Up-regulation of microRNA-210 inhibits proliferation of hepatocellular carcinoma cells by targeting YES1.

AIM: To determine the expression of microRNA-210 (miR-210) in hepatocellular carcinoma (HCC) and to examine its role using HCC cells. METHODS: The expression of miR-210 was determined in 21 pairs of HCC samples and the corresponding surrounding non-tumor tissues. The effects of miR-210 on proliferation and cell cycle progression were examined using HepG2 and HuH7 cells. Over-expression and inhibition of miR-210 was achieved by transfection of the cells with miR-210 mimic or inhibitor. Luciferase reporter constructs were used to identify the miR-210 interacting site on Yes1. Yes1 expression was examined after miR-210 transfection, as well as in the HCC samples. RESULTS: miR-210 was significantly up-regulated by 3.4 fold (P < 0.01) in the tumor samples. The over-expression of miR-210 significantly reduced cell proliferation compared to the mock-treated cells (68.9% ± 7.4% and 53.6% ± 5.0%, P < 0.05 for the HepG2 and HuH7 cells respectively). Analysis of the HuH7 cells transfected with miR-210 mimic by flow cytometry showed that the cells took a longer time to reach the G2/M phase. The interaction between miR-210 and the 3'UTR of the Yes1 transcript was confirmed using a luciferase reporter assay. Over-expression of miR-210 reduced the expression of Yes1 protein in both HuH7 and HepG2 cells. Tumors with a greater than four-fold increase in the expression of miR-210 showed consistently lower expressions of Yes1 in the tumors. In nocodazole-treated cells with a significant G2/M cell population, Yes1 protein was significantly reduced and pre-inhibition of miR-210 in HuH7 cells was able to prevent the reduction of Yes1 protein expression. Knock-down of Yes1 by siRNA also led to reduced cell proliferation (70.8% ± 7.5%, P < 0.05 in the HuH7 cells). CONCLUSION: Up-regulation of miR-210 inhibits cell proliferation. Yes1 is a target of miR-210 and affects cell proliferation in HCC.

miR-106b-25/miR-17-92 clusters: polycistrons with oncogenic roles in hepatocellular carcinoma.

MicroRNAs are small endogenously expressed RNA molecules which are involved in the process of silencing gene expression through translational regulation. The polycistronic miR-17-92 cluster is the first microRNA cluster shown to play a role in tumorigenesis. It has two other paralogs in the human genome, the miR-106b-25 cluster and the miR-106a-363 cluster. Collectively, the microRNAs encoded by these clusters can be further grouped based on the seed sequences into four families, namely the miR-17, the miR-92, the miR-18 and the miR-19 families. Over-expression of the miR-106b-25 and miR-17-92 clusters has been reported not only during the development of cirrhosis but also subsequently during the development of hepatocellular carcinoma. Members of these clusters have also been shown to affect the replication of hepatitis B and hepatitis C viruses. Various targets of these microRNAs have been identified, and these targets are involved in tumor growth, cell survival and metastasis. In this review, we first describe the regulation of these clusters by c-Myc and E2F1, and how the members of these clusters in turn regulate E2F1 expression forming an auto-regulatory loop. In addition, the roles of the various members of the clusters in affecting relevant target gene expression in the pathogenesis of hepatocellular carcinoma will also be discussed.

A novel MLL5 isoform that is essential to activate E6 and E7 transcription in HPV16/18-associated cervical cancers.

Human papillomavirus (HPV) is the primary cause of human cervical cancer. The viral proteins E6 and E7 are essential to transform noncancerous epithelial cells into cancerous carcinomas by targeting key tumor suppressors p53 and retinoblastoma (Rb) proteins, respectively, but the cellular factors involved in E6 and E7 transcription themselves are incompletely understood. In this study, we defined a novel isoform of the mixed lineage leukemia 5 gene (MLL5ß) as a specific and critical regulator of E6 and E7 transcription in cervical carcinoma cells. MLL5ß is present in HPV16/18-positive cells including human primary cervical carcinoma specimens. Interaction of MLL5ß with the AP-1-binding site at the distal region of the HPV18 long control region led to activation of E6/E7 transcription. Conversely, RNA interference-mediated knockdown of MLL5ß downregulated both E6 and E7 expression. MLL5ß downregulation was sufficient to restore p53 protein levels and reduce Rb phosphorylation, thereby reactivating apoptosis and cell-cycle checkpoints. By defining this novel MLL5ß isoform and its specific critical role in activating E6/E7 gene transcription in HPV16/18-induced cervical cancers, our work highlights the potential of MLL5ß as a biomarker and new therapeutic target in primary HPV-induced cervical cancers.

A global transcriptomic view of the multifaceted role of glutathione peroxidase-1 in cerebral ischemic-reperfusion injury.

Transient cerebral ischemia often results in secondary ischemic/reperfusion injury, the pathogenesis of which remains unclear. This study provides a comprehensive, temporal description of the molecular events contributing to neuronal injury after transient cerebral ischemia. Intraluminal middle cerebral artery occlusion (MCAO) was performed to induce a 2-h ischemia with reperfusion. Microarray analysis was then performed on the infarct cortex of wild-type (WT) and glutathione peroxidase-1 (a major antioxidant enzyme) knockout (Gpx1(-/-)) mice at 8 and 24h postreperfusion to identify differential gene expression profile patterns and potential alternative injury cascades in the absence of Gpx1, a crucial antioxidant enzyme, in cerebral ischemia. Genes with at least ±1.5-fold change in expression at either time point were considered significant. Global transcriptomic analyses demonstrated that 70% of the WT-MCAO profile overlapped with that of Gpx1(-/-)-MCAO, and 28% vice versa. Critical analysis of the 1034 gene probes specific to the Gpx1(-/-)-MCAO profile revealed regulation of additional novel pathways, including the p53-mediated proapoptotic pathway and Fas ligand (CD95/Apo1)-mediated pathways; downplay of the Nrf2 antioxidative cascade; and ubiquitin-proteasome system dysfunction. Therefore, this comparative study forms the foundation for the establishment of screening platforms for target definition in acute cerebral ischemia intervention.

NRAMP1 and hGPX1 gene polymorphism and response to bacillus Calmette-Guérin therapy for bladder cancer.

BACKGROUND: The natural resistance-associated macrophage protein 1 (NRAMP1) gene is associated with susceptibility to Mycobacterium tuberculosis in humans and to bacillus Calmette-Guérin (BCG) in mice. The detoxification enzyme, human glutathione peroxidase 1 (hGPX1), is associated with recurrence of bladder cancer (BCa). OBJECTIVE: To determine whether NRAMP1 and hGPX1 gene polymorphisms correlate with response to BCG immunotherapy for non-muscle-invasive BCa (NMIBC). DESIGN, SETTING, AND PARTICIPANTS: DNA was obtained from the peripheral blood of 99 NMIBC patients who were prospectively randomized to receive postresection intravesical BCG (81 mg [n=50] or 27 mg [n=19]) or BCG (27 mg) with interferon alpha (IFN-α; n=30). The median follow-up time was 60 mo. INTERVENTION: Intravesical BCG or BCG-IFN-α. MEASUREMENTS: Restriction fragment length polymorphism (RFLP) analysis was performed to identify polymorphisms in the NRAMP1 promoter region (GT repeat number) and at position 543 (aspartate [D] and/or asparagine [N] expression) within the NRAMP1 protein (D543N) and position 198 (proline and/or leucine expression) within the hGPX1 protein (Pro198Leu). Data were analyzed using χ(2) analysis, multivariate analysis, and Kaplan-Meier curves. RESULTS AND LIMITATIONS: On univariate analysis, the NRAMP1 D543N G:G genotype had decreased cancer-specific survival (CSS; p=0.036). The hGPX1 CT genotype (Pro-Leu) had decreased recurrence time (p=0.03) after BCG therapy. On multivariate analysis, patients with the NRAMP1 D543N G:G genotype and allele 3 (GT)n polymorphism had decreased recurrence time (p=0.014 and p=0.03) after BCG therapy. The limitation of this study was its small sample size. CONCLUSIONS: Polymorphisms of the NRAMP1 and hGPX1 genes may be associated with recurrence of BCa after BCG immunotherapy.

Gene profiling reveals hydrogen sulphide recruits death signaling via the N-methyl-D-aspartate receptor identifying commonalities with excitotoxicity.

Recently the role of hydrogen sulphide (H(2) S) as a gasotransmitter stimulated wide interest owing to its involvement in Alzheimer's disease and ischemic stroke. Previously we demonstrated the importance of functional ionotropic glutamate receptors (GluRs) by neurons is critical for H(2) S-mediated dose- and time-dependent injury. Moreover N-methyl-D-aspartate receptor (NMDAR) antagonists abolished the consequences of H(2) S-induced neuronal death. This study focuses on deciphering the downstream effects activation of NMDAR on H(2) S-mediated neuronal injury by analyzing the time-course of global gene profiling (5, 15, and 24 h) to provide a comprehensive description of the recruitment of NMDAR-mediated signaling. Microarray analyses were performed on RNA from cultured mouse primary cortical neurons treated with 200 µM sodium hydrosulphide (NaHS) or NMDA over a time-course of 5-24 h. Data were validated via real-time PCR, western blotting, and global proteomic analysis. A substantial overlap of 1649 genes, accounting for over 80% of NMDA global gene profile present in that of H(2) S and over 50% vice versa, was observed. Within these commonly occurring genes, the percentage of transcriptional consistency at each time-point ranged from 81 to 97%. Gene families involved included those related to cell death, endoplasmic reticulum stress, calcium homeostasis, cell cycle, heat shock proteins, and chaperones. Examination of genes exclusive to H(2) S-mediated injury (43%) revealed extensive dysfunction of the ubiquitin-proteasome system. These data form a foundation for the development of screening platforms and define targets for intervention in H(2) S neuropathologies where NMDAR-activated signaling cascades played a substantial role.

Role of the miR-106b-25 microRNA cluster in hepatocellular carcinoma.

MicroRNAs are tiny RNA molecules which serve as important post-transcriptional regulators of gene expression. Dysregulated expression of microRNAs has been observed in human cancers, indicating that microRNAs may function as oncogenes or as tumor suppressors. To date, the microRNAs encoded by the oncogenic miR-17-92 cluster, and its paralog the miR-106b-25 cluster, are among those which are differentially expressed in human cancers. In this study, we examined and confirmed the over-expression of these clusters in hepatocellular carcinoma and in hepatoma-derived cells. At least 50% of the tumor samples showed a greater than two-fold increase in the expression for miR-18 and for the miR-106b-25 cluster when compared with the corresponding paired non-tumor samples. Knock-down studies for the miR-106b-25 cluster, which includes miR-106b, miR-93 and miR-25, showed that the expression of the cluster is necessary for cell proliferation and for anchorage-independent growth. In tumors with high expression of this cluster, reduced expression of the BH3-only protein Bim, a miR-25 target, was observed. We further identified the transcription factor E2F1 as a target gene for miR-106b and miR-93 and it is likely that one of the roles of the miR-106b-25 cluster is to prevent excessively high E2F1 expression, which may then cause apoptosis. We conclude that there is aberrant expression of microRNAs encoded by the oncogenic miR-17-92 cluster and the miR-106b-25 cluster in hepatocellular carcinoma. The consistent overexpression of the miR-106b-25 cluster and its role in cell proliferation and anchorage-independent growth points to the oncogenic potential of this cluster.

Molecular and functional characterization of drug-metabolizing enzymes and transporter expression in the novel spontaneously immortalized human hepatocyte line HC-04.

In toxicological research, immortalized human hepatocytes provide a useful alternative to primary hepatocytes because interindividual variability in the expression of drug-metabolizing enzymes and drug transporters can largely be eliminated. However, it is essential that the cell line retain the original phenotype. The purpose of this study was to characterize a novel spontaneously immortalized human hepatocyte cell line, HC-04, with respect to the transcript and functional protein expression profile for the major drug-metabolizing enzymes and transmembrane transporters. HC-04 cells retained hepatocyte-specific function including albumin production and ornithine transcarbamoylase and glucose-6-phosphatase activity. Most of the major CYP forms were expressed at basal levels and responsive to inducing agents. In particular, CYP3A4 was expressed abundantly, and HC-04 cells were able to metabolize the CYP3A4 probe, midazolam, at a rate similar to primary human hepatocytes. Furthermore, the major human sulfotransferase and UDP-glucuronosyltransferase forms, as well as members of the ABC and SLC transporter superfamilies, nuclear receptors, and hepatic transcription factors were also expressed. HC-04 cells readily responded to standard hepatotoxicants that are dependent on CYP-mediated bioactivation, while another, tumor-derived cell line remained refractory to the drug challenge. Collectively, HC-04 cells provide a reliable, stable, and reproducible model for biomechanistic studies in drug toxicology.

In vitro cytotoxicity of Stealth liposomes co-encapsulating doxorubicin and verapamil on doxorubicin-resistant tumor cells.

Multidrug resistance (MDR) is a major obstacle to successful clinical cancer chemotherapy. A novel doxorubicin anti-resistant Stealth liposomes (DARSLs), prepared by co-encapsulating doxorubicin (DOX) and verapamil (VER) into stealth liposomes, has been developed. The average particle size of DARSLs was 118.1+/-22.3 nm. Encapsulation efficiencies of DOX and VER in DARSLs were greater than 95% and 70%, respectively. The IC(50) of DARSLs as measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay in multidrug resistant rat prostate cancer Mat-LyLu-B2 (MLLB2) cells was 0.079+/-0.017 microM, 13 fold less than that for liposomal DOX with free VER (LDFV 0.96+/-0.46 microM) but only about 2 times less than FDFV. The IC(50) cytotoxicity on MLLB2 cells of the various formulations was as follows: DARSLs approximately LDLV

Multidrug resistance protein 4 (MRP4/ABCC4) mediates efflux of bimane-glutathione.

Multidrug resistance proteins (MRPs) are ATP-dependent export pumps that mediate the export of organic anions. ABCC1 (MRP1), ABCC2 (MRP2) and ABCC3 (MRP3) are all able to facilitate the efflux of anionic conjugates including glutathione (GSH), glucuronide and sulfate conjugates of xenobiotics and endogenous molecules. Earlier studies showed that ABCC4 functions as an ATP-driven export pump for cyclic AMP and cyclic GMP, as well as estradiol-17-beta-D-glucuronide. However, it was unclear if other conjugated metabolites can be transported by ABCC4. Hence in this study, a fluorescent substrate, bimane-glutathione (bimane-GS) was used to further examine the transport activity of ABCC4. Using cells stably overexpressing ABCC4, this study shows that ABCC4 can facilitate the efflux of the glutathione conjugate, bimane-glutathione. Bimane-glutathione efflux increased with time and >85% of the conjugate was exported after 15min. This transport was abolished in the presence of 2.5microM carbonylcyanide m-chlorophenylhydrasone (CCCP), an uncoupler of oxidative phosphorylation. Inhibition was also observed with known inhibitors of MRP transporters including benzbromarone, verapamil and indomethacin. In addition, 100microM methotrexate, an ABCC4 substrate or 100microM 6-thioguanine (6-TG), a compound whose monophosphate metabolite is an ABCC4 substrate, reduced efflux by >40%. A concentration-dependent inhibition of bimane-glutathione efflux was observed with 1-chloro-2,4-dinitrobenzene (CDNB) which is metabolized intracellularly to the glutathione conjugate, 2,4-dinitrophenyl-glutathione (DNP-GS). The determination that ABCC4 can mediate the transport of glucuronide and glutathione conjugates indicates that ABCC4 may play a role in the cellular extrusion of Phase II detoxification metabolites.

Genetic determinants of tetracycline resistance in Vibrio harveyi.

Isolates of Vibrio harveyi, a prawn pathogen, have demonstrated multiple antibiotic resistance to commonly used antimicrobial agents, such as oxytetracycline. In this paper, we describe the cloning and characterization of two tetracycline resistance determinants from V. harveyi strain M3.4L. The first resistance determinant, cloned as a 4,590-bp fragment, was identical to tetA and flanking sequences encoded on transposon Tn10 from Shigella flexneri. The second determinant, cloned as a 3,358-bp fragment in pATJ1, contains two open reading frames, designated tet35 and txr. tet35 encodes a 369-amino-acid protein that was predicted to have nine transmembrane regions. It is a novel protein which has no homology to any other drug resistance protein but has low levels of homology (28%) to Na(+)/H(+) antiporters. Transposon mutagenesis showed that tet35 and txr were required for tetracycline resistance in a heterologous Escherichia coli host. Tetracycline accumulation studies indicate that E. coli carrying tet35 and txr can function as an energy-dependent tetracycline efflux pump but is less efficient than TetA.

Role of glutathione in the multidrug resistance protein 4 (MRP4/ABCC4)-mediated efflux of cAMP and resistance to purine analogues.

Multidrug resistance protein 4 (MRP4/ABCC4) is a member of the MRP subfamily, which in turn is a member of the superfamily of ATP-binding-cassette (ABC) transporters. Within the MRP subfamily, ABCC4,ABCC5 (MRP5), ABCC11 (MRP8) and ABCC12 (MRP9) have similar predicted membrane topologies. All lack the additional transmembrane domain, TMD(0), which is present in the other MRPs. Using cells stably overexpressing ABCC4, this study shows that ABCC4 exports GSH. ABCC4 also facilitates the efflux of cAMP. Depletion of intracellular GSH with DL-buthionine-(S,R)-sulphoximine led to decreased export of cAMP and a corresponding increase in intracellular cAMP was observed. ABCC4 also mediates resistance to purine analogues 9-(2-phosphonylmethoxyethyl)-adenine and 6-thioguanine. This resistance can be reversed by the presence of DL-buthionine-(S,R)-sulphoximine. We conclude that as well as nucleotide and nucleoside analogues, ABCC4 can mediate the export of GSH. In addition, GSH plays an important role in the function of ABCC4. Depletion of intracellular GSH adversely affects the export of cAMP by ABCC4. Resistance to nucleoside analogues is also adversely affected by depletion of cellular GSH.