
  5. Regulation of signaling networks by microRNAs
  6. Clinical potentials of microRNAs
  7. Concluding remarks and future perspectives
  8. Acknowledgments
  9. References

MicroRNAs (miRNAs) are small non-coding RNAs of 19–23 nucleotides that negatively regulate gene expression through binding to the 3′-untranslated regions of target messenger RNAs (mRNAs). Although the miRNA family constitutes only a minor fraction of the human genome, they hold fundamental importance in diverse physiological and developmental processes due to their pleiotropic effects on the post-transcriptional regulation of many vital genes. This class of regulatory RNAs has also emerged as important players in carcinogenesis; most, if not all, cancer types have abnormal miRNA expression patterns. In hepatocellular carcinoma (HCC), miRNA dysregulation plays a key role in mediating the pathogenicity of several etiologic risk factors and, more importantly, they promote a number of cancer-inducing signaling pathways. Recent studies have also demonstrated their potential values in the clinical management of HCC patients as some miRNAs may be used as prognostic or diagnostic markers. The significance of miRNAs in liver carcinogenesis emphasizes their values as therapeutic targets, while technological advances in the delivery of miRNA has shed new possibilities for their use as novel therapeutic agents against HCC.


MicroRNA: A tiny molecule with enormous impacts

  1. Top of page
  2. Abstract
  3. MicroRNA: A tiny molecule with enormous impacts
  4. Involvements of microRNAs in HCC etiologic factors
  5. Regulation of signaling networks by microRNAs
  6. Clinical potentials of microRNAs
  7. Concluding remarks and future perspectives
  8. Acknowledgments
  9. References

In the past few decades, genome research has established the fundamental importance of genetic and epigenetic alterations of oncogenes and tumor suppressor genes (TSGs) in the initiation and progression of human neoplasms. The recent discovery of microRNA (miRNA) put forward an alternate regulatory element, in which the actions of miRNAs regulate cancer-inducing cellular genes post-transcriptionally.

Discovery of miRNA

The founding member of miRNA, lin-4, was discovered in the larval development of Caenorhabditis elegans in 1993.1 Nevertheless, the role of small RNA in gene expression regulation had to await the discovery of a second miRNA member, let-7, 7 years later.2 Pioneering studies further revealed let-7 as a negative regulator of the RAS oncogene in human tumor cells.3 This discovery soon aroused tremendous efforts into the research of cancer-related miRNAs. By now, miRNAs have been reported in a variety of organisms, ranging from viruses to mammals. To facilitate miRNA research, a miRNA registry (miRBase) has been established and is currently maintained by the University of Manchester.4 So far, 940 human miRNAs have been reported (miRBase release 15) and the list is still expanding.

Biogenesis and functions of miRNA

The family of miRNA constitutes about 1–3% of the human genome. Most miRNA genes are situated within the intergenic regions and have their own transcription units. About a quarter are located within exons or introns of other coding genes where their transcription is controlled by the host genes. MiRNAs can be transcribed as monocistronic transcripts or in polycistronic clusters; the latter involves several miRNAs situated on a single transcript being controlled by the same promoter (Fig. 1).

image

Figure 1. Micro-RNA Biogenesis. In the nucleus, miRNAs are transcribed as either monocistronic or polycistronic pri-miRNAs by Pol II. Pri-miRNAs cleaved by Drosha and Pasha to pre-miRNAs are exported to the cytoplasm by exportin 5. In the cytoplasm, pre-miRNAs are excised to double-stranded miRNA : miRNA* duplex of 20–23 nucleotides by Dicer. The miRNA duplex unwinds to single-stranded mature miRNA, and incorporates into RNA-induced silencing complex (RISC), which is composed of Argonaute proteins. The miRNA/RISC complex binds to the 3′-untranslated region (3′-UTR) of target cellular gene and negatively regulates gene expression with a mechanism depending on the complementarity between miRNA and its target mRNA. Perfect complementarity triggers mRNA degradation, while partial complementarity results in translational repression.

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In the nucleus, miRNA genes are transcribed as primary-miRNAs (pri-miRNAs) by RNA polymerase II (PolII). Structurally, pri-miRNAs consist of a 5′-7-methyl guanylate (m7G) cap, a characteristic imperfect stem-loop secondary structure, and a 3′-poly(A) tail. Pri-miRNAs are cleaved to precursor miRNAs (pre-miRNAs) of about 50–150 nucleotides by Drosha, an endoribonuclease III (RNAse III), and its cofactor RNA-binding protein Pasha (DGCR8). The pre-miRNAs are then exported to the cytoplasm by exportin 5. This is further excised to double-stranded duplices of 20–23 nucleotides by Dicer, an RNAse III enzyme. The miRNA duplex later separates into single-stranded mature miRNA, and incorporates into the RNA-induced silencing complex (RISC), which is composed of Argonaute proteins. This complex binds to the 3′-untranslated region (3′-UTR) of its target transcript and negatively regulates protein translation by a mechanism that depends on the complementarity between the miRNA and target messenger RNA. Partial complementarity results in translational repression, while complete complementarity triggers mRNA degradation.

An important feature of miRNA is that a single miRNA can regulate multiple target mRNAs. This pleiotropic property enables miRNAs to exert wide control on a network of genes. It has been demonstrated that overexpression of a single miRNA can downregulate over 100 mRNAs.5 Further, bioinformatic analysis predicts miRNAs can affect up to 30% of all human genes.6 It is therefore not surprising that miRNAs are involved in diverse physiological and developmental processes. These range from cell survival, differentiation, responses to external stress and morphogenesis. In cancer, an imbalance of miRNA expression was first described in B-cell chronic lymphocytic leukemia,7 and by now virtually all examined tumor types display abnormal miRNA expression patterns.

MiRNA and cancer

Studies on cancer miRNA have shown that they are often downregulated in tumor tissues, irrespective of cell type.8 This general phenomenon may be exemplified by the observation of common downregulation of Dicer, a key enzyme in the miRNA biogenesis, in many human neoplasms, including hepatocellular carcinoma (HCC),9 ovarian cancer,10 gastric cancer,11 and lung adenocarcinoma.12 In addition, reduced Dicer expression has been shown to correlate with shorter postoperative survival of patients with non-small-cell lung carcinoma.13 A study correlating the expression of precursor and mature miRNAs further showed that a vast number of miRNAs was transcribed but not processed to mature forms in cancer cell lines.14 Taken together, these findings seem to indicate that defective miRNA processing promotes transformation of normal cells to cancer, and that the miRNAome, as a whole, plays a critical role in tumor suppression.

This review will focus on the miRNA dysregulation in HCC. The first part summarizes the involvement of miRNAs in the pathogenicity of HCC risk factors. The second part highlights the relation of miRNAs to HCC-associated signaling pathways, which encompass the apoptotic pathway, cell cycle regulation, receptor tyrosine kinase (RTK)-mediated pathway, and transforming growth factor β (TGF-β) signaling. The final part explores the clinical value of miRNA as prognostic and diagnostic markers.

Involvements of microRNAs in HCC etiologic factors

  1. Top of page
  2. Abstract
  3. MicroRNA: A tiny molecule with enormous impacts
  4. Involvements of microRNAs in HCC etiologic factors
  5. Regulation of signaling networks by microRNAs
  6. Clinical potentials of microRNAs
  7. Concluding remarks and future perspectives
  8. Acknowledgments
  9. References

Hepatocellular carcinoma is a highly aggressive tumor that currently ranks the fifth most prevalent cancer worldwide. Although few studies have reported on promising treatment strategies for HCC, the dismal outcome remains unchanged; the median survival of most patients is still 6–9 months from diagnosis.15 Epidemiological studies have firmly established a number of etiologic risk factors in the development of HCC. These can be broadly divided into host factors and environmental factors. Host factors include male gender and genetic metabolic defects contributing to obesity, whereas environmental factors entail viral hepatitis (types B and C) infections, excessive chronic alcohol intake, dietary aflatoxin B1 exposure, and cigarette smoking.15 Complex interactions among these factors ultimately lead to chronic liver disease and/or liver cirrhosis, and the increased risk of HCC development.

Several studies have begun to examine for specific miRNA deregulation in hepatitis B virus (HBV)-related and hepatitis C virus (HCV)-related HCCs. While a microarray profiling study in 25 HCC specimens showed no significant difference in miRNA expression between HBV and HCV-associated cases,16 another complementary study measuring the expression of 188 miRNAs in 12 HBV-related and 14 HCV-related HCCs identified 19 miRNAs that could differentiate the HBV and HCV groups.17 Thirteen miRNAs exhibited a decreased expression in the HCV group (including miR-190, miR-134, miR-151), and six showed specific reduced expression in the HBV group (including miR-23a, miR-142-5p, miR-34c).17 Concordantly, several studies have also identified miRNAs that were differentially regulated by HBV or HCV. Transfection of the HCV genome resulted in downregulation of 10 miRNAs and upregulation of 23 miRNAs, amongst which elevated miR-193b expression was apparent.18 On the other hand, miR-96 was reported to be distinctively upregulated in HBV-associated HCC tumors.19 Interactions between host miRNAs and HCV have also been studied. MiR-122, a liver-specific miRNA, is abundantly expressed in liver tissues and accounts for 70% of the total liver miRNA population.20 Host miR-122 was found to accelerate ribosome binding to HCV RNA, which in turn stimulated viral translation.21 Functional inactivation of miR-122 could lead to 80% reduction of HCV RNA replication in HCC cell lines.22 In this connection, repression of miR-122 in HCC might represent a compensatory mechanism that confers resistance to HCV replication in HCC cells.22

Hepatocellular carcinoma predominantly affects men, with an incidence typically two to four times higher than in women.23 In an attempt to elucidate the role of miRNAs in this gender disparity, the expression profile of a panel of 17 frequently deregulated miRNAs was compared between male and female HCC patients.24 Elevated miR-18a expression appeared to be statistically associated with female gender (female to male ratio: 4.58). Reporter assay readouts later confirmed estrogen receptor-α (ERα) as a target of miR-18a. Since it is known that estrogen protects females from the development of HCC, it is plausible that miR-18a weakens the protective effects of estrogen by suppressing the translation of ERα, thereby increasing the risk of HCC development in women.24

Chronic heavy alcohol consumption is another risk factor in the development of HCC. In a miRNA microarray study, miR-126* was shown to be specifically downregulated in alcohol-related HCC.25 This downregulation, however, was not evident in non-tumoral tissues, implicating that miR-126* repression might be directly linked to alcohol-induced hepatocarcinogenesis.25

Obesity is becoming an important risk factor for HCC in recent years. Obese patients are prone to develop non-alcoholic fatty liver disease (NAFLD), which is deposition of fat in liver cells unrelated to alcohol consumption. The spectrum of NAFLD ranges from fatty liver, to non-alcoholic steatohepatitis (NASH), and finally cirrhosis, which predisposes to HCC development. MiRNAs have been shown to be involved in the pathogenesis of NASH. Unsaturated fatty acids have been shown to increase miR-21 expression, which affects phosphatase and tensin homolog (PTEN) expression and consequentially induces steatosis.26 The pathophysiological relevance of this phenomenon was further verified by the observation of an increased miR-21 level and PTEN downregulation in the livers of Wistar rats fed with a high-fat diet, and in human liver biopsies of patients with steatosis.26 In mice administered a choline-deficient and amino acid-defined (CDAA) diet that promoted NASH-induced hepatocarcinogenesis, microarray analysis identified 30 differential expressed miRNAs.27 Among these, miR-155 was consistently upregulated during the course of CDAA intake. In RAW 264.7 cells, miR-155 was reported to target CCAAT/enhancer binding protein beta (C/EBPβ),28 a transcription factor with tumor suppressive activity. Transfection of miR-155 readily decreased C/EBPβ expression and promoted cell viability in Hep3B and HepG2 cells.27 The results of these studies imply considerable importance for both miR-155 and miR-21 in NASH-associated HCC.

MiRNAs may also potentiate the actions of hepato-carcinogens. For instance, tamoxifen, an estrogen receptor antagonist commonly used in the clinical treatment of breast cancer, has been shown to induce HCC in rats.29 Long-term exposure of tamoxifen to female rodents perturbed miRNA expression and induced oncogenic miRNAs expression, including miR-17-92 cluster, miR-106a, and miR-34. These changes in miRNA could have predisposed to malignant liver transformation.29

Regulation of signaling networks by microRNAs

  1. Top of page
  2. Abstract
  3. MicroRNA: A tiny molecule with enormous impacts
  4. Involvements of microRNAs in HCC etiologic factors
  5. Regulation of signaling networks by microRNAs
  6. Clinical potentials of microRNAs
  7. Concluding remarks and future perspectives
  8. Acknowledgments
  9. References

Under normal physiological conditions, the balance between cell proliferation and programmed cell death is tightly regulated in order to maintain tissue homeostasis. Alteration in genes that play critical roles in cell division, cell death, and DNA repair tilts this balance and results in unrestrained cell proliferation that predisposes to cancer. MiRNAs have been closely associated with these cellular genes, and found to exert a critical role in regulating the complex signaling networks of liver carcinogenesis. A list of commonly dysregulated miRNAs in HCC tumors has been summarized in Table 1.

Table 1.  Common dysregulated MiRNAs identified in hepatocellular carcinoma (HCC)
miRNAConfirmed target(s)Involvement in cellular processesReferences
  1. ADAM, a disintegrin and metaloprotease; API-5, apoptosis inhibitor 5; CDK, cyclin-dependent kinase; DDIT4, DNA damage-inducible transcript 4; ER, estrogen receptor; FNDC3B, fibronectin type III domain containing 3B; PPP2R2A, protein phosphatase 2A subunit B; PTEN, phosphatase and tensin homolog; RhoGDIA, RhoGDP dissociation inhibitor; TIMP3, tissue inhibitors of metalloproteinases 3; ZEB, zinc finger E-box binding homeobox; n/a, not available.

Upregulation   
 miR-15bn/an/a24,30,31
 miR-18n/aProliferation16,30,32
 miR-18aERalpha24Proliferation24,31,33,34
 miR-19an/aProliferation30,33,35
 miR-21PTEN26,36,37Proliferation, anchorage-independent growth, apoptosis, migration, invasion19,24,30,32,35,36,38–41
 miR-34aNOTCH1,29 c- Met,42 E2F337Proliferation, migration, invasion30,36,41
 miR-93E2F133Proliferation30,33,34,41
 miR-96n/an/a38,40,41
 miR-106bE2F133Proliferation32,41,49,83
 miR-130bn/an/a24,30,32
 miR-151RhoGDIA43Migration, invasion30,40,43
 miR-182n/an/a30,38–40
 miR-183n/aApoptosis30,38,40
 miR-185n/an/a30,38,44
 miR-210n/an/a30,34,36,41
 miR-222PTEN,45 TIMP3,45 PPP2R2A38Migration, invasion19,24,30,31,34,36,38,40,41,45
 miR-221p57/Kip2,46 p27/Kip1,46 Bmf,47 PTEN,45 TIMP3,45 DDIT441Proliferation, colony formation, apoptosis, migration,24,30–32,36,40,41,45–48
 miR-224API-540Proliferation, apoptosis16,19,24,31,34,38,40,41
 miR-301n/an/a24,30,32,38,40
 miR-374n/an/a30,38,40
Down-regulation   
 let-7cc-Myc49,50Proliferation24,41,48
 let-7gtype I collagen α2,51 c-Myc52Proliferation, colony formation, migration30,44,48,51,52
 miR-101FOS,39 Mcl-I34Colony formation, apoptosis, migration, invasion, tumorigenicity31,32,34,39
 miR-122Cyclin G,48 Bcl-w,53 ADAM17,54 ADAM1055Proliferation, colony formation, apoptosis, migration, invasion, anchor-independent growth, drug resistance, angiogenesis, hepatic metastasis19,24,30,36,44,48,54,55
 miR-125an/an/a16,30,36,38
 miR-125bn/aProliferation31,34,36,38,44
 miR-126n/an/a17,30,44
 miR-139n/an/a32,38,40
 miR-145n/an/a24,30,38,40,48
 miR-148an/an/a30,39,44
 miR-150n/an/a41,47,72,84
 miR-195Cyclin D1,56 CDK6,56 E2F356Proliferation, colony formation16,24,30,34,38,48,56
 miR-199an/an/a24,32,36,38
 miR-199a-5pn/an/a16,30,32,34,38,48
 miR-199bn/an/a24,32,38,48
 miR-200bZEB1,37 ZEB237n/a32,38,48
 miR-214n/an/a30,32,38,40,48
 miR-223Stathmin130Proliferation24,30,32,34,48

Apoptotic pathway

Apoptosis is mediated through two main routes, namely the perturbation of mitochondria membrane permeability (intrinsic pathway) and the activation of death receptors (extrinsic pathway). Both pathways converge to induce the activation of caspases, which act as the final executioners of cell death (Fig. 2).

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Figure 2. Regulation of apoptosis by miRNA in hepatocellular carcinoma (HCC). MiRNA dysregulation in HCC allows cancer cells to evade apoptosis and permits them to survive in adverse conditions. (a) The intrinsic pathway involves the Bcl-2 family, which governs mitochondrial membrane permeability. Several members of the Bcl-2 family are regulated by miRNAs. An imbalance between the pro- and anti-apoptotic members of Bcl-2 family results in the release of cytochrome c from the mitochondria into cytoplasm and elicits apoptosis. (b) In the extrinsic pathway, apoptosis is activated upon ligands binding to the death receptors. Overexpression of miR-221-222 cluster has been shown to confer resistance to tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL)-induced apoptosis in HCC cells. On the other hand, activation of Fas receptor induced miR-491-5p expression, which can sensitize HCC cells to TNF-α-induced apoptosis. PARP, poly (ADP-ribose) polymerase.

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Intrinsic pathway

A number of miRNAs have been shown to be involved in mitochondria-mediated apoptosis; they act by targeting the Bcl-2 family. In this connection, pro-apoptotic members Bmf47 and Bim33 could be inhibited by miR-221 and miR-25, respectively. In HCC, elevated levels of miR-221 and miR-25 are found in 50–70% of patients. Functionally, miR-221 overexpression conferred resistance to anoikis in HCC cell lines. In vitro studies further revealed that miR-221 silencing increased the number of dead cells in non-adherent culture; the process was accompanied by induction of Bmf expression and caspase 3 cleavage.47

MiR-25 is a member of the miR-106b-25 cluster (which encompasses miR-106b/miR-93/miR-25). In primary HCC tumors, miR-25 upregulation correlated inversely with Bim expression.33 Knockdown of miR-25 decreased HCC cell viability and anchorage independent growth, although these inhibitory effects were more profound when combined with the other two members of the cluster, miR-93 and miR-106b.33 Conversely, anti-apoptotic members Mcl-1 and Bcl-w could be targeted by miR-10134 and miR-122,53 respectively. Furthermore, miR-29 has been shown to repress two anti-apoptotic Bcl-2 family members, Mcl-1 and Bcl-2.57 Restoration of miR-101 or miR-29 expression could sensitize HCC cells to serum starvation- or chemotherapeutic drug-induced apoptosis; it also abolished tumorigenicity in a xenograft mouse model.34,57 Downregulation of these miRNAs in HCC cells enabled them to evade apoptosis and survive in nutrient-depleted and hypoxic environments.

Extrinsic pathway

There are relatively few studies investigating the association of miRNAs and death-receptor mediated apoptosis in HCC. In a murine model with Fas receptor activation, induction of miR-491-5p was suggested from miRNA profiling.58In vitro study demonstrated that miR-491-5p sensitized HCC cells to TNF-α-induced apoptosis, possibly through decreasing the levels of miR-491-5p predicted targets, including α-fetoprotein, heat shock protein-90 and nuclear factor-kappa B (NF-κB).58 Though many of the direct target associations await further confirmation by reporter assays, this study signified the involvement of miR-491-5p in the crosstalk between Fas receptor- and TNF-α mediated apoptosis. In HCC, overexpression of the miR-221-222 cluster has been shown to negatively regulate PTEN and tissue inhibitors of metalloproteinases 3 (TIMP3), as well as to increase resistance to TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis as revealed from Annexin V and caspase3/7 activity analyses.45 Moreover, silencing of PTEN or TIMP3 phenocopied the effects of miR-221-222 cluster on TRAIL resistance and was accompanied by a reduction of caspases activation and poly (ADP-ribose) polymerase (PARP) cleavage.45 This further corroborated the anti-apoptotic role of miR-221-222.

Cell cycle regulators

Several miRNAs have been reported to play a role in the control of cell cycle (Fig. 3), in particular at the G1/S checkpoint. MiR-26a targets G1/S cyclins (both cyclin D2 and E2) in murine liver cancer,59 whereas miR-195 targets multiple genes (including cyclin D1, CDK6, and E2F3) of the G1/S transition in primary HCC.56 Both miR-26a and miR-195 have been found to be frequently downregulated in HCC, where they have been shown to cooperate in overcoming the G1/S cell cycle blockade through repression of E2F transcription.56,59 In addition, miR-221 in HCC has also been reported to target CDKN1B/p27/Kip1 and CDKN1C/p57/Kip2, both of which are CDK inhibitors.46 Taken together with the above described anti-apoptotic properties,45,47 these findings indicate that miR-221 can simultaneously affect multiple oncogenic pathways in HCC.

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Figure 3. Regulation of cell cycle by miRNA. Several miRNAs take part in the control of cell cycle in hepatocellular carcinoma (HCC). (a) MiR-223 has been shown to target stathmin1 (STMN1), which is a regulatory protein involved in destabilizing the microtubules and plays a role in maintaining the dynamic nature of mitotic spindles at the G2/M transition. (b) MiR-26a can target both cyclin D2 and E2 while miR-195 has been shown to negatively regulate multiple genes, including cyclin D1, CDK6, and E2F3. In addition, miR-221 has been reported to target CDK inhibitors, p27 and p57. Members of miR-106b-25 cluster, miR-106b and miR-93, can repress E2F1 expression, whereby preventing excessive E2F1 accumulation that may paradoxically result in apoptosis.

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Intriguingly, both miR-106b and miR-93 can also target E2F1.33 While this may seemingly contradict the oncogenic property of miR-106b-25 cluster in HCC, it is plausible that since high levels of E2F1 can cause apoptosis,60 upregulation of miR-106b and miR-93 may serve to prevent excessive accumulation of E2F1. Thus, together with miR-25 targeting of pro-apoptotic Bim protein,33 members of the miR-106b-25 cluster might coordinate the curtailment of apoptosis in HCC.

Our previous work demonstrated that miR-223 is commonly downregulated in HCC.30 Re-expression of miR-223 revealed a consistent inhibitory effect on cell viability. We also showed that miR-223 could target stathmin1 (STMN1), which is a microtubule destabilizer that sequesters tubulin for depolymerization and affects microtubule assembly.30 As microtubules have an important role in the segregation of metaphase chromosomes during mitosis, it is probable that miR-223 has a function in regulating the G2/M transition.

RTK-mediated pathways

Receptor tyrosine kinases are cell surface receptors that transmit extracellular stimuli to intracellular signaling responses. RTK transduction activates a series of proteins and elicits downstream signaling cascades that eventually alter transcription of a myriad of genes involved in cellular processes, such as proliferation, apoptosis and survival (Fig. 4).

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Figure 4. Involvement of miRNA in the receptor tyrosine kinase (RTK)-mediated pathways. MiRNAs are involved in a number of RTK-mediated pathways in hepatocellular carcinoma (HCC), including the Ras-Raf-MEK-ERK cascade and PI3K-Akt pathway. MiR-101 and let-7c can target c-Fos and c-myc, respectively. In HCC, phosphatase and tensin homolog (PTEN) can be negatively regulated by miR-21 and miR-222. Moreover, miR-222 has been shown to target PP2A. MiR-1, miR-34a and miR-23b have been reported to target c-Met and the expressions of these miRNAs are commonly downregulated in HCC. EGF, epidermal growth factor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor.

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c-Met-activated signaling

Hepatocyte growth factor receptor (HGFR; also known as c-Met) is a RTK, whose oncogenic function has been extensively documented in HCC.61,62 It has been shown that c-Met can be regulated by a number of miRNAs. miR-1-1, which is often silenced by methylation in HCC tissues and cell lines, represents one such miRNA that could target c-Met.63 Forced expression of miR-1-1 in HepG2 cells decreased cell viability and colony forming ability, with evident G2/M arrest and apoptosis.63In vitro studies in HepG2 cells also demonstrated that miR-34a could reduce both c-Met RNA and protein levels, and strong inhibitory effects on HepG2 cell migratory and invasive properties.42 c-Met could also be suppressed by miR-23b, which, when expressed, could repress HCC cell migration and proliferative capabilities.64 Moreover, evidence obtained on miR-23b also showed that it could target urokinase-type plasminogen activator (uPA), an enzyme involved in the proteolytic cascade and promotion of liver metastases.64

PI3K-Akt

Various studies have linked miRNAs to the phosphoinositide 3-kinase (PI3K)-Akt pathway. In this regard, our previous study uncovered that miR-222 could target the protein phosphatase 2A subunit B (PPP2R2A), which is the regulatory subunit B of the protein phosphatase 2A (PP2A).38 Loss-of-function study of miR-222 demonstrated decreased phospho-Akt levels and suppressed HCC cell motility through reduced filopodia formation.38 Together with miR-221, miR-222 could also repress PTEN, a well-characterized antagonist of PI3K activity and negative regulator of the PI3K/Akt signaling path.45 These two miRNAs could also simultaneously target TIMP, which, if repressed, could confer positive advantages on HCC cell migration, invasion, cell growth and resistance to apoptosis.45 Besides the miR-222-221 cluster, luciferase reporter assay also confirmed target association between miR-21 and PTEN in HCC.26,36 Inhibition of miR-21 in cultured HCC cells increased PTEN expression, and this corresponded to considerably decreased tumor cell proliferation, migration and invasion through the PTEN negative modulation of Akt and focal adhesion kinase (FAK) activities.26,36 In contrast to the miR-222, miR-221 and miR-21 oncogenic functions, miR-125b acted as a tumor-suppressor miRNA in HCC by decreasing Akt phosphorylation and suppressing HCC cell proliferation.31

Ras-Raf-Mek-Erk cascade and downstream transcription factors

MiRNAs targeting the Ras-Raf-Mek-Erk cascade, together with upregulated immediate early genes (IEGs) noted during liver regeneration, have been reported in HCC and other cancer types.3,39,65 In HCC, miR-101 has been shown to target c-Fos, a component of AP-1, and this affected tumor spread.39 On the other hand, members of the let-7 family are known to repress vital oncogenes in the Ras-Raf-Mek-Erk cascade, such as K-Ras in lung cancer3 and c-Myc in Burkitt lymphoma cells.65 Sustained activation of peroxisome proliferator-activated receptor alpha (PPAR-α) could lead to the development of HCC.66 In a rodent model of activated PPAR-α, liver oncogenesis was found to be promoted through inhibition of let-7c expression.49 It was also shown that loss of let-7c targeting on c-Myc led to subsequent increase in the expression of the miR-17-92 cluster.49 Knockdown of oncogenic miR-17-92 polycistron (which encompasses miR-17/miR-19a/miR-20/miR-92) caused 50% reduction in both hepatocyte proliferation and anchorage-independent growth, implicating its involvement in the malignant transformation of hepatocytes.35 Together, these observations reveal a let-7c signaling cascade critical for the PPAR-α-induced liver tumorigenesis.

TGF-β signaling pathway

Transforming growth factor β plays a paradoxical role in cancer (Fig. 5). In HCC, TGF-β has been shown to induce specific miRNA expression.35,67–69 MiRNA profiling of TGF-β-stimulated HCC cells revealed upregulation of 12 miRNAs and downregulation of nine miRNAs.67 An induction of the miR-23a-27a-24 cluster, as confirmed by quantitative PCR, was directly influenced by Small mother against decapentaplegic (SMAD) 2, 3, and 4. Transfection of the miR-23a-27a-24 cluster into Huh7 cells attenuated the anti-proliferative and pro-apoptotic effects of TGF-β. These findings would suggest a novel mechanism through which TGF-β induced specific miRNA expression to escape from its suppressive effects.67

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Figure 5. Involvement of miRNA in transforming growth factor-β (TGF-β) signaling. TGF-β has been shown to induce specific miRNA expression in hepatocellular carcinoma (HCC). TGF-β-treated HCC cells can induce expression of miR-23a-27a-24 cluster, which in turn attenuates the anti-proliferative and pro-apoptotic effects of TGF-β in an auto-regulatory manner. Furthermore, TGF-β can increase expression of miR-181b, which targets TIMP3 and promotes proliferation, migration, invasion and tumorigenicity of HCC cells.

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In another study of mice fed with CDAA diet, miRNA expression profiling of HCC tumors showed significant upregulation of miR-181b and miR-181d.68 Increased expression of hepatic TGF-β and downstream mediators SMAD2, 3 and 4 correlated with elevated miR-181b/d. Exposure of hepatic cells to TGF-β augmented the level of precursor and mature miR-181b, whereas silencing of Smad4 significantly reversed this induction, implicating the direct involvement of TGF-β signaling pathway in the miR-181 expression. Functionally, repressed TIMP3, a validated target of miR-181, enhanced metallopeptidase 2 (MMP2) and MMP9 activities and promoted growth, clonogenic survival, motility of HCC cells and tumorigenicity in vivo.68 In addition, members of the miR106b-25 and miR-17-92 clusters have been shown to abrogate cell cycle arrest and apoptosis induced by TGF-β signaling.69 Since these miRNAs have physiological functions in the control of cell cycle and apoptosis, in line with the early reports it is probable that miRNA-based homeostatic mechanisms can be seized by cancer cells to resist the TGF-β tumor suppressive actions.69

Metastasis pathways

MiRNA expression profiling has identified miRNAs that underscore the metastatic potential of HCC. One of these studies reported on a 20-miRNA metastasis signature based on the profiling of 131 HCC patients. This signature significantly predicted HCC tissues with venous metastases from solitary tumors.44 Further substantiation of its independent predictive value was obtained in an independent cohort of 110 cases.44

Some miRNAs function as suppressors of the metastasis process. For instance, the liver-specific miR-122 was significantly downregulated in liver cancers, particularly in those with intrahepatic metastases.54 Restoration of miR-122 significantly reduced migration, invasion and anchorage-independent growth of Mahlavu and SK-Hep1 cells. The role of miR-122 in metastasis was further validated in both subcutaneous and orthotopic murine models of liver cancer. MiR-122 significantly decreased the tumor volume and suppressed metastasis by reducing blood vessel formation. Integrated analysis combining expression array and in silico prediction revealed that miR-122 had 45 potential mRNA targets. Thirty-two of these cellular genes have been validated by reporter assays, and shown to be involved in functional ontologies, mainly “cell morphology” and “cell movement”. Subsequent experiments illustrated that silencing of one such gene, a disintegrin and metalloprotease 17 (ADAM17), showed similar phenotypes to that when miR-122 was restored.54 Furthermore, the level of let-7g was found to be significantly lowered in metastatic HCC compared with metastasis-free HCC.51 Transfection of let-7g significantly inhibited HCC cell migration but not invasion. While in silico prediction showed that 11 collagen genes contained 3′-UTR binding sites for let-7g, type I collagen α2 (COL1A2) was experimentally validated as a direct target. Moreover, addition of COL1A2 counteracted the inhibitory effect of let-7g on cell migration. It would therefore be suggested that let-7g suppressed HCC metastasis, at least in part, through targeting COL1A2.51

A number of miRNAs, including miR-9, miR-143, miR-30d and miR-151, have been shown to function as promoters of HCC metastasis. In this respect, miR-9 was found to be commonly upregulated in metastatic HCC tumors.70 MiR-9 inhibition reduced SK-Hep1 cell invasion with re-expression of E-cadherin, an epithelial adhesion molecule.70 Downregulation of E-cadherin decreases the strength of cellular adhesion resulting in an increase in cell motility, which is characteristic of epithelial-mesenchymal transition (EMT).71 On the other hand, miR-143 favored the invasive and metastatic behavior of liver tumor cells in both in vitro and in vivo models, an effect exerted by targeting the fibronectin type III domain containing 3B (FNDC3B).72 MiR-143 induced by NF-κB was found to be significantly upregulated in metastatic HBV-related HCC tumors.72 Moreover, marked upregulation of miR-30d in metastatic HCC has been shown to enhance migration and invasion of HCC cells, and to promote intrahepatic and distal pulmonary metastasis in an orthotopic mouse model.73 Luciferase activity assays have confirmed the target association of miR-30d with Galphai2 (GNAI2), a G protein α subunit that inhibits adenylate cyclase activity.73

MiR-151, located within the intronic region of FAK on Chr8q24.3, was found to be frequently overexpressed in HCC.43 Upregulation of miR-151 promoted HCC cell migration and invasion both in vitro and in vivo by targeting RhoGDP dissociation inhibitor (RhoGDIA). The RhoGTPases, including RhoA, Rac1, Cdc42, are important regulators of cell migration. RhoGDIA binds to the GDP-bound form of RhoGTPase and prevents the activation of metastasis-promoting Rho pathway.43

Signaling pathways discussed in the previous sections, including c-Met, PI3K-Akt and TGF-β pathways also take part in the migratory and invasion processes of HCC. Table 2 summarizes the miRNAs reported in the metastatic potential of HCC.

Table 2.  A summary of miRNAs reported in association with metastatic behavior of hepatocellular carcinoma (HCC)
MicroRNADysregulation in metastatic HCCConfirmed targetsReferences
  1. ADAM17, a disintegrin and metaloprotease 17; FNDC3B, fibronectin type III domain containing 3B; mTOR, mammalian target of rapamycin; PPP2R2A, protein phosphatase 2A subunit B; PTEN, phosphatase and tensin homolog; RhoGDIA, RhoGDP dissociation inhibitor; TIMP3, tissue inhibitors of metalloproteinases 3; uPA, urokinase-type plasminogen activator; n/a, not available.

miR-9UpregulatedE-cadherin70
miR-17-5pUpregulatedn/a31,74
miR-21UpregulatedPTEN26,48
miR-25UpregulatedBim31,33
miR-30dUpregulatedGalphai231,73
miR-92Upregulatedn/a31
miR-93Upregulatedn/a31
miR-99bUpregulatedn/a31
miR-106aUpregulatedn/a31
miR-106bUpregulatedn/a31
miR-143UpregulatedFNDC3B72
miR-151UpregulatedRhoGDIA43
miR-181bUpregulatedTIMP368
miR-185Upregulatedn/a44
miR-207Upregulatedn/a44
miR-219-1Upregulatedn/a44
miR-221UpregulatedPTEN, TIMP345
miR-222UpregulatedPPP2R2A, PTEN, TIMP338,45
miR-296Upregulatedn/a31
miR-338Upregulatedn/a44
let-7gDownregulatedtype I collagen α244,51
miR-1-2Downregulatedn/a44
miR-9-2Downregulatedn/a44
miR-15aDownregulatedn/a44
miR-19aDownregulatedn/a44
miR-23bDownregulatedc-Met, uPA64
miR-30aDownregulatedn/a44
miR-30c-1Downregulatedn/a44
miR-30eDownregulatedn/a44
miR-34aDownregulatedc-Met42,44,75
miR-98Downregulatedn/a31
miR-122DownregulatedADAM1731,44,54
miR-124a-2Downregulatedn/a44
miR-125b-2Downregulatedn/a44
miR-126Downregulatedn/a44
miR-148aDownregulatedn/a44
miR-148bDownregulatedn/a44
miR-181aDownregulatedOsteopontin76
miR-194Downregulatedn/a44
miR-199a-3pDownregulatedmTOR, c-Met77
miR-202Downregulatedn/a31
miR-210Downregulatedn/a31
miR-424Downregulatedn/a31
miR-516-3pDownregulatedn/a31

