Upregulation of RelB in the miR-122 knockout mice contributes to increased levels of proinflammatory chemokines/cytokines in the liver and macrophages.

OBJECTIVE: MicroRNA-122 (miR-122) is the most abundant miRNA in the liver and it plays an important role in regulating liver metabolism and tumor formation. Previous studies also reveal an anti-inflammatory function of miR-122; however, relatively little is known about the mechanisms by which miR-122 suppresses inflammation. This study aims to search the effect of miR-122 on proinflammatory chemokines/cytokines production in mice. METHODS: Quantitative real-time PCR, Western blot analysis, and ELISA were performed to examine gene expression. TargetScan, miRanda, and microT v3.0 were used to search for possible miR-122 target sites in the 3'-untranslated regions (3'-UTR) of candidate genes. Luciferase reporter assay and site-directed mutagenesis were applied to verify miR-122 target sequences. LPS was applied to peritoneal macrophages and mice to evaluate inflammatory response. RESULTS: The expression of proinflammatory chemokines, including Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10, and Relb in the livers of miR-122 knockout (KO) mice was increased. We identified Relb as a direct miR-122 target. Overexpressing RelB in the mouse liver increased the expression of Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10. Peritoneal macrophages from miR-122 KO mice had a higher level of RelB, and they showed a stronger NF-κB activation and more TNF-α and IL-6 secretion after LPS stimulation. Overexpression of RelB in a macrophage cell line augmented LPS-induced TNF-α and IL-6 production. miR-122 KO mice showed a greatly increased mortality rate and generated a stronger and lasting inflammatory response to LPS. CONCLUSIONS: Deletion of miR-122 caused an upregulation of proinflammatory chemokines and RelB in the liver. Increased RelB may contribute to increases in these chemokine in the liver. Intriguingly, deletion of miR-122 also enhanced the sensitivity of macrophages and mice to LPS. Our results reveal that reducing RelB expression is a new mechanism by which miR-122 regulates inflammation.

Pten Haplodeficiency Accelerates Liver Tumor Growth in miR-122a-Null Mice via Expansion of Periportal Hepatocyte-Like Cells.

This study aimed to shed light on the molecular and cellular mechanisms responsible for initiation and progression of liver malignancies by examining the role of phosphatase and tensin homolog on chromosome 10 (Pten) in liver tumor progression in miR-122a (Mir122a)-null mice. We generated and monitored liver tumor initiation in Mir122a-null Pten heterozygous (Mir122a-/-;Pten+/- and Mir122a-/-;Alb-Cre;Ptenfx/+) mice and compared the results with those in Mir122a-/- mice. Both Mir122a-/-;Pten+/- and Mir122a-/-;Alb-Cre;Ptenfx/+ mice developed visible liver tumor nodules at 6 months of age. In premalignant livers of Mir122a-/-;Pten+/- mice, decreased PTEN and increased phosphorylated AKT were specifically observed in periportal cells, associated with inflammatory and fibrotic microenvironments. Furthermore, IL-1ß and tumor necrosis factor-α levels significantly increased in Mir122a-/-;Pten+/- premalignant livers at 6 months of age. Oval cells expressing A6, epithelial cell adhesion molecule, keratin (K) 8, K19, and SRY (sex determining region Y)-box 9 (SOX9) were present in both Mir122a-/- and Mir122a-/-;Pten+/- livers. Interestingly, a hybrid hepatocyte-like population with intermediate levels of K8, HNF4α, and SOX9 was located proximally to the oval cells in Mir122a-/-;Pten+/- livers. Lineage-tracing experiments revealed that these intermediate levels of K8 hepatocyte-like cells may be the cells of origin for Mir122a-/-;Pten+/- liver tumors. These findings suggest that inflammatory microenvironments in the periportal area of Mir122a-null mice may locally cause Pten down-regulation and expand tumor-initiating cells, causing hepatocellular carcinoma.

MicroRNA-224 down-regulates Glycine N-methyltransferase gene expression in Hepatocellular Carcinoma.

Glycine N-methyltransferase (GNMT) is a tumor suppressor for HCC. It is down-regulated in HCC, but the mechanism is not fully understood. MicroRNA-224 (miR-224) acts as an onco-miR in HCC. This study is the first to investigate miR-224 targeting the coding region of GNMT transcript. The GNMT-MT plasmid containing a miR-224 binding site silent mutation of the GNMT coding sequence can escape the suppression of miR-224 in HEK293T cells. Expression of both exogenous and endogenous GNMT was suppressed by miR-224, while miR-224 inhibitor enhanced GNMT expression. miR-224 counteracts the effects of GNMT on the reduction of cell proliferation and tumor growth. The levels of miR-224 and GNMT mRNA showed a significant inverse relationship in tumor specimens from HCC patients. Utilizing CCl4-treated hepatoma cells and mice as a cell damage of inflammatory or liver injury model, we observed that the decreased expression levels of GNMT were accompanied with the elevated expression levels of miR-224 in hepatoma cells and mouse liver. Finally, hepatic AAV-mediated GNMT also reduced CCl4-induced miR-224 expression and liver fibrosis. These results indicated that AAV-mediated GNMT has potential liver protection activity. miR-224 can target the GNMT mRNA coding sequence and plays an important role in GNMT suppression during liver tumorigenesis.

Flux balance analysis predicts Warburg-like effects of mouse hepatocyte deficient in miR-122a.

The liver is a vital organ involving in various major metabolic functions in human body. MicroRNA-122 (miR-122) plays an important role in the regulation of liver metabolism, but its intrinsic physiological functions require further clarification. This study integrated the genome-scale metabolic model of hepatocytes and mouse experimental data with germline deletion of Mir122a (Mir122a-/-) to infer Warburg-like effects. Elevated expression of MiR-122a target genes in Mir122a-/-mice, especially those encoding for metabolic enzymes, was applied to analyze the flux distributions of the genome-scale metabolic model in normal and deficient states. By definition of the similarity ratio, we compared the flux fold change of the genome-scale metabolic model computational results and metabolomic profiling data measured through a liquid-chromatography with mass spectrometer, respectively, for hepatocytes of 2-month-old mice in normal and deficient states. The Ddc gene demonstrated the highest similarity ratio of 95% to the biological hypothesis of the Warburg effect, and similarity of 75% to the experimental observation. We also used 2, 6, and 11 months of mir-122 knockout mice liver cell to examined the expression pattern of DDC in the knockout mice livers to show upregulated profiles of DDC from the data. Furthermore, through a bioinformatics (LINCS program) prediction, BTK inhibitors and withaferin A could downregulate DDC expression, suggesting that such drugs could potentially alter the early events of metabolomics of liver cancer cells.

miR-122-mediated translational repression of PEG10 and its suppression in human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC), a primary liver malignancy, is the most common cancer in males and fourth common cancer in females in Taiwan. HCC patients usually have a poor prognosis due to late diagnosis. It has been classified as a complex disease because of the heterogeneous phenotypic and genetic traits of the patients and a wide range of risk factors. Micro (mi)RNAs regulate oncogenes and tumor suppressor genes that are known to be dysregulated in HCC. Several studies have found an association between downregulation of miR-122, a liver-specific miRNA, and upregulation of paternally expressed gene 10 (PEG10) in HCC; however, the correlation between low miR-122 and high PEG10 levels still remains to be defined and require more investigations to evaluate their performance as an effective prognostic biomarker for HCC. METHODS: An in silico approach was used to isolate PEG10, a potential miR-122 target implicated in HCC development. miR-122S binding sites in the PEG10 promoter were evaluated with a reporter assay. The regulation of PEG10 by miR-122S overexpression was examined by quantitative RT-PCR, western blotting, and immunohistochemistry in miR-122 knockout mice and liver tissue from HCC patients. The relationship between PEG10 expression and clinicopathologic features of HCC patients was also evaluated. RESULTS: miR-122 downregulated the expression of PEG10 protein through binding to 3'-untranslated region (UTR) of the PEG10 transcript. In miR-122 knockout mice and HCC patients, the deficiency of miR-122 was associated with HCC progression. The expression of PEG10 was increased in 57.3 % of HCC as compared to paired non-cancerous tissue samples. However, significant upregulation was detected in 56.5 % of patients and was correlated with Okuda stage (P = 0.05) and histological grade (P = 0.001). CONCLUSIONS: miR-122 suppresses PEG10 expression via direct binding to the 3'-UTR of the PEG10 transcript. Therefore, while PEG10 could not be an ideal diagnostic biomarker for HCC but its upregulation in HCC tissue still has predictive value for HCC prognosis.

Evaluation of the medicinal herb Graptopetalum paraguayense as a treatment for liver cancer.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third most common cause of cancer-related death worldwide. Sorafenib is the only drug for patients with advanced-stage hepatocellular carcinoma (HCC) that has been shown to confer a survival benefit to patients with HCC; however, it has many side effects. Thus, alternate therapeutic strategies with improved safety and therapeutic efficacy for the management of HCC should be developed. METHODS AND FINDINGS: We demonstrate that an extract of Graptopetalum paraguayense (GP) down-regulated the expression levels of several onco-proteins, including AURKA, AURKB, and FLJ10540, in HCC cells. To isolate the active components in the GP extracts, we prepared extracts fractions and assessed their effects on the expression of onco-proteins in HCC cells. The fraction designated HH-F3 was enriched in active ingredients, exhibited cytotoxic effects, and suppressed the expression of the onco-proteins in HCC cells. The structure of the main active compound in HH-F3 was found to be similar to that of the proanthocyanidin compounds derived from Rhodiola rosea. In addition, a distinct new compound rich in 3, 4, 5-trihydroxy benzylic moieties was identified in the HH-F3 preparations. Mechanistic studies indicated that HH-F3 induced apoptosis in HCC cells by promoting the loss of mitochondrial membrane potential and the production of reactive oxygen species. HH-F3 also enhanced PTEN expression and decreased AKT phosphorylation at Ser473 in a concentration-dependent manner in HCC cells. Moreover combination of GP or HH-F3 and sorafenib synergistically inhibits the proliferation of Huh7 cells. The treatment of a rat model with diethylnitrosamine (DEN)-induced liver cancer with extracts of GP and HH-F3 decreased hepatic collagen contents and inhibited tumor growth. CONCLUSIONS: These results indicate that GP extracts and HH-F3 can protect the liver by suppressing tumor growth; consequently, these compounds could be considered for the treatment of HCC.

Gluconeogenesis, lipogenesis, and HBV replication are commonly regulated by PGC-1α-dependent pathway.

PGC-1α, a major metabolic regulator of gluconeogenesis and lipogenesis, is strongly induced to coactivate Hepatitis B virus (HBV) gene expression in the liver of fasting mice. We found that 8-Br-cAMP and glucocorticoids synergistically induce PGC-1α and its downstream targets, including PEPCK and G6Pase. Also, HBV core promoter activity was synergistically enhanced by 8-Br-cAMP and glucocorticoids. Graptopetalum paraguayense (GP), a herbal medicine, is commonly used in Taiwan to treat liver disorders. Partially purified fraction of GP (named HH-F3) suppressed 8-Br-cAMP/glucocorticoid-induced G6Pase, PEPCK and PGC-1α expression and suppressed HBV core promoter activity. HH-F3 blocked HBV core promoter activity via inhibition of PGC-1α expression. Ectopically expressed PGC-1α rescued HH-F3-inhibited HBV surface antigen expression, HBV mRNA production, core protein levels, and HBV replication. HH-F3 also inhibited fatty acid synthase (FASN) expression and decreased lipid accumulation by down-regulating PGC-1α. Thus, HH-F3 can inhibit HBV replication, gluconeogenesis and lipogenesis by down-regulating PGC-1α. Our study indicates that targeting PGC-1α may be a therapeutic strategy for treatment of HBV infections. HH-F3 may have potential use for the treatment of chronic hepatitis B patients with associated metabolic syndrome.

Integrated analyses to reconstruct microRNA-mediated regulatory networks in mouse liver using high-throughput profiling.

BACKGROUND: MicroRNAs (miRNAs) simultaneously target many transcripts through partial complementarity binding, and have emerged as a key type of post-transcriptional regulator for gene expression. How miRNA accomplishes its pleiotropic effects largely depends on its expression and its target repertoire. Previous studies discovered thousands of miRNAs and numerous miRNA target genes mainly through computation and prediction methods which produced high rates of false positive prediction. The development of Argonaute cross-linked immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) provides a system to effectively determine miRNA target genes. Likewise, the accuracy of dissecting the transcriptional regulation of miRNA genes has been greatly improved by chromatin immunoprecipitation of the transcription factors coupled with sequencing (ChIP-Seq). Elucidation of the miRNA target repertoire will provide an in-depth understanding of the functional roles of microRNA pathways. To reliably reconstruct a miRNA-mediated regulatory network, we established a computational framework using publicly available, sequence-based transcription factor-miRNA databases, including ChIPBase and TransmiR for the TF-miRNA interactions, along with miRNA-target databases, including miRTarBase, TarBase and starBase, for the miRNA-target interactions. We applied the computational framework to elucidate the miRNA-mediated regulatory network in the Mir122a⁻/⁻ mouse model, which has an altered transcriptome and progressive liver disease. RESULTS: We applied our computational framework to the expression profiles of miRNA/mRNA of Mir122a⁻/⁻ mutant mice and wild-type mice. The miRNA-mediated network involves 40 curated TFs contributing to the aberrant expression of 65 miRNAs and 723 curated miRNA target genes, of which 56% was found in the differentially-expressed genes of Mir122a--mice. Hence, the regulatory network disclosed previously-known and also many previously-unidentified miRNA-mediated regulations in mutant mice. Moreover, we demonstrate that loss of imprinting at the chromosome 12qF1 region is associated with miRNA overexpression in human hepatocellular carcinoma and stem cells, suggesting initiation of precancerous changes in young mice deficient in miR-122. A group of 9 miRNAs was found to share miR-122 target genes, indicating synergy between miRNAs and target genes by way of multiplicity and cooperativity. CONCLUSIONS: The study provides significant insight into miRNA-mediated regulatory networks. Based on experimentally verified data, this network is highly reliable and effective in revealing previously-undetermined disease-associated molecular mechanisms. This computational framework can be applied to explore the significant TF-miRNA-miRNA target interactions in any complex biological systems with high degrees of confidence.

Large-scale investigation of human TF-miRNA relations based on coexpression profiles.

Noncoding, endogenous microRNAs (miRNAs) are fairly well known for regulating gene expression rather than protein coding. Dysregulation of miRNA gene, either upregulated or downregulated, may lead to severe diseases or oncogenesis, especially when the miRNA disorder involves significant bioreactions or pathways. Thus, how miRNA genes are transcriptionally regulated has been highlighted as well as target recognition in recent years. In this study, a large-scale investigation of novel cis- and trans-elements was undertaken to further determine TF-miRNA regulatory relations, which are necessary to unravel the transcriptional regulation of miRNA genes. Based on miRNA and annotated gene expression profiles, the term "coTFBS" was introduced to detect common transcription factors and the corresponding binding sites within the promoter regions of each miRNA and its coexpressed annotated genes. The computational pipeline was successfully established to filter redundancy due to short sequence motifs for TFBS pattern search. Eventually, we identified more convinced TF-miRNA regulatory relations for 225 human miRNAs. This valuable information is helpful in understanding miRNA functions and provides knowledge to evaluate the therapeutic potential in clinical research. Once most expression profiles of miRNAs in the latest database are completed, TF candidates of more miRNAs can be explored by this filtering approach in the future.

MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis.

MicroRNA-122 (miR-122), which accounts for 70% of the liver's total miRNAs, plays a pivotal role in the liver. However, its intrinsic physiological roles remain largely undetermined. We demonstrated that mice lacking the gene encoding miR-122a (Mir122a) are viable but develop temporally controlled steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). These mice exhibited a striking disparity in HCC incidence based on sex, with a male-to-female ratio of 3.9:1, which recapitulates the disease incidence in humans. Impaired expression of microsomal triglyceride transfer protein (MTTP) contributed to steatosis, which was reversed by in vivo restoration of Mttp expression. We found that hepatic fibrosis onset can be partially attributed to the action of a miR-122a target, the Klf6 transcript. In addition, Mir122a(-/-) livers exhibited disruptions in a range of pathways, many of which closely resemble the disruptions found in human HCC. Importantly, the reexpression of miR-122a reduced disease manifestation and tumor incidence in Mir122a(-/-) mice. This study demonstrates that mice with a targeted deletion of the Mir122a gene possess several key phenotypes of human liver diseases, which provides a rationale for the development of a unique therapy for the treatment of chronic liver disease and HCC.

Analysis of NQO1 polymorphisms and p53 protein expression in patients with hepatocellular carcinoma.

NAD(P)H: quinone oxidoreductase 1 (NQO1), a cytosolic enzyme which catalyzes the two-electron reduction of quinone compounds, has been suggested to prevent the generation of semiquinone free radicals and reactive oxygen species, thus protecting cells from oxidative damage. However, the enzymatic activity of NQO1 strongly depends on the individual genetic polymorphism of the NQO1 gene. A common NQO1 polymorphism is a C to T transition at position 609, which results in an inactive enzyme. Recent studies showed that NQO1 is an important enzyme for stabilizing p53 protein, which is involved in anti-tumorigenesis. Thus, the lack of enzymatic activity in the homozygous C609T NQO1 polymorphism may play a pivotal role in tumor development. This study aimed to investigate the relationship between C609T NQO1 polymorphism and p53 expression in human hepatocellular carcinoma (HCC). Genotyping of NQO1 was performed on 100 HCC specimens by PCR-RFLP analysis. In addition, NQO1 and p53 protein expression in HCC samples at different TNM stages was determined by immunohistochemistry. Our data showed that (1) the frequency of C609T NQO1 was significantly increased in TNM stage III HCC patients; (2) no significant association was found between p53 expression and C609T polymorphism of NQO1 gene; and (3) a tumor/non-tumor (T/N) ratio > 1.27 of NQO1 expression revealed by real-time qPCR analyses was positively correlated with poorer survival in patients with tumors >5 cm, suggesting that an increase of NQO1 expression may be an indicator of advanced tumor progression. This study provides important information about NQO1 genotypes and its expression to HCC tumor development and progression.

MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma.

UNLABELLED: MicroRNAs (miRNAs), which are inhibitors of gene expression, participate in diverse biological functions and in carcinogenesis. In this study, we show that liver-specific microRNA-122 (miR-122) is significantly down-regulated in liver cancers with intrahepatic metastasis and negatively regulates tumorigenesis. Restoration of miR-122 in metastatic Mahlavu and SK-HEP-1 cells significantly reduced in vitro migration, invasion, and anchorage-independent growth as well as in vivo tumorigenesis, angiogenesis, and intrahepatic metastasis in an orthotopic liver cancer model. Because an inverse expression pattern is often present between an miRNA and its target genes, we used a computational approach and identified multiple miR-122 candidate target genes from two independent expression microarray datasets. Thirty-two target genes were empirically verified, and this group of genes was enriched with genes regulating cell movement, cell morphology, cell-cell signaling, and transcription. We further showed that one of the miR-122 targets, ADAM17 (a disintegrin and metalloprotease 17) is involved in metastasis. Silencing of ADAM17 resulted in a dramatic reduction of in vitro migration, invasion, in vivo tumorigenesis, angiogenesis, and local invasion in the livers of nude mice, which is similar to that which occurs with the restoration of miR-122. CONCLUSION: Our study suggests that miR-122, a tumor suppressor microRNA affecting hepatocellular carcinoma intrahepatic metastasis by angiogenesis suppression, exerts some of its action via regulation of ADAM17. Restoration of miR-122 has a far-reaching effect on the cell. Using the concomitant down-regulation of its targets, including ADAM17, a rational therapeutic strategy based on miR-122 may prove to be beneficial for patients with hepatocellular carcinoma.

miRNAMap 2.0: genomic maps of microRNAs in metazoan genomes.

MicroRNAs (miRNAs) are small non-coding RNA molecules that can negatively regulate gene expression and thus control numerous cellular mechanisms. This work develops a resource, miRNAMap 2.0, for collecting experimentally verified microRNAs and experimentally verified miRNA target genes in human, mouse, rat and other metazoan genomes. Three computational tools, miRanda, RNAhybrid and TargetScan, were employed to identify miRNA targets in 3'-UTR of genes as well as the known miRNA targets. Various criteria for filtering the putative miRNA targets are applied to reduce the false positive prediction rate of miRNA target sites. Additionally, miRNA expression profiles can provide valuable clues on the characteristics of miRNAs, including tissue specificity and differential expression in cancer/normal cell. Therefore, quantitative polymerase chain reaction experiments were performed to monitor the expression profiles of 224 human miRNAs in 18 major normal tissues in human. The negative correlation between the miRNA expression profile and the expression profiles of its target genes typically helps to elucidate the regulatory functions of the miRNA. The interface is also redesigned and enhanced. The miRNAMap 2.0 is now available at http://miRNAMap.mbc.nctu.edu.tw/.

DDX3, a DEAD box RNA helicase with tumor growth-suppressive property and transcriptional regulation activity of the p21waf1/cip1 promoter, is a candidate tumor suppressor.

DDX3 is a DEAD box RNA helicase with diverse biological functions. Using colony formation assay, our results revealed that DDX3 inhibited the colony formation ability of various tumor cells, and this inhibition might be due to a reduced growth rate caused by DDX3. Additionally, we identified p21(waf1/cip1), a cyclin-dependent kinase inhibitor, as a target gene of DDX3, and the up-regulation of p21(waf1/cip1) expression accounted for the colony-suppressing activity of DDX3. Moreover, DDX3 exerted its transactivation function on p21(waf1/cip1) promoter through an ATPase-dependent but helicase-independent mechanism, and the four Sp1 sites located within the -123 to -63 region, relative to the transcription start site of p21(waf1/cip1) promoter, were essential for the response to DDX3. Furthermore, DDX3 interacted and cooperated with Sp1 to up-regulate the promoter activity of p21(waf1/cip1). To determine the relevance of DDX3 in clinical cancers, the expression profile of DDX3 in various tumors was also examined. A declined expression of DDX3 mRNA and protein was found in approximately 58% to 73% of hepatoma specimens, which led to the reduction of p21(waf1/cip1) expression in a manner independent of p53 status. Additionally, an alteration of subcellular localization from nuclei to cytoplasm was also observed in >70% of cutaneous squamous cell carcinoma samples. Because DDX3 exhibits tumor suppressor functions, such as a growth-suppressive property and transcriptional activation of the p21(waf1/cip1) promoter, and is inactivated through down-regulation of gene expression or alteration of subcellular localization in tumor cells, all these features together suggest that DDX3 might be a candidate tumor suppressor.

Effects of Scutellaria baicalensis Georgi on macrophage-hepatocyte interaction through cytokines related to growth control of murine hepatocytes.

The aim of this study is to elucidate the effects of Scutellaria baicalensis Georgi (SbG) extract and its constituents on macrophage-hepatocyte interaction in primary cultures. By using trans-well primary Kupffer cell culture or conditioned medium (CM) from murine macrophage RAW264.7 cell line (RAW cells), effects of SbG on hepatocyte growth were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and trypan blue exclusion assay. Cytokine production, antibody-neutralization studies, and molecular mechanisms of transforming growth factor (TGF)-beta1 gene expression were elucidated on SbG-treated RAW264.7 cells. In addition, recombinant human TGF-beta1 (r-human TGF-beta1) was added to elucidate the mechanisms of SbG effects on cultured hepatocytes. Immunohistochemistry using anti-NF-kappaB antibody was used to determine the possible signal transduction pathways in primary hepatocyte culture. The results showed that SbG stimulated the proliferation of cultured hepatocytes, possibly through NF-kappaB, but not of Toll-like receptor 4 activation; whereas SbG-RAW-CM and SbG in trans-well significantly suppressed the proliferation of hepatocytes. Antibody-neutralization studies revealed that TGF-beta1 was the main antimitotic cytokine in SbG-treated RAW cells CM. The growth stimulation effect of SbG on cultured hepatocytes was inhibited by exogenous administration of r-human TGF-beta1. Furthermore, SbG induced NF-kB translocation into the nuclei of cultured cells. In the RAW264.7 line, SbG and baicalin stimulated TGF-beta1 gene expression via NF-kappaB and protein kinase C activation. We conclude that SbG stimulates hepatocyte growth via activation of the NF-kappaB pathway and induces TGF-beta1 gene expression through the Kupffer cell-hepatocyte interaction, which subsequently results in the inhibition of SbG-stimulated hepatocyte growth.

