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Chloride intracellular channel gene knockdown induces insect cell lines death and level increases of intracellular calcium ions.

ABSTRACT: Chloride intracellular channel (CLIC) is a member of the chloride channel protein family for which growing evidence supports a pivotal role in fundamental cellular events. However, the physiological function of CLIC in insects is still rarely uncovered. The ovary-derived High Five (Hi-5) cell line isolated from the cabbage looper (Trichoplusia ni) is widely used in laboratories. Here, we studied both characteristics and functions of CLIC in Hi-5 cells (TnCLIC). We identified the TnCLIC gene in Hi-5 cells and annotated highly conserved CLIC proteins in most insect species. After RNA interference of TnCLIC, the phenomenon of significantly increased cell death suggests that the TnCLIC protein is essential for the survival of Hi-5 cells. The same lethal effect was also observed in Spodoptera frugiperda 9 and Drosophila melanogaster Schneider 2 cells after CLIC knockdown. Furthermore, we found that this kind of cell death was accompanied by increases in intracellular calcium ions after TnCLIC knockdown with the transcriptomic analyses and the detection of calcium levels. Our results provide insights into insect CLIC as a key factor for cell survival and lay the foundation for the cell death mechanism.

SUBMITTER: Song J

PROVIDER: S-EPMC10356983 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Chloride intracellular channel gene knockdown induces insect cell lines death and level increases of intracellular calcium ions.

Song Jiqiang J Yu Yanping Y Yan Zhichao Z Xiao Shan S Zhao Xianxin X Wang Fang F Fang Qi Q Ye Gongyin G

Frontiers in physiology 20230706

Chloride intracellular channel (CLIC) is a member of the chloride channel protein family for which growing evidence supports a pivotal role in fundamental cellular events. However, the physiological function of CLIC in insects is still rarely uncovered. The ovary-derived High Five (Hi-5) cell line isolated from the cabbage looper (Trichoplusia ni) is widely used in laboratories. Here, we studied both characteristics and functions of CLIC in Hi-5 cells (TnCLIC). We identified the <i ...[more]

PMID: 37485065

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Project description:Chloride intracellular channels (CLICs) are a unique family of evolutionarily conserved metamorphic proteins, switching between stable conformations based on redox conditions. CLICs have been implicated in a wide variety biological processes including ion channel activity, apoptosis, membrane trafficking, and enzymatic oxidoreductase activity. Understanding the molecular mechanisms by which CLICs engage in these activities is an area of active research. Here, the sole Drosophila melanogaster ortholog, Clic, was targeted for RNAi knockdown to identify genes and biological processes associated with Clic expression. Clic knockdown had a substantial impact on global transcription, altering expression of over 9% of transcribed Drosophila genes. Overrepresentation analysis of differentially expressed genes identified enrichment of 23 Gene Ontology terms including Cytoplasmic Translation, Oxidation-Reduction Process, Heme Binding, Membrane, Cell Junction, and Nucleolus. The top term, Cytoplasmic Translation, was enriched almost exclusively with downregulated genes. Drosophila Clic and vertebrate ortholog Clic4 have previously been tied to ethanol sensitivity and ethanol-regulated expression. Clic knockdown-responsive genes from the present study were found to overlap significantly with gene sets from 4 independently published studies related to ethanol exposure and sensitivity in Drosophila. Bioinformatic analysis of genes shared between these studies revealed an enrichment of genes related to amino acid metabolism, protein processing, oxidation-reduction processes, and lipid particles among others. To determine whether the modulation of ethanol sensitivity by Clic may be related to co-regulated oxidation-reduction processes, we evaluated the effect of hyperoxia on ethanol sedation in Clic knockdown flies. Consistent with previous findings, Clic knockdown reduced acute ethanol sedation sensitivity in flies housed under nomoxia. However, this effect was reversed by exposure to hyperoxia, suggesting a common set of molecular-genetic mechanism may modulate each of these processes. This study suggests that Drosophila Clic has a major influence on regulation of oxidative stress signaling and that this function overlaps with the molecular mechanisms of acute ethanol sensitivity in the fly.

Project description:BACKGROUND:Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K+ channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells. METHODS:We transduced C2C12 myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student's t test or two-way ANOVAs and SAS software as indicated. RESULTS:Expression of human ERG1A in C2C12 myotubes increased basal intracellular calcium concentration 51.7% (p?<?0.0001; n?=?177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p?<?0.08; n?=?24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p?<?0.05; n?=?6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p?<?0.05; n?=?6) in calpastatin which could contribute to enhanced calpain activity. CONCLUSIONS:Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.

