Cdc2 Research Articles

A new layer of regulation of chromosomal passenger complex (CPC) translocation in budding yeast.

The conserved chromosomal passenger complex (CPC) consists of Ipl1Aurora-B, Sli15INCENP, Bir1Survivin, and Nbl1Borealin, and localizes at the kinetochore/centromere to correct kinetochore attachment errors and to prevent checkpoint silencing. After anaphase entry, the CPC moves from the kinetochore/centromere to the spindle. In budding yeast, CPC subunit Sli15 is phosphorylated by both cyclin-dependent kinase (CDK) and Ipl1 kinase. Following anaphase onset, activated Cdc14 phosphatase reverses Sli15 phosphorylation imposed by CDK to promote CPC translocation. Although abolished Sli15 phosphorylation imposed by Ipl1 also causes CPC translocation, the regulation of Ipl1-imposed Sli15 phosphorylation remains unclear. In addition to Sli15, Cdc14 also dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. Here, we present evidence supporting the notion that kinetochore-localized Fin1-PP1 likely reverses Ipl1-imposed Sli15 phosphorylation to promote CPC translocation from the kinetochore/centromere to the spindle. Importantly, premature Fin1 kinetochore localization or phospho-deficient sli15 mutation causes checkpoint defects in response to tensionless attachments, resulting in chromosome missegregation. In addition, our data indicate that reversion of CDK- and Ipl1-imposed Sli15 phosphorylation shows an additive effect on CPC translocation. Together, these results reveal a previously unidentified pathway to regulate CPC translocation, which is important for accurate chromosome segregation.

Cdc14 phosphatase counteracts Cdk-dependent Dna2 phosphorylation to inhibit resection during recombinational DNA repair

Cyclin-dependent kinase (Cdk) stimulates resection of DNA double-strand breaks ends to generate single-stranded DNA (ssDNA) needed for recombinational DNA repair. Here we show in Saccharomyces cerevisiae that lack of the Cdk-counteracting phosphatase Cdc14 produces abnormally extended resected tracts at the DNA break ends, involving the phosphatase in the inhibition of resection. Over-resection in the absence of Cdc14 activity is bypassed when the exonuclease Dna2 is inactivated or when its Cdk consensus sites are mutated, indicating that the phosphatase restrains resection by acting through this nuclease. Accordingly, mitotically activated Cdc14 promotes Dna2 dephosphorylation to exclude it from the DNA lesion. Cdc14-dependent resection inhibition is essential to sustain DNA re-synthesis, thus ensuring the appropriate length, frequency, and distribution of the gene conversion tracts. These results establish a role for Cdc14 in controlling the extent of resection through Dna2 regulation and demonstrate that the accumulation of excessively long ssDNA affects the accurate repair of the broken DNA by homologous recombination.

Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast

Timely completion of genome replication is a prerequisite for mitosis, genome integrity, and cell survival. A challenge to this timely completion comes from the need to replicate the hundreds of untranscribed copies of rDNA that organisms maintain in addition to the copies required for ribosome biogenesis. Replication of these rDNA arrays is relegated to late S phase despite their large size, repetitive nature, and essentiality. Here, we show that, in Saccharomyces cerevisiae, reducing the number of rDNA repeats leads to early rDNA replication, which results in delaying replication elsewhere in the genome. Moreover, cells with early-replicating rDNA arrays and delayed genome-wide replication aberrantly release the mitotic phosphatase Cdc14 from the nucleolus and enter anaphase prematurely. We propose that rDNA copy number determines the replication time of the rDNA locus and that the release of Cdc14 upon completion of rDNA replication is a signal for cell cycle progression.

Cdc14 phosphatase contributes to cell wall integrity and pathogenesis in Candida albicans.

The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. Here, we identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm ) as the sole source of Cdc14 proliferated like the wild-type parent strain but exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a novel and conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reduced Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity is important for C. albicans cell wall integrity and pathogenesis and suggest that Cdc14 may be worth future exploration as an antifungal drug target.

Downregulation of CDC25C in NPCs Disturbed Cortical Neurogenesis.

Cell division regulators play a vital role in neural progenitor cell (NPC) proliferation and differentiation. Cell division cycle 25C (CDC25C) is a member of the CDC25 family of phosphatases which positively regulate cell division by activating cyclin-dependent protein kinases (CDKs). However, mice with the Cdc25c gene knocked out were shown to be viable and lacked the apparent phenotype due to genetic compensation by Cdc25a and/or Cdc25b. Here, we investigate the function of Cdc25c in developing rat brains by knocking down Cdc25c in NPCs using in utero electroporation. Our results indicate that Cdc25c plays an essential role in maintaining the proliferative state of NPCs during cortical development. The knockdown of Cdc25c causes early cell cycle exit and the premature differentiation of NPCs. Our study uncovers a novel role of CDC25C in NPC division and cell fate determination. In addition, our study presents a functional approach to studying the role of genes, which elicit genetic compensation with knockout, in cortical neurogenesis by knocking down in vivo.