Clinical potentials of microRNAs

  1. Top of page
  2. Abstract
  3. MicroRNA: A tiny molecule with enormous impacts
  4. Involvements of microRNAs in HCC etiologic factors
  5. Regulation of signaling networks by microRNAs
  6. Clinical potentials of microRNAs
  7. Concluding remarks and future perspectives
  8. Acknowledgments
  9. References

The prognostication of HCC patients remains a major challenge for clinicians, and emerging evidence indicates that the outcome varies with underlying molecular pathology.78 To this end, profiling of miRNA expression have been informative in cancer risk prediction, diagnosis, prognosis, and responses to therapy.79–82

Polymorphisms in miRNAs and their targets have proved useful in predicting cancer risk. For instance, a G>C polymorphism in miR-146a precursor (rs2910164) predicted HCC development.80 This polymorphism located in the stem region opposite the mature miR-146a resulted in a change from G : U pair to C : U mismatch. Male individuals with GG genotype were twofold more susceptible to HCC compared with those with CC genotype. In this context, the G-allelic miR-146a precursor displayed an increased production of mature miR-146a than the C-allelic one. Further investigations revealed that miR-146a significantly promoted cell proliferation and colony formation in NIH/3T3 cells.80

Another study also reported that polymorphisms present in the 3′-UTRs of mRNAs could affect miRNA binding. A polymorphism with insertion of ‘TTCA’ in the 3′UTR of interleukin-1 alpha (IL1A) (rs3783553) disrupted miR-122 and miR-378 binding, resulting in an increased expression of IL1A.81 The presence of this polymorphism likely contributed to HCC susceptibility as IL1A affects tumor growth, invasiveness, and also the interactions between malignant cells and the host's immune system.81

Hepatocellular carcinoma tissues secrete various tumor-related proteins into the blood, and these may serve as circulating biomarkers for early diagnosis of HCC. Although serum alpha fetoprotein (AFP) is widely used as a biomarker for HCC, recent research proposed new molecular biomarkers that are more specific.78 Serum miR-500 level has been shown to be commonly elevated in HCC patients and values returned to normal after surgical treatment.82 Certain miRNAs are associated with HCC subtypes, implying their potential in patient stratification for prognosis. Apart from the 20-miRNA metastasis signature that was shown to be associated with patient survival,44 another study demonstrated a set of 19-miRNAs correlated with HCC disease outcome.32 Proteins involved in cell cycle progression have been predicted to be targets of this 19-miRNA signature.32 It is also noteworthy that upregulation of the miR-221-222 cluster38,47 and downregulation of miR-26,83 miR-29,57 miR-12284 and miR-125b31 have been validated in independent studies to be associated with poor prognosis and shorter disease-free survival of HCC patients.

Aside from their clinical usefulness as diagnostic and prognostic markers, miRNAs have also been shown to influence sensitivity of tumors to chemotherapeutic drugs. Depletion of miR-181b could sensitize SK-Hep1 cells to doxorubicin, implicating that antagomirs targeting miR-181b might be useful in increasing drug efficacy.68 Furthermore, patients with low miR-26 expression showed better responses to interferon therapy.83 MiR-122 restoration also sensitized HCC cells to doxorubicin,84 as well as multi-kinase inhibitor Sorafenib,55 indicating miR-122 mimic in combination with anticancer drugs could be a promising therapeutic regimen against HCC.

Concluding remarks and future perspectives

  1. Top of page
  2. Abstract
  3. MicroRNA: A tiny molecule with enormous impacts
  4. Involvements of microRNAs in HCC etiologic factors
  5. Regulation of signaling networks by microRNAs
  6. Clinical potentials of microRNAs
  7. Concluding remarks and future perspectives
  8. Acknowledgments
  9. References

The discovery of miRNA has substantially altered conventional concepts on gene regulation and this class of tiny non-coding RNAs has emerged as novel players in the control of genes expression in cancer. Studies on miRNA profiling have revealed characteristic miRNA dysregulations in different tumor types and unveiled the importance of miRNA involvement in carcinogenesis. Functional and target association studies on dysregulated miRNAs in HCC have enabled us to gain a more comprehensive understanding on their roles in the oncogenic signaling pathways. Nevertheless, the mechanistic cause of miRNA dysregulation remains to be fully explored and the characterization of many of the differential expressed miRNAs and their molecular and cell biological targets is still in progress.

From a clinical point of view, preliminary studies have highlighted the value of miRNAs in the diagnosis and prognosis of HCC. Differential expressed miRNA patterns may be useful in the stratification of patients to predict disease outcome and recurrence. Recently, there has been considerable interest in the potential use of antagomiRs as anticancer agents, especially for HCC because of their predominant uptake by the liver and enhanced hepatic stability.85 Technological advances have also demonstrated the feasibility of utilizing adeno-associated virus to administer miRNAs in a murine HCC model.59 In addition, treatment of chimpanzees with locked nucleic acid (LNA)-modified oligonucleotide suppressed HCV infection.86 The success of miRNA delivery in these animal models may hold promise in the further development of miRNA targeted therapy, which may represent a new avenue for the treatment of HCC.

References

  1. Top of page
  2. Abstract
  3. MicroRNA: A tiny molecule with enormous impacts
  4. Involvements of microRNAs in HCC etiologic factors
  5. Regulation of signaling networks by microRNAs
  6. Clinical potentials of microRNAs
  7. Concluding remarks and future perspectives
  8. Acknowledgments
  9. References