Project description:Chloride intracellular channels (CLICs) are a unique family of evolutionarily conserved metamorphic proteins, switching between stable conformations based on redox conditions. CLICs have been implicated in a wide variety biological processes including ion channel activity, apoptosis, membrane trafficking, and enzymatic oxidoreductase activity. Understanding the molecular mechanisms by which CLICs engage in these activities is an area of active research. Here, the sole Drosophila melanogaster ortholog, Clic, was targeted for RNAi knockdown to identify genes and biological processes associated with Clic expression. Clic knockdown had a substantial impact on global transcription, altering expression of over 7% of transcribed Drosophila genes. Overrepresentation analysis of differentially expressed genes identified enrichment of Gene Ontology terms including Cytoplasmic Translation, Oxidation-Reduction Process, Heme Binding, Membrane, Cell Junction, and Nucleolus. The top term, Cytoplasmic Translation, was enriched almost exclusively with downregulated genes. Drosophila Clic and vertebrate ortholog Clic4 have previously been tied to ethanol sensitivity and ethanol-regulated expression. Clic knockdown-responsive genes from the present study were found to overlap significantly with gene sets from 4 independently published studies related to ethanol exposure and sensitivity in Drosophila. Bioinformatic analysis of genes shared between these studies revealed an enrichment of genes related to amino acid metabolism, protein processing, oxidation-reduction processes, and lipid particles among others. To determine whether the modulation of ethanol sensitivity by Clic may be related to co-regulated oxidation-reduction processes, we evaluated the effect of hyperoxia on ethanol sedation in Clic knockdown flies. Consistent with previous findings, Clic knockdown reduced acute ethanol sedation sensitivity in flies housed under normoxia. However, this effect was reversed by exposure to hyperoxia, suggesting a common set of molecular-genetic mechanism may modulate each of these processes. This study suggests that Drosophila Clic has a major influence on regulation of oxidative stress signaling and that this function overlaps with the molecular mechanisms of acute ethanol sensitivity in the fly.

Project description:To investigate the effect of chloride intracellular channel 1 (CLIC1) on the cell proliferation, apoptosis, migration and invasion of gastric cancer cells.CLIC1 expression was evaluated in human gastric cancer cell lines SGC-7901 and MGC-803 by real time polymerase chain reaction (RT-PCR). Four segments of small interference RNA (siRNA) targeting CLIC1 mRNA and a no-sense control segment were designed by bioinformatics technology. CLIC1 siRNA was selected using Lipofectamine 2000 and transfected transiently into human gastric cancer SGC-7901 and MGC-803 cells. The transfected efficiency was observed under fluorescence microscope. After transfection, mRNA expression of CLIC1 was detected with RT-PCR and Western blotting was used to detect the protein expression. Proliferation was examined by methyl thiazolyl tetrazolium and apoptosis was detected with flow cytometry. Polycarbonate membrane transwell chamber and Matrigel were used for the detection of the changes of invasion and migration of the two cell lines.In gastric cancer cell lines SGC-7901 and MGC-803, CLIC1 was obviously expressed and CLIC1 siRNA could effectively suppress the expression of CLIC1 protein and mRNA. Proliferation of cells transfected with CLIC1 siRNA3 was enhanced notably, and the highest proliferation rate was 23.3% (P = 0.002) in SGC-7901 and 35.55% (P = 0.001) in MGC-803 cells at 48 h. The G2/M phase proportion increased, while G0/G1 and S phase proportions decreased. The apoptotic rate of the CLIC1 siRNA3 group obviously decreased in both SGC-7901 cells (62.24%, P = 0.000) and MGC-803 cells (52.67%, P = 0.004). Down-regulation of CLIC1 led to the inhibition of invasion and migration by 54.31% (P = 0.000) and 33.62% (P = 0.001) in SGC-7901 and 40.74% (P = 0.000) and 29.26% (P = 0.002) in MGC-803. However, there was no significant difference between the mock group cells and the negative control group cells.High CLIC1 expression can efficiently inhibit proliferation and enhance apoptosis, migration and invasion of gastric cancer cells in vitro. CLIC1 might be a promising target for the treatment of gastric cancer.

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Chloride intracellular channel gene knockdown induces insect cell lines death and level increases of intracellular calcium ions.

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<i>Chloride intracellular channel</i> gene knockdown induces insect cell lines death and level increases of intracellular calcium ions.

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