RASAL1 is a potent regulator of hepatic stellate cell activity and liver fibrosis.

Liver fibrosis, leading to cirrhosis and liver failure, can occur after chronic liver injury. The transition of hepatic stellate cells (HSCs) from quiescent cells into proliferative and fibrogenic cells is a central event in liver fibrosis. Here, we show that RAS protein activator like-1 (RASAL1), a RAS-GTPase-activating protein, which switches off RAS activity, is significantly decreased during HSC activation, and that HSC activation can be antagonized by forced expression of the RASAL1 protein. We demonstrate that RASAL1 suppresses HSC proliferation by regulating the Ras-MAPK pathway, and that RASAL1 suppresses HSC fibrogenic activity by regulating the PKA-LKB1-AMPK-SRF pathway by interacting with angiotensin II receptor, type 1. We also show that RASAL1-deficient mice are more susceptible to liver fibrosis. These data demonstrate that deregulated RASAL1 expression levels and the affected downstream intracellular signaling are central mediators of perpetuated HSC activation and fibrogenesis in the liver.

Repression of MicroRNA Function Mediates Inflammation-associated Colon Tumorigenesis.

BACKGROUND & AIMS: Little is known about the mechanisms by which chronic inflammation contributes to carcinogenesis, such as the development of colon tumors in patients with inflammatory bowel diseases. Specific microRNA (miRNAs) can function as suppressors or oncogenes, and widespread alterations in miRNA expression have been associated with tumorigenesis. We studied whether alterations in miRNA function contribute to inflammation-associated colon carcinogenesis. METHODS: We studied the effects of inflammatory cytokines, such as tumor necrosis factor, interleukin-1α (IL1A), and IL1ß (IL1B), on miRNA function, measured by activity of reporter constructs containing miRNA-binding sites in their 3' untranslated regions, in human 293T embryonic kidney, Caco-2, HT29, and HCT116 colon carcinoma cells, as well as dicer+/+ and dicer-/-, and Apobec3+/+ and Apobec3-/- mouse embryonic fibroblasts. Cells were analyzed by immunoblots, immunohistochemistry, and flow cytometry. We generated transgenic mice expressing reporter constructs regulated by LET7B, MIR122, and MIR29b response elements; some mice were given injections of miRNA inhibitors (anti-MIR122 or anti-LET7B), a negative control, or tumor necrosis factor. Liver tissues were collected and analyzed by immunoblotting. Reporter mice were given azoxymethane followed by dextran sulfate sodium to induce colitis and colon tumors; some mice were given the ROCK inhibitor fasudil along with these agents (ROCK inhibitors increase miRNA function). Colon tissues were collected and analyzed by immunohistochemistry, immunoblots, and fluorescence microscopy. RESULTS: Incubation of cell lines with inflammatory cytokines reduced the ability of miRNAs to down-regulate expression from reporter constructs; dicer was required for this effect, so these cytokines relieve miRNA-dependent reductions in expression. The cytokines promoted degradation of APOBEC3G, which normally promotes miRNA loading into argonaute 2-related complexes. Mice with colitis had reduced miRNA function, based on increased expression of reporter genes. Administration of fasudil to mice did not reduce the severity of colitis that developed but greatly reduced the numbers of colon tumors formed (mean 2 tumors/colon in mice given fasudil vs 9 tumors/colon in mice given control agent). We made similar observations in IL10-deficient mice. CONCLUSIONS: We found inflammatory cytokines to reduce the activities of miRNAs. In mice with colitis, activities of miRNAs are reduced; administration of an agent that increases miRNA function prevents colon tumor formation in these mice. This pathway might be targeted to prevent colon carcinogenesis in patients with inflammatory bowel diseases.

MicroRNAs and liver disease.

The biological roles of microRNAs (miRNAs) have been extensively studied. miRNA122 represents more than half of the miRNAs expressed in the liver and has various physiological and pathological functions, which include enhancing hepatitis virus replication, regulating lipid metabolism and suppressing hepatocellular carcinoma. miRNAs, whether globally or individually, have been linked with hepatocarcinogenesis. Furthermore, some miRNAs have been shown to be involved in the pathogenesis of nonalcoholic steatohepatitis. Using nucleotide-based strategies, these miRNAs may be developed as potential therapeutic targets. Because changes in miRNA expression can be measured in sera, they may be used as non-invasive biomarkers if they correctly reflect the pathological state of the liver. In this review, we show the biological roles of representative miRNAs in liver disease and discuss the current issues that remain to be clarified for future clinical applications.

Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7.

The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection.

Circulating RNAs as new biomarkers for detecting pancreatic cancer.

Pancreatic cancer remains difficult to treat and has a high mortality rate. It is difficult to diagnose early, mainly due to the lack of screening imaging modalities and specific biomarkers. Consequently, it is important to develop biomarkers that enable the detection of early stage tumors. Emerging evidence is accumulating that tumor cells release substantial amounts of RNA into the bloodstream that strongly resist RNases in the blood and are present at sufficient levels for quantitative analyses. These circulating RNAs are upregulated in the serum and plasma of cancer patients, including those with pancreatic cancer, compared with healthy controls. The majority of RNA biomarker studies have assessed circulating microRNAs (miRs), which are often tissue-specific. There are few reports of the tumor-specific upregulation of other types of small non-coding RNAs (ncRNAs), such as small nucleolar RNAs and Piwi-interacting RNAs. Long ncRNAs (lncRNAs), such as HOTAIR and MALAT1, in the serum/plasma of pancreatic cancer patients have also been reported as diagnostic and prognostic markers. Among tissue-derived RNAs, some miRs show increased expression even in pre-cancerous tissues, and their expression profiles may allow for the discrimination between a chronic inflammatory state and carcinoma. Additionally, some miRs and lncRNAs have been reported with significant alterations in expression according to disease progression, and they may thus represent potential candidate diagnostic or prognostic biomarkers that may be used to evaluate patients once detection methods in peripheral blood are well established. Furthermore, recent innovations in high-throughput sequencing techniques have enabled the discovery of unannotated tumor-associated ncRNAs and tumor-specific alternative splicing as novel and specific biomarkers of cancers. Although much work is required to clarify the release mechanism, origin of tumor-specific circulating RNAs, and selectivity of carrier complexes, and technical advances must also be achieved, such as creating a consensus normalization protocol for quantitative data analysis, circulating RNAs are largely unexplored and might represent novel clinical biomarkers.

ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.

The reduced expression levels and functional impairment of global miRNAs are related to various human diseases, including cancers. However, relatively little is known about how global miRNA function may be upregulated. Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors. The regulation of miRNA function by ROCK inhibitors is mediated, at least in part, by poly(A)-binding protein-interacting protein 2 (PAIP2), which enhances poly(A)-shortening of miRNA-targeted mRNAs and leads to global upregulation of miRNA function. In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A. Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription. ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.

Novel therapeutic approaches for hepatitis B virus covalently closed circular DNA.

Hepatitis B virus (HBV) infection is a major global health problem. Although current therapies, such as the use of nucleos(t)ide analogs, inhibit HBV replication efficiently, they do not eliminate covalently closed circular DNA (cccDNA), which persists in hepatocyte nuclei. As HBV cccDNA is a viral transcription template, novel therapeutic approaches to directly target HBV cccDNA are necessary to completely eradicate persistent HBV infections. HBV cccDNA levels in HBV-infected human liver cells are extremely low; thus, more reliable and simple measurement methods are needed to correctly monitor their levels during therapeutic treatment. Although reverse transcription-polymerase chain reaction or Southern blot procedures are currently used in research studies, these methods are not completely reliable and are also time-consuming and labor-intensive. Genome editing technologies, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which are designed to target specific DNA sequences, represent highly promising potential therapeutic tools. In particular, the CRISPR/Cas9 system is an easily customizable sequence-specific nuclease with high flexibility and may be the most feasible approach to target HBV cccDNA. Further research to develop easier, safer, and more effective protocols should be pursued.

Decreased miR122 in hepatocellular carcinoma leads to chemoresistance with increased arginine.

Reduced expression of microRNA122 (miR122), a liver-specific microRNA, is frequent in hepatocellular carcinoma (HCC). However, its biological significances remain poorly understood. Because deregulated amino acid levels in cancers can affect their biological behavior, we determined the amino acid levels in miR122-silenced mouse liver tissues, in which intracellular arginine levels were significantly increased. The increased intracellular arginine levels were through upregulation of the solute carrier family 7 (SLC7A1), a transporter of arginine and a direct target of miR122. Arginine is the substrate for nitric oxide (NO) synthetase, and intracellular NO levels were increased in miR122-silenced HCC cells, with increased resistance to sorafenib, a multikinase inhibitor. Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib. Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib. These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset.

Diagnostic and therapeutic application of noncoding RNAs for hepatocellular carcinoma.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression posttranscriptionally, targeting thousands of messenger RNAs. Long noncoding RNAs (lncRNAs), another class of noncoding RNAs, have been determined to be also involved in transcription regulation and translation of target genes. Since deregulated expression levels or functions of miRNAs and lncRNAs in hepatocellular carcinoma (HCC) are frequently observed, clinical use of noncoding RNAs for novel diagnostic and therapeutic applications in the management of HCCs is highly and emergently expected. Here, we summarize recent findings regarding deregulated miRNAs and lncRNAs for their potential clinical use as diagnostic and prognostic biomarkers of HCC. Specifically, we emphasize the deregulated expression levels of such noncoding RNAs in patients' sera as noninvasive biomarkers, a field that requires urgent improvement in the clinical surveillance of HCC. Since nucleotide-based strategies are being applied to clinical therapeutics, we further summarize clinical and preclinical trials using oligonucleotides involving the use of miRNAs and small interfering RNAs against HCC as novel therapeutics. Finally, we discuss current open questions, which must be clarified in the near future for realistic clinical applications of these new strategies.

The flavonoid apigenin inhibits hepatitis C virus replication by decreasing mature microRNA122 levels.

Despite recent progress in the development of direct-acting antivirals against hepatitis C virus (HCV), chronic HCV infection remains an important health burden worldwide. MicroRNA122 (miR122), a liver-specific microRNA (miRNA), positively regulates HCV replication, and systemic application of antisense oligonucleotides against miR122 led to the long-lasting suppression of HCV viremia in human clinical trials. Here, we report that apigenin, a flavonoid and an inhibitor of maturation of a subset of miRNAs, inhibits HCV replication in vitro. Apigenin decreased the expression levels of mature miR122 without significantly affecting cell growth. Because supplementation of synthesized miR122 oligonucleotides or overexpression of constitutively active TRBP blocked these effects, the inhibitory effects of apigenin on HCV replication seemed to be dependent on the reduction of mature miR122 expression levels through inhibition of TRBP phosphorylation. Thus, apigenin intake, either through regular diet or supplements, may decrease HCV replication in chronically infected patients.

Specific delivery of microRNA93 into HBV-replicating hepatocytes downregulates protein expression of liver cancer susceptible gene MICA.

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). To date, the lack of efficient in vitro systems supporting HBV infection and replication has been a major limitation of HBV research. Although primary human hepatocytes support the complete HBV life cycle, their limited availability and difficulties with gene transduction remain problematic. Here, we used human primary hepatocytes isolated from humanized chimeric uPA/SCID mice as efficient sources. These hepatocytes supported HBV replication in vitro. Based on analyses of mRNA and microRNA (miRNA) expression levels in HBV-infected hepatocytes, miRNA93 was significantly downregulated during HBV infection. MiRNA93 is critical for regulating the expression levels of MICA protein, which is a determinant for HBV-induced HCC susceptibility. Exogenous addition of miRNA93 in HBV-infected hepatocytes using bionanocapsules consisted of HBV envelope L proteins restored MICA protein expression levels in the supernatant. These results suggest that the rescued suppression of soluble MICA protein levels by miRNA93 targeted to HBV-infected hepatocytes using bionanocapsules may be useful for the prevention of HBV-induced HCC by altering deregulated miRNA93 expression.

The role of microRNAs in hepatocarcinogenesis: current knowledge and future prospects.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression post-transcriptionally through complementary base pairing with thousands of messenger RNAs. Although the precise biological functions of individual miRNAs are still unknown, miRNAs are speculated to play important roles in diverse biological processes through fine regulation of their target gene expression. A growing body of data indicates the deregulation of miRNAs during hepatocarcinogenesis. In this review, we summarize recent findings regarding deregulated miRNA expression and their possible target genes in hepatocarcinogenesis, with emphasis on inflammation-related hepatocarcinogenesis. Because miRNA-based strategies are being applied to clinical therapeutics, precise knowledge of miRNA functions is crucial both scientifically and clinically. We discuss the current open questions from these points of view, which must be clarified in the near future.

Unique haploinsufficient role of the microRNA-processing molecule Dicer1 in a murine colitis-associated tumorigenesis model.

A widespread downregulated expression of microRNAs (miRNAs) is commonly observed in human cancers. Similarly, deregulated expression of miRNA-processing pathway components, which results in the reduction of global miRNA expression, may also be associated with tumorigenesis. Here, we show that specific ablation of Dicer1 in intestinal epithelial cells accelerates intestinal inflammation-associated tumorigenesis. This effect was apparent only when a single copy of Dicer1 was deleted, but not with complete Dicer1 ablation. DICER expression and subsequent mature miRNA levels were inversely correlated with the number of intact Dicer1 alleles. Because the expression levels of DICER were retained in tumors and its surrounding tissues even after induction of colitis-associated tumors, the effects of Dicer1 deletion were cell-autonomous. Although the expression levels of representative oncogenes and tumor suppressor genes were in most cases inversely correlated with the expression levels of DICER, some genes were not affected by Dicer1 deletion. Thus, deregulating the delicate balance between the expression levels of tumor-promoting and -suppressive genes may be crucial for tumorigenesis in this unique haploinsufficient case.

Regulation of the expression of the liver cancer susceptibility gene MICA by microRNAs.

Hepatocellular carcinoma (HCC) is a threat to public health worldwide. We previously identified the association of a single nucleotide polymorphism (SNP) at the promoter region of the MHC class I polypeptide-related sequence A (MICA) gene with the risk of hepatitis-virus-related HCC. Because this SNP affects MICA expression levels, regulating MICA expression levels may be important in the prevention of HCC. We herein show that the microRNA (miR) 25-93-106b cluster can modulate MICA levels in HCC cells. Overexpression of the miR 25-93-106b cluster significantly suppressed MICA expression. Conversely, silencing of this miR cluster enhanced MICA expression in cells that express substantial amounts of MICA. The changes in MICA expression levels by the miR25-93-106b cluster were biologically significant in an NKG2D-binding assay and an in vivo cell-killing model. These data suggest that the modulation of MICA expression levels by miRNAs may be a useful method to regulate HCCs during hepatitis viral infection.

The flavonoid apigenin improves glucose tolerance through inhibition of microRNA maturation in miRNA103 transgenic mice.

Polyphenols are representative bioactive substances with diverse biological effects. Here, we show that apigenin, a flavonoid, has suppressive effects on microRNA (miRNA) function. The effects were mediated by impaired maturation of a subset of miRNAs, probably through inhibition of the phosphorylation of TRBP, a component of miRNA-generating complexes via impaired mitogen-activated protein kinase (MAPK) Erk activation. While glucose intolerance was observed in miRNA103 (miR103)-overexpressing transgenic mice, administration of apigenin improved this pathogenic status likely through suppression of matured miR103 expression levels. These results suggest that apigenin may have favorable effects on the pathogenic status induced by overexpression of miRNA103, whose maturation is mediated by phosphorylated TRBP.

Inhibition of microRNA122 decreases SREBP1 expression by modulating suppressor of cytokine signaling 3 expression.

While inhibition of microRNA122 (miR122) function in vivo results in reduced serum cholesterol and fatty acid levels, the molecular mechanisms underlying the link between miR122 function and lipid metabolism remains unclear. Because the expression of SREBP1, a central transcription factor involved in lipid metabolism, is known to be increased by suppressor of cytokine signaling 3 (SOCS3) expression, and because we previously found that SOCS3 expression is regulated by miR122, in this study, we examined the correlation between miR122 status and the expression levels of SOCS3 and SREBP1. SREBP1 expression decreased when SOCS3 expression was reduced by miR122 silencing in vitro. Conversely, SREBP1 expression in miR122-silenced cells was restored by enforced expression of SOCS3. Such correlations were observed in human liver tissues with different miR122 expression levels. These signaling links may explain one of the molecular mechanisms linking inhibition of miR122 function or decreased expression of miR122 to decreased fatty acid and cholesterol levels, in the inhibition of miR122 function, or in pathological status in chronic liver diseases.

MicroRNA-140 acts as a liver tumor suppressor by controlling NF-κB activity by directly targeting DNA methyltransferase 1 (Dnmt1) expression.