Gene expression profiling predicts liver responses to a herbal remedy after partial hepatectomy in mice.

The aim of this study was to demonstrate that regenerating liver responses to a herbal remedy could be presented by gene expression profiling. Compositions of the ingredients in the remedy containing Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Wild (S/B remedy) were analyzed and quantified by high performance liquid chromatography. By using a 70% partial hepatectomy in BALB/c mice as an in vivo model, the effects of high dose (50 mg/kg) and low dose (1 mg/kg) S/B remedy were evaluated by cDNA microarray, followed by RT-PCR and real-time PCR confirmation. Factors affecting proliferative activities of mouse hepatocytes were measured by DNA flow cytometry, BrdU incorporation assay and serum interleukin-6 (IL-6) level. Based on global gene expression profiles, the results showed that the low dose S/B remedy down-regulated expression of immediate early genes and cell cycle-related genes, whereas the high dose had opposite effects. The gene expression was further verified by real-time RT-PCR. Proliferative activities, in terms of synthetic phase fractions and G2/M phase fractions, in vehicle, low dose, and high dose groups were 18.45+/-2.56%, 14.65+/-1.06%; 9.27+/-0.85%, 7.80+/-0.11%; and 18.90+/-2.17%, 22.95+/-0.25%, respectively. The serum IL-6 level was also dose-dependent in both low and high dose S/B remedy-treated mice. We conclude that in vivo gene expression profiling correlates with liver responses to a herbal remedy, which provides a new direction for pharmaceutical studies on human diseases.

Phosphorylation and stabilization of HURP by Aurora-A: implication of HURP as a transforming target of Aurora-A.

Aurora-A, a mitotic serine/threonine kinase with oncogene characteristics, has recently drawn intense attention because of its association with the development of human cancers and its relationship with mitotic progression. Using the gene expression profiles of Aurora-A as a template to search for and compare transcriptome expression profiles in publicly accessible microarray data sets, we identified HURP (encodes hepatoma upregulated protein) as one of the best Aurora-A-correlated genes. Empirical validation indicates that HURP has several characteristics in common with Aurora-A. These two genes have similar expression patterns in hepatocellular carcinoma, liver regeneration after partial hepatectomy, and cell cycle progression and across a variety of tissues and cell lines. Moreover, Aurora-A phosphorylated HURP in vitro and in vivo. Ectopic expression of either the catalytically inactive form of Aurora-A or the HURP-4P mutant, in which the Aurora-A phosphorylation sites were replaced with Ala, resulted in HURP instability and complex disassembly. In addition, HURP-wild-type stable transfectants were capable of growing in low-serum environments whereas HURP-4P grew poorly under low-serum conditions and failed to proliferate. These studies together support the view that the ability to integrate evidence derived from microarray studies into biochemical analyses may ultimately augment our predictive power when analyzing the potential role of poorly characterized proteins. While this combined approach was simply an initial attempt to answer a range of complex biological questions, our findings do suggest that HURP is a potential oncogenic target of Aurora-A.

Integrative genomics based identification of potential human hepatocarcinogenesis-associated cell cycle regulators: RHAMM as an example.

DNA microarray has been widely used to examine gene expression profile of different human tumors. The information generated from microarray analysis usually represents the overall range of cancer-associated abnormality associated with gene regulation. In order to identify key regulatory genes involved in carcinogenesis of human cancer, hypothesis driven data mining of the microarray data plus experimental validation becomes a critical approach in the post-genome era. Here, we present an integrative genomic analysis of published microarray data and homolog gene database. Over 20,000 genes were examined to reveal 16 genes specific to vertebrates, cell cycle G2/M regulated, and overexpressed in human HCC. Using Affymetrix microarray analysis, we found that all 16 genes were up-regulated in human HCC. Among these 16 genes, we experimentally validated the up-regulation of receptor for hyaluronan-mediated motility (RHAMM) in different cell model systems. We first confirmed elevation of RHAMM in the G2/M phase of synchronized HeLa cells. We also found that RHAMM had an elevated level of expression in all the HCC samples we examined and it was induced during the G2/M phase of regenerating mouse hepatocytes after partial hepatectomy. Thus, the expression of RHAMM appears to be tightly regulated during mammalian cell cycle G2/M progression. The ectopic overexpression of RHAMM in 293T cells resulted in the accumulation of cells at G2/M phase. RHAMM-induced mitotic arrest of cells was predominantly in the prophase. Taken together, using an integrated functional genomic approach, we have uncovered a set of genes that may play specific roles in cell cycle progression and in HCC development. To elucidate the function of these genes in cell cycle regulation may shed light on the control mechanism of human HCC in the future.

Overexpression of a novel imprinted gene, PEG10, in human hepatocellular carcinoma and in regenerating mouse livers.

Many of the promising applications of the microarray technology are pertinent to identifying abnormalities in gene expression that contribute to malignant progression. We developed a bioinformatics tool to identify differentially expressed genes in human hepatocellular carcinoma (HCC). This involved the construction of a liver EST database (http://lestdb.nhri.org.tw) and in silico verification of differentially expressed genes with a human hepatoma microarray database. The stringency of the search was reinforced with a statistical analysis. A novel imprinted gene, paternally expressed 10(PEG10) was identified as having an elevated level of expression in the majority of the HCC samples and was also induced to express during G2/M phase of regenerating mouse liver. Ectopic expression of PEG10 in 293T cells affects cell cycle progression. PEG10 is distributed in the cytosol and associates with the nuclear membrane. This is the first time that an imprinted gene has been found to reexpress in both human HCC and in the regenerating mouse liver. This result indicates that the induction of the paternally imprinted gene may play an important role during liver regeneration or carcinogenesis of the human hepatocyte. Understanding the molecular basis of the abnormal imprinting of PEG10 will shed new light on the process that leads to liver disease.

Selenite-induced survival of HuH7 hepatoma cells involves activation of focal adhesion kinase-phosphatidylinositol 3-kinase-Akt pathway and Rac1.

Selenium has been shown to sustain the growth of selected human hepatocellular carcinoma cell lines under serum-free conditions, but the detailed mechanism remained undetermined. In the present study, the molecular mechanism(s) involving sodium selenite (Na2SO3, Se) as a survival agent were determined. Selenite not only protects HuH7 cells from serum deprivation-induced apoptosis, it also supports its long-term growth in sodium selenite (10(-7)m) supplemented serum-free medium. The anti-apoptotic effect correlates with activation of focal adhesion kinase and the phosphatidylinositol 3-kinase (PI3K)-Akt kinase pathway. Using HuH7 cells stably transfected with a constitutively active Akt kinase and PI3K inhibitor LY294002, selenite-induced cell survival was shown to be PI3K-Akt-dependent. Parallel changes included a significant reduction in the intracellular reactive oxygen species content, the reversal of DNA fragmentation, and the suppression of caspase and apoptosis signal-regulating kinase 1 activities. HuH7 cells stably expressing a Rac1 mutant N17 (Rac1N17-HuH7) are refractory to selenite treatment. In these cells selenite supplement neither triggers Akt activation nor supports cell proliferation. Participation of Rac1 activation in this event is supported by the fact that selenite treatment drastically enhanced activation of Rac1. The exact link between selenite treatment, Rac1 activation, and activation of the focal adhesion kinase-PI 3-kinase, however, remains to be characterized. The mitogenic signaling mediated by selenite may involve unconventional growth stimuli including higher glutathione peroxidase 1 activity and higher transcription levels of selenoprotein P. The selenium-HuH7 system we have established thus provides a unique tool that will allow the biological role of selenite in growth regulation of hepatocytes to be studied in detail.