String/Cdc25 phosphatase is a suppressor of Tau-associated neurodegeneration.

Tau pathology is defined by the intracellular accumulation of abnormally phosphorylated Tau (MAPT) and is prevalent in several neurodegenerative disorders. The identification of modulators of Tau abnormal phosphorylation and aggregation is key to understanding disease progression and developing targeted therapeutic approaches. In this study, we identified String (Stg)/Cdc25 phosphatase as a suppressor of abnormal Tau phosphorylation and associated toxicity. Using a Drosophila model of tauopathy, we showed that Tau dephosphorylation by Stg/Cdc25 correlates with reduced Tau oligomerization, brain vacuolization and locomotor deficits in flies. Moreover, using a disease mimetic model, we provided evidence that Stg/Cdc25 reduces Tau phosphorylation levels independently of Tau aggregation status and delays neurodegeneration progression in the fly. These findings uncover a role for Stg/Cdc25 phosphatases as regulators of Tau biology that extends beyond their well-characterized function as cell-cycle regulators during cell proliferation, and indicate Stg/Cdc25-based approaches as promising entry points to target abnormal Tau phosphorylation.

A novel human multiple myeloma cell line with a 1q21 gain genetic abnormality and CKS1B overexpression.

Multiple myeloma (MM) is an incurable hematologic malignancy mainly due to its cytogenetic abnormalities. Therefore, it is important to establish permanent malignant MM cell lines as tools to develop more effective therapies. Pleural effusion cells of a 70-year-old patient was collected to establish the CZ2 cell line. Characterization of CZ2 was determined with nephelometry, flow cytometry, fluorescence in situ hybridization (FISH), and chromosomal microarray analysis (CMA). Western blotting analysis was adopted to determine protein expression. Cell viability was measured by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We established and characterized a new MM cell line, CZ2. Using nephelometry and flow cytometry, cells with typical plasma cell morphology but not classical plasma cell phenotype were found to be non-immunoglobulin-secretary cells. FISH analysis of cells revealed a unique characteristic, namely, that there was only gain of the 1q21 region (1q21+). No other common cytogenetic abnormalities in MM, such as deletion of 17p (17p-), deletion of 13q (13q-), or translocation of immunoglobulin heavy chain (IgH), were observed. In addition, the original cell line maintains its single cytogenetic abnormality. Meanwhile, we observed through western blotting that CDC28 protein kinase regulatory subunit 1B (CKS1B), an adverse prognostic gene located in the 1q21 region, was highly expressed in CZ2. Knockdown of CKS1B reduced cell viability and also increased the levels of cleaved-poly(ADP-ribose) polymerase (cleaved-PARP) and cleaved-caspase3. CZ2 provides a suitable material for cellular and molecular studies of MM with only a 1q21 abnormality. This cell line is characterized by a gain of 1q21, and the high expression of CKS1B is an important model for studies of myeloma cell growth and drug resistance during therapy.

Abstract 2160: Human Cdc14 Phosphatase Stimulation by a

Abstract 2160: Human Cdc14 Phosphatase Stimulation by a

Mammalian CDC14 phosphatases control exit from stemness in pluripotent cells.

Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal.

CDC25B Inhibition by Menadione: A Potential New Therapeutical Approach

Gastric cancer (GC) is the fifth most common type of tumor and the third leading cause of cancer death worldwide. The evolution of gastric carcinogenesis is still poorly understood and, for this reason, preclinical research protocols were established that included the development of gastric cancer cell lines and the establishment of models of gastric carcinogenesis in non-human primates such as Sapajus apella. A comprehensive literature search was performed in relevant databases such as PubMed, ResearchGate, and Google Scholar to identify studies related to the topic. After an in-depth study of these reports, significant data were collected and compiled under appropriate headings. The main result of the studies carried out by the group on GC is the demonstration of the MYC gene overexpression as a common phenomenon in stomach carcinogenesis. Furthermore, we revealed that reducing the expression of the CDC25B gene, regulated by the MYC protein, is a therapeutic strategy against stomach tumors. This review article reveals preclinical evidence that treatment with menadione in experimental models of gastric tumorigenesis, in vivo and in vitro, inhibits the action of the phosphatase CDC25B and, consequently, prevents cell proliferation, invasion, and migration.

A quantitative and spatial analysis of cell cycle regulators during the fission yeast cycle

We have carried out a systems-level analysis of the spatial and temporal dynamics of cell cycle regulators in the fission yeast Schizosaccharomyces pombe. In a comprehensive single-cell analysis, we have precisely quantified the levels of 38 proteins previously identified as regulators of the G2 to mitosis transition and of 7 proteins acting at the G1- to S-phase transition. Only 2 of the 38 mitotic regulators exhibit changes in concentration at the whole-cell level: the mitotic B-type cyclin Cdc13, which accumulates continually throughout the cell cycle, and the regulatory phosphatase Cdc25, which exhibits a complex cell cycle pattern. Both proteins show similar patterns of change within the nucleus as in the whole cell but at higher concentrations. In addition, the concentrations of the major fission yeast cyclin-dependent kinase (CDK) Cdc2, the CDK regulator Suc1, and the inhibitory kinase Wee1 also increase in the nucleus, peaking at mitotic onset, but are constant in the whole cell. The significant increase in concentration with size for Cdc13 supports the view that mitotic B-type cyclin accumulation could act as a cell size sensor. We propose a two-step process for the control of mitosis. First, Cdc13 accumulates in a size-dependent manner, which drives increasing CDK activity. Second, from mid-G2, the increasing nuclear accumulation of Cdc25 and the counteracting Wee1 introduce a bistability switch that results in a rapid rise of CDK activity at the end of G2 and thus, brings about an orderly progression into mitosis.

Regulation of oocyte maturation: Role of conserved ERK signaling.

During oogenesis, oocytes arrest at meiotic prophase I to acquire competencies for resuming meiosis, fertilization, and early embryonic development. Following this arrested period, oocytes resume meiosis in response to species-specific hormones, a process known as oocyte maturation, that precedes ovulation and fertilization. Involvement of endocrine and autocrine/paracrine factors and signaling events during maintenance of prophase I arrest, and resumption of meiosis is an area of active research. Studies in vertebrate and invertebrate model organisms have delineated the molecular determinants and signaling pathways that regulate oocyte maturation. Cell cycle regulators, such as cyclin-dependent kinase (CDK1), polo-like kinase (PLK1), Wee1/Myt1 kinase, and the phosphatase CDC25 play conserved roles during meiotic resumption. Extracellular signal-regulated kinase (ERK), on the other hand, while activated during oocyte maturation in all species, regulates both species-specific, as well as conserved events among different organisms. In this review, we synthesize the general signaling mechanisms and focus on conserved and distinct functions of ERK signaling pathway during oocyte maturation in mammals, non-mammalian vertebrates, and invertebrates such as Drosophila and Caenorhabditis elegans.

Comprehensive Expression Profiling and Molecular Basis of CDC28 Protein Kinase Regulatory Subunit 2 in Cervical Cancer.

More and more evidence suggests the oncogenic function of overexpressed CDC28 protein kinase regulatory subunit 2 (CKS2) in various human cancers. However, CKS2 has rarely been studied in cervical cancer. Herein, taking advantage of massive genetics data from multicenter RNA-seq and microarrays, we were the first group to perform tissue microarrays for CKS2 in cervical cancer. We were also the first to evaluate the clinical significance of CKS2 with large samples (980 cervical cancer cases and 422 noncancer cases). We further excavated the mechanism of the tumor-promoting activities of CKS2 in cervical cancer through analysis of genetic mutation profiles, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) significant enrichment of genes coexpressed with CKS2. According to the results, expression data from multilevels unanimously supported the overexpression of CKS2 in cervical cancer. Patients with cervical cancer in stage II from inhouse microarrays had significantly higher expression of CKS2, and CKS2 overexpression had an adverse impact on the disease-free survival status of cervical cancer patients in GSE44001. Both mutation types of mRNA high and mRNA low appeared in cervical cancer cases from the TCGA Firehose project. Gene coexpressed with CKS2 participated in pathways including the cell cycle, estrogen signaling pathway, and DNA replication. In summary, upregulated CKS2 is closely associated with the malignant clinical development of cervical cancer and might serve as a valuable therapeutic target in cervical cancer.

Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast.

Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog slp1. While depletion of slp1 readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55Pab1 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56Par1 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55Pab1 and PP2A-B56Par1, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56Par1 in a mechanism independent of CDK.

Identification of a Pseudosubstrate Stimulatory Motif Uncovers Novel Roles of Cdc14 Phosphatase in Fungal Cell Wall Integrity

Fungal pathogens are a growing threat to human health and global food security as they are becoming increasingly resistant to the few existing antifungal treatments. Cdc14 is a protein phosphatase primarily known for its essential role in controlling mitotic exit in the model yeast Saccharomyces cerevisiae. However, in other fungal species Cdc14 is non‐essential and the reasons for its strict conservation across Dikarya remain unclear. Recently, Cdc14 was shown to be required for host infection by several plant pathogenic fungi. Here, we show that Cdc14 is also required for virulence in the human pathogen, Candida albicans.These observations, coupled with our structural and biochemical knowledge of Cdc14 phosphatases, suggest it could be a useful target for antifungal development. In our search for differences between fungal and animal Cdc14 enzymes that could be exploited for antifungal design, we discovered an invariant motif in the otherwise disordered C‐terminal tail of fungal Cdc14 orthologs. The motif resembles the recognition sequence of optimal Cdc14 substrates. We provide evidence from molecular modeling and enzyme kinetic studies that this motif is a “pseudosubstrate” that binds the Cdc14 active site and functions to accelerate the rate‐limiting catalytic step, providing a plausible mechanism for dynamic control of intracellular Cdc14 activity. The turnover rate for Cdc14 lacking a functional pseudosubstrate motif is reduced by an order of magnitude compared to the wild‐type enzyme. Both S. cerevisiaeand C. albicanscells expressing Cdc14 variants with point mutations in this motif exhibit a specific and pronounced sensitivity to cell wall stresses, including treatment with echinocandin antifungal drugs that inhibit cell wall synthesis. Moreover, signaling through the cell wall integrity pathway is chronically elevated in these cells, indicative of a cell wall structural defect. Preliminary evidence in synchronized S. cerevisiaecultures with reduced Cdc14 activity suggests that cell wall integrity is compromised around the time of cytokinesis and septation, consistent with its known functions in these processes. We are currently testing if the pseudosubstrate motif is required for virulence of C. albicans.Our findings reveal 1) a novel contribution to catalysis of a structural element outside the conserved catalytic core of Cdc14 enzymes and 2) novel roles of Cdc14 in promoting cell wall integrity and pathogenesis. More detailed characterization of the mechanisms by which Cdc14 contributes to cell wall integrity and host infection in human pathogenic fungi should help assess its future value for antifungal development.

A Comprehensive Overview of the Developments of Cdc25 Phosphatase Inhibitors.

Cdc25 phosphatases have been considered promising targets for anticancer development due to the correlation of their overexpression with a wide variety of cancers. In the last two decades, the interest in this subject has considerably increased and many publications have been launched concerning this issue. An overview is constructed based on data analysis of the results of the previous publications covering the years from 1992 to 2021. Thus, the main objective of the current review is to report the chemical structures of Cdc25s inhibitors and answer the question, how to design an inhibitor with better efficacy and lower toxicity?

Anti-hepatoma Effect of DC2.4 Cells Transfected with Tumor-Associated Antigen Cdc25C In Vitro.

Cell division cyclin 25 homolog C (Cdc25C) is a tumor-associated antigen candidate gene, and this may be used as an effective target in cancer treatment. The present study aims to evaluate the lysis effect of cytotoxic T lymphocytes (CTLs) induced by dendritic cell line DC2.4 overexpressing Cdc25C, and the feasibility of Cdc25C as a component in hepatoma immunotherapy. The mouse Cdc25C gene was ligated into a lentiviral vector, and transfected into DC2.4 cells. The DC2.4 cell phenotype and cytokine secretion were determined by flow cytometry and ELISA, respectively. CD8+ T cells were sorted from the spleens of C57BL/6 mice using a magnetic bead sorting kit obtained from Miltenyi Biotech, Germany, and co-cultured with DC2.4 cells for one week as effector cells. Then, IL-2, granzyme B and perforin were detected in the CTL culture medium by ELISA. Next, time-resolved fluorescence immunoassay was used to detect the immune killing effect of Cdc25C-specific CTLs on target cells. Meanwhile, the effect of blocking MHC-I sites on target cells with a monoclonal anti-MHC-I antibody was evaluated. The results revealed that Cdc25C could be stably overexpressed in DC2.4 cells by LV-Cdc25C infection. DC2.4 cells transfected with LV-Cdc25C secreted more IL-6, IL-12, TNF-α and IFN-γ, and had higher expression levels of CD40, CD86, CCR7 and MHC-II than unaltered DC2.4 cells. The elevated Cdc25C in dendritic cells also further increased the secretion of IL-2, granzyme B and perforin to elicit Cdc25C-specific CTLs, and induced the higher cytotoxicity in Hepa1-6 cell lines (P<0.05), but this had no effect on the target cells when MHC-I monoclonal antibodies were blocked. DC2.4 cells transfected with LV-Cdc25C can induce specific CTLs, and result in a strong cellular immune response. The dendritic cells that overexpress Cdc25C may be useful for hepatoma immunotherapy.

Cdc14 spatiotemporally dephosphorylates Atg13 to activate autophagy during meiotic divisions.

Autophagy is a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues. Autophagy is essential for the meiotic exit and sporulation in budding yeast, but the underlying molecular mechanisms remain unknown. Here, we show that autophagy is maintained during meiosis and stimulated in anaphase I and II. Cells with higher levels of autophagy complete meiosis faster, and genetically enhanced autophagy increases meiotic kinetics and sporulation efficiency. Strikingly, our data reveal that the conserved phosphatase Cdc14 regulates meiosis-specific autophagy. Cdc14 is activated in anaphase I and II, accompanying its subcellular relocation from the nucleolus to the cytoplasm, where it dephosphorylates Atg13 to stimulate Atg1 kinase activity and thus autophagy. Together, our findings reveal a meiosis-tailored mechanism that spatiotemporally controls meiotic autophagy activity to ensure meiosis progression, exit, and sporulation.

BN82002 alleviated tissue damage of septic mice by reducing inflammatory response through inhibiting AKT2/NF-κB signaling pathway

BN82002 is well-known as an inhibitor of the CDC25 phosphatase. However, it was recently reported that BN82002 also selectively suppressed AKT2 and reduced inflammatory responses in lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells. Therefore, in this study, we evaluated the alleviating efficacy of BN82002 in sepsis in vivo. BN82002 (50 μM) suppressed the mRNA levels of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in LPS-treated peritoneal macrophages without cytotoxicity. The septic in vivo mouse model was established on the basis of the endotoxin model using poly(I:C) (10 mg/kg) and LPS (54 mg/kg). In histological analysis, peritoneal injection of BN82002 (20 mg/kg) significantly reduced lung, kidney, and liver damage. Lung edema and serum alanine transaminase (ALT), aspartate transaminase (AST), TNF-α, IL-1β, and nitric oxide (NO) levels also were decreased by BN82002 (20 mg/kg). In addition, BN82002 (20 mg/kg) suppressed the mRNA levels of TNF-α in lung and liver tissues. Gene expression levels of IL-1β and IL-6 were decreased in lung, kidney, and liver in the BN82002 (20 mg/kg) group. Furthermore, p-AKT2 and p-IκBα levels were reduced by BN82002 (20 mg/kg). Finally, all septic mice died 7 days after poly(I:C)/LPS-injection, whereas 4 mice in the BN82002 (20 mg/kg) group, survived strongly suggesting that BN82002 reduces sepsis mortality. In conclusion, we verified that pre-treatment with BN82002 protects against tissue damage and increases survival by inhibiting AKT2-NF-κB signaling in septic mice. These results suggest that BN82002 could be utilized in the treatment of sepsis.

Regulation of microRNA miR-197-3p/CDC28 protein kinase regulatory subunit 1B (CKS1B) axis by Circular RNA hsa_circ_0000285 promotes glioma progression

ABSTRACT Circular RNA circ_0000285 is differentially expressed in several malignancies; however, its role in gliomas is under investigation. Reverse transcription quantitative polymerase chain reaction was conducted to evaluate the expression of circ_0000285, miR-197-3p, and CDC28 protein kinase regulatory subunit 1B (CKS1B) in glioma tissues and cells. Cell Counting Kit-8 and Transwell invasion assays coupled with Western blotting analysis using anti-Bax and anti-Bcl-2 antibodies were performed to evaluate cell proliferation, invasion, and apoptosis. Luciferase reporter and AGO2 RNA immunoprecipitation assays were conducted to verify the interaction between miR-197-3p and circ_0000285 or CKS1B. Xenograft tumor growth was evaluated in mice. We noted that circ_0000285 was highly expressed in glioma tissues and cells and that circ_0000285-silencing retarded tumor growth both in vitro and in vivo. This effect was mediated by the binding of circ_0000285 to miR-197-3p, which silenced CKS1B, an essential driver of glioma cell proliferation and invasion. Thus, circ_0000285 boosted glioma progression by regulating the miR-197-3p/CKS1B axis, highlighting a novel competing endogenous RNA circuit of glioma progression.