UNLABELLED: MicroRNAs (miRNAs) are small RNAs that regulate the expression of specific target genes. While deregulated miRNA expression levels have been detected in many tumors, whether miRNA functional impairment is also involved in carcinogenesis remains unknown. We investigated whether deregulation of miRNA machinery components and subsequent functional impairment of miRNAs are involved in hepatocarcinogenesis. Among miRNA-containing ribonucleoprotein complex components, reduced expression of DDX20 was frequently observed in human hepatocellular carcinomas, in which enhanced nuclear factor-κB (NF-κB) activity is believed to be closely linked to carcinogenesis. Because DDX20 normally suppresses NF-κB activity by preferentially regulating the function of the NF-κB-suppressing miRNA-140, we hypothesized that impairment of miRNA-140 function may be involved in hepatocarcinogenesis. DNA methyltransferase 1 (Dnmt1) was identified as a direct target of miRNA-140, and increased Dnmt1 expression in DDX20-deficient cells hypermethylated the promoters of metallothionein genes, resulting in decreased metallothionein expression leading to enhanced NF-κB activity. MiRNA-140-knockout mice were prone to hepatocarcinogenesis and had a phenotype similar to that of DDX20 deficiency, suggesting that miRNA-140 plays a central role in DDX20 deficiency-related pathogenesis. CONCLUSION: These results indicate that miRNA-140 acts as a liver tumor suppressor, and that impairment of miRNA-140 function due to a deficiency of DDX20, a miRNA machinery component, could lead to hepatocarcinogenesis.

Current status of miRNA-targeting therapeutics and preclinical studies against gastroenterological carcinoma.

Expanding knowledge about the crucial roles of microRNAs (miRNAs) in human diseases has led to the idea that miRNAs may be novel, promising therapeutic targets against various pathological conditions. The recent success of a human clinical trial using anti-miR-122 oligonucleotides against chronic hepatitis C virus has paved the way for this approach. In this review, we summarize briefly the current status of clinical trials of miRNA-targeting therapy and several representative preclinical trials against hepato-gastrointestinal carcinoma. In addition, we describe the currently available technologies for modification and delivery of oligonucleotides, which are essential in providing efficient, specific and safe approaches to targeting miRNAs.

Silencing of microRNA-122 enhances interferon-α signaling in the liver through regulating SOCS3 promoter methylation.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. Although novel drugs against HCV are under development, the current standard therapy consists principally of interferon (IFN). To improve the response to IFN treatment by enhancing interferon-stimulated response element (ISRE)-mediated gene transcription, we screened 75 microRNAs highly expressed in hepatocytes for their ability to modulate ISRE activity. Overexpression of microRNA-122 (miR122) significantly suppressed ISRE activity. Conversely, silencing of miR122 function enhanced IFN-induced ISRE activity, by decreasing expression of suppressor of cytokine signaling 3 (SOCS3). This decrease in SOCS3 level was not mediated by microRNA target gene suppression, but rather by enhanced methylation at SOCS3 gene promoter. Taken together, our data, along with the fact that antisense oligonucleotides of miR122 also directly inhibit HCV replication, suggest that a combination therapy comprising IFN and silencing of miR122 function may be a promising therapeutic option in the near future.

A miRNA machinery component DDX20 controls NF-κB via microRNA-140 function.

Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide, but the molecular mechanisms in tumorigenesis remain largely unknown. Previously, a DEAD-box protein DDX20, a component of microRNA-containing ribonucleoprotein complexes, was identified as a liver tumor suppressor candidate in an oncogenomics-based in vivo RNAi screen. However, the molecular mechanisms were unknown. Here, we show that deficiency of DDX20 results in the enhancement of NF-κB activity, a crucial intracellular signaling pathway closely linked with hepatocarcinogenesis. While DDX20 normally suppresses NF-κB activity by regulating NF-κB-suppressing miRNA-140 function, this suppressive effect was lost in DDX20-deficient cells. The impairment of miRNA function due to DDX20 deficiency appears to be miRNA species-specific at the point of loading miRNAs into the RNA-induced silencing complex. These results indicate that DDX20 deficiency enhances NF-κB activity by impairing the NF-κB-suppressive action of microRNAs, and suggest that dysregulation of the microRNA machinery components may also be involved in pathogenesis in various human diseases.