Molecular Mechanism Of RNAi Suppression Of Mitochondrial Intracellular Chloride Channel During Hydrogen Peroxide-induced Rat Glial Cell Injury

Along with the worldwide population aging, more and more old people suffer from neural degenerative diseases such as Alzheimer's disease, ischemic /hypoxic brain injuries and Humtington's chorea disease. The pathogenisis of the diseases is not very clear, but a lot of research data have demonstrated that the neurocytes apoptosis happens during these diseases development. According to the recent research that many signaling cascades involved the process, and in a recent comprehensive review that the ion channel plays an very important role during the apoptosis.We refer mostly to these anion channels as Cl–channels, because Cl– is the most abundant anion and the predominant permeating species in all organisms. In contrast with cation channels, they are not involved in the initiation or spread of excitation, but in the regulation of excitability, transmembrane transport,pH regulation of intracellular organelles and volume regulation. Chloride channels are classified based on structural differences. The six major classes are chloride channnel family (CLCs), cystic fibrosis transmembrane regulator (CFTR), swelling-activated chloride channels (ClVol), calcium- activated chloride channels (ClCa), Chloride intracellular channels (CLICs) and ligand-gated chloride channels.The CLIC family represents a separate and relatively newly discovered seven-member family of intracellular chloride channels. Intracellular chloride channel are present in membranes of a variety of organelles. In mitochondria, only exits intracellular chloride channel 4(CLIC4), so we also name CLIC4 as mitochondrial chloride intracellular channel (mtCLIC). Besides mitochondria, CLIC4 are present such as the nucleus, cytoplasm, the Golgi apparatus, the trans-Golgi network, endoplasmic reticulum, and the recycling endosomes.Intracellular chloride channel are present in membranes of a variety of organelles. These channels facilitate electrogenic cation transport in organelles to maintain the internal pH more acidic than the cytoplasm. Cl- transport maintains volume in organelles and critical volume dependent functions such as stress-induced mitochondrial swelling and release of enzymes across the inner mitochondrial membrane are influenced by chloride channel activity. Maintaining acidic pH within organelles is crucial for proper processing of proteins that are destined to localize to the integral membrane or to be secreted, and organellar pH is especially important for endosomes and trans-Golgi network. The proton gradient within intracellular organelles is maintained by the action of ATPases that generate positive electrical potential, coincidentally promoting acidification. The large positive electric potential inside the organelle created by ATPase proton-pumps ultimately limits further transport of protons, thus preventing further reduction of pH. Additional pH regulation can be achieved by activation of chloride channels on the organellar membrane to allow Cl- to diffuse from the cytoplasm into the organelle. This shunting procedure reduces the positive electric potential, and allows ATPases to transport more protons to optimize organellar pH. Thus, chloride conductance is necessary for proton transport, and proton-mediated acidification can vary depending on the activity of Cl- in intracellular organelles. Acidification mediated by intracellular chloride channels can either initiate or further amplifies apoptotic signals by activating pH-dependent endonucleases (DNA fragmentation) and accelerating chromatin condensation in the nucleus, enhancing the process of apoptosis.Fernandez-Salas E found CLIC4/mtCLIC participates in the apoptotic response,Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis.The putative nuclear localization signal (NLS) on the C terminus of CLIC4 suggested that the NLS may be involved in the nuclear translocation and help CLIC4 translocate from cytoplasmic to the nucleus. The contribution of the nucear-targeted CLIC4 for the cellular metabolism regulation is still futher investigation.RNA interference (RNAi) is the process of sequence-specific posttranscriptional gene silencing triggered by double-stranded RNAs(dsRNAs) homologous to the silenced gene. This phenomenon was first observed from studies in Caenorhabditis elegans and Drosophila melanogaster and was subsequently found in other organisms.Whether artificial synthetic or Small RNA (siRNAs) transcripted from plasmids can target complementary mRNAs for degradation RNAi. Because there still has no specific inhibitors for chloride channels, we use DNA-vector-based RNAi approach for establishing a long-term siRNA strategy to blocking CLIC4 expression in glioma cells and to determine the role of CLIC4 during Hydrogen Peroxide-induced Glioma Cell Injuries. To explore the role and molecular mechanism of RNAi suppression of the mitochondrial intracellular chloride channel during H2O2-induced glial cell injury.Methods:(1) To detect the CLIC4 localization in C6 cells by cytoimmunochemical staining and identify the function of CLIC4 by MTT,RT-PCR and Western Blotting.(2) Sequence and analyze the CLIC4 cDNA through constructing pMD18-T-CLIC4 plasmid.(3) To construct intracellular chloride channel 4 (CLIC4) gene-specific small interfering RNA(siRNA) expression vectors and detect their silencing effects.(4) RT-PCR and Western blotting analysis were used to detect the CLIC4-mRNA and CLIC4 protein level and the contribution during H2O2-induced glial cell injury.Results:(1) CLIC4 localizes to the mitochondria, nucleus and cytoplasm of glioma C6 cells detected by immunocytochemical staining.(2) During the H2O2-induced glioma C6 cells injuries,CLIC4 protein as well as the Caspase3 and VDAC1 proteins expression increased significantly. At the same time the ratio of Bax/Bcl-2 mRNA and protein increased obviously.(3) We have identified the cDNA nucleotide and deduced amino acid sequences of CLIC4 in SD rattus glioma cells (GenBankTM accession number EF397567) for the first time.(4) The DNA fragments encoding CLIC4-targeted siRNA were subcloned into the pSilencer 3.1-H1 plasmids and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR analysis revealed a strongly decreased level of CLIC4 mRNA in C6 cells stably transfected with the pSilencer 3.1-H1 constructs of siRNA compared with the negative control or untransfected group.(5) RNAi suppression of the CLIC4 enhanced H₂O₂-induced glioma cell apoptosis.Conclusions: In this study, we have identified for the first time the cDNA nucleotide and deduced amino acid sequences of CLIC4 in SD rattus glioma cells (GenBankTM accession number EF397567) and successfully constructed a recombinant siRNA expression plasmid which can significantly inhibit the CLIC4 gene expression. We found that the mitochondrial-pathway apoptosis plays a very important role during the H₂O₂-induced glioma C6 cells apoptosis. RNAi suppression of the CLIC4 enhanced H₂O₂-induced glioma cell apoptosis and may caused the CLIC4 to translocate to the nucleus.These suggest that CLIC4 influences a variety of important cellular controls for proliferation and viability that are involved in cancer pathogenesis, and CLIC4 protein should be considered as potential molecular targets for cancer therapy.