Chi Chiu Wang

Therapeutic potentials of gene silencing by RNA interference: principles, challenges, and new strategies.

During recent decades there have been remarkable advances in biology, in which one of the most important discoveries is RNA interference (RNAi). RNAi is a specific post-transcriptional regulatory pathway that can result in silencing gene functions. Efforts have been done to translate this new discovery into clinical applications for disease treatment. However, technical difficulties restrict the development of RNAi, including stability, off-target effects, immunostimulation and delivery problems. Researchers have attempted to surmount these barriers and improve the bioavailability and safety of RNAi-based therapeutics by optimizing the chemistry and structure of these molecules. This paper aimed to describe the principles of RNA interference, review the therapeutic potential in various diseases and discuss the new strategies for in vivo delivery of RNAi to overcome the challenges.

Lipid peroxidation in cord blood at birth: the effect of labour

Objective To determine the effect of labour on free oxygen radical activity in the fetus, as reflected by lipid peroxide levels in umbilical cord arterial blood.

Redox Control in Mammalian Embryo Development

The development of an embryo constitutes a complex choreography of regulatory events that underlies precise temporal and spatial control. Throughout this process the embryo encounters ever changing environments, which challenge its metabolism. Oxygen is required for embryogenesis but it also poses a potential hazard via formation of reactive oxygen and reactive nitrogen species (ROS/RNS). These metabolites are capable of modifying macromolecules (lipids, proteins, nucleic acids) and altering their biological functions. On one hand, such modifications may have deleterious consequences and must be counteracted by antioxidant defense systems. On the other hand, ROS/RNS function as essential signal transducers regulating the cellular phenotype. In this context the combined maternal/embryonic redox homeostasis is of major importance and dysregulations in the equilibrium of pro- and antioxidative processes retard embryo development, leading to organ malformation and embryo lethality. Silencing the in vivo expression of pro- and antioxidative enzymes provided deeper insights into the role of the embryonic redox equilibrium. Moreover, novel mechanisms linking the cellular redox homeostasis to gene expression regulation have recently been discovered (oxygen sensing DNA demethylases and protein phosphatases, redox-sensitive microRNAs and transcription factors, moonlighting enzymes of the cellular redox homeostasis) and their contribution to embryo development is critically reviewed.

The roles of glutathione peroxidases during embryo development

Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency – in contrast to all other GPx family members – leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on GPx4.

A paradoxical teratogenic mechanism for retinoic acid

Retinoic acid, an active metabolite of vitamin A, plays essential signaling roles in mammalian embryogenesis. Nevertheless, it has long been recognized that overexposure to vitamin A or retinoic acid causes widespread teratogenesis in rodents as well as humans. Although it has a short half-life, exposure to high levels of retinoic acid can disrupt development of yet-to-be formed organs, including the metanephros, the embryonic organ which normally differentiates into the mature kidney. Paradoxically, it is known that either an excess or a deficiency of retinoic acid results in similar malformations in some organs, including the mammalian kidney. Accordingly, we hypothesized that excess retinoic acid is teratogenic by inducing a longer lasting, local retinoic acid deficiency. This idea was tested in an established in vivo mouse model in which exposure to excess retinoic acid well before metanephric rudiments exist leads to failure of kidney formation several days later. Results showed that teratogen exposure was followed by decreased levels of Raldh transcripts encoding retinoic acid-synthesizing enzymes and increased levels of Cyp26a1 and Cyp26b1 mRNAs encoding enzymes that catabolize retinoic acid. Concomitantly, there was significant reduction in retinoic acid levels in whole embryos and kidney rudiments. Restoration of retinoic acid levels by maternal supplementation with low doses of retinoic acid following the teratogenic insult rescued metanephric kidney development and abrogated several extrarenal developmental defects. This previously undescribed and unsuspected mechanism provides insight into the molecular pathway of retinoic acid-induced teratogenesis.

Anti-angiogenic effects of green tea catechin on an experimental endometriosis mouse model

BACKGROUND The development of new blood vessels plays an essential role in growth and survival of endometriosis. Epigallocatechin gallate (EGCG) from green tea has powerful anti-angiogenic properties and our aim was to evaluate these properties in experimental endometriosis. METHODS AND RESULTS Eutopic endometrium from endometriosis patients was transplanted s.c. to severely compromised immunodeficient mice, randomly treated i.p. with EGCG (anti-angiogenic and -oxidant), Vitamin E (a non-angiogenic antioxidant) or saline for 2 weeks. The endometrial implant, including adjacent host outer skin and subcutaneous layers plus inner abdominal muscle and peritoneum, was collected. New microvessels were determined by species-specific immunohistochemistry. Angiogenic factors in lesions and abdominal muscle were detected by quantitative real-time PCR. Apoptosis was studied by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling and quantitative real-time PCR. In saline control, endometrial implants developed new blood vessels with proliferating glandular epithelium and were tightly adhered to host subcutaneous and abdominal muscle layers. After EGCG, endometriotic lesions were smaller than control (P < 0.05), and glandular epithelium was smaller and eccentrically distributed. Angiogenesis in lesions from the implant and adjacent tissues was under-developed, and microvessel size and density were lower (both P < 0.01) than control. mRNA for angiogenic vascular endothelial growth factor A, but not hypoxia inducible factor 1, alpha subunit, was significantly down-regulated in lesions after EGCG (P < 0.05). In addition, apoptosis in the lesions was more obvious, and nuclear factor kappa B and mitogen activated protein kinase 1 mRNA levels were up-regulated (P < 0.05) after EGCG treatment. No differences were observed with Vitamin E treatment. CONCLUSIONS EGCG significantly inhibits the development of experimental endometriosis through anti-angiogenic effects.

The Role of Phospholipid Hydroperoxide Glutathione Peroxidase Isoforms in Murine Embryogenesis

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice.

MicroRNA-182 plays an onco-miRNA role in cervical cancer.

OBJECTIVES The purposes of this study were to identify aberrantly expressed miRNAs and investigate their pathogenic roles in cervical cancer. METHODS miRNA expression was assessed in cervical cancer cell lines, micro-dissected normal cervical epithelium cells and primary cervical carcinoma by TaqMan RT-PCR. Spatial expression of miR-182 in cervical carcinoma and normal cervix was explored by in situ hybridization. HeLa xenograft mice model was used for evaluation of the effect on tumor growth of miR-182 inhibitor. Western blot, flow cytometry and gene expression analysis were used for identification of the functional role of miR-182 in HeLa cells. RESULTS Two up-regulated (miR-182 and -183) and nine down-regulated (miR-211, 145, 223, 150, 142-5p, 328, 195, 199b, 142-3p) microRNAs were consistently identified in cervical cancer cell lines. Further investigation confirmed the most up-regulated miRNA (miR-182) was significantly elevated in primary cervical carcinoma and discovered a significant correlation between the increased expression of miR-182 and advanced stages of cervical cancer. In HeLa xenograft mouse model, we demonstrated that inhibition of the miR-182 could exert the effect of tumor growth regression. Western blot, flow cytometry and pathway analysis for the HeLa cells with miR-182 over/down-expression in vitro showed that miR-182 was involved in apoptosis and cell cycle pathways, it also associated with the regulation of FOXO1. CONCLUSIONS Our findings indicated that miR-182 plays an onco-miRNA role in cervical cancer and its alteration is associated with cervical cancer pathogenesis by disrupting cell proliferation.

Partial neuroprotective effect of pretreatment with tanshinone IIA on neonatal hypoxia-ischemia brain damage.

Tanshinone IIA is a compound purified from the Chinese herb Danshen (Radix Salviae Miltiorrhiza Bge). The neuroprotective effect of tanshinone IIA was investigated in a neonatal rat model of hypoxia-ischemia brain damage. Hypoxia-ischemia encephalopathy was induced in rats at day 7 of postnatal age by ligation of the right common carotid artery, followed by 2 h of hypoxia. Tanshinone IIA (10 mg/kg, i.p.) was injected daily from day 2 before surgery for 9 or 16 d. Our results demonstrated significant and sustained brain damage in the hypoxia-ischemia– and vehicle-treated groups at 1 and 3 wk after surgery. Treatment with tanshinone IIA significantly reduced the severity of brain injury, as indicated by the increase in ipsilateral brain weight and neuron density, compared with those of sham-operated animals. The recovery of sensorimotor function and histology was observed in animals that received tanshinone IIA. The plasma of tanshinone IIA–treated rats exhibited higher antioxidant activities, as reflected by the oxygen radical absorbance capacity assay, compared with the vehicle-treated rats. In the neural progenitor cell line C17.2 that was subjected to 2,2′-azobis (2-amidino propane hydrochloride)–induced oxidative stress, tanshinone IIA increased cell viability and protected against mitochondrial damage (JC-1 assay). Our results suggest that tanshinone IIA has antioxidative activities and that treatment that is started before a hypoxic-ischemic insult is partially neuroprotective. Further studies are required to elucidate whether rescue treatment with tanshinone IIA is effective and to determine whether its protective effect is also associated with secondary cooling of the brain.

Comparison of peritoneal oxidative stress during laparoscopy and laparotomy

STUDY OBJECTIVE To identify oxidative stress in peritoneum during laparoscopic and open surgery by measuring products of lipid peroxidation, and to determine whether surgical approach influences the type of oxidative metabolite synthesized. DESIGN Retrospective analysis (Canadian Task Force classification II-2). SETTING University-affiliated hospital. PATIENTS Twenty-eight consecutive women with uterine myomas or ovarian cysts. INTERVENTION Laparoscopic or open surgery (14 patients each). MEASUREMENTS AND MAIN RESULTS We obtained 1 x 1-cm squares of peritoneum at the beginning and end of surgical procedures away from sites of surgery. 8-Isoprostaglandin F(2alpha), hydroxyeicosatetranoic acids (HETEs), and malondyaldehyde (MDA) were measured by enzyme-immunoassay, high-performance liquid chromatography, and thiobarbituric acid adduction method, respectively. Comparisons showed significant increases in 5-HETE and 8-prostane in the laparoscopy group, which were correlated with duration of pneumoperitoneum and volume of carbon dioxide (CO(2)) insufflated, respectively. In the laparotomy group only MDA rose significantly related to duration of surgery. CONCLUSIONS Lipid peroxidation was observed in peripheral peritoneum during laparoscopic surgery, mediated through noncyclooxygenase and lipoxygenase pathways, and appears to be due to effects of CO(2) pneumoperitoneum. Biochemical reactions were also observed in the laparotomy group, but are thought to be related to mechanisms other than lipid peroxidation.