Background/Aims: Treatment of breast cancer remains a clinical challenge. This study aims to validate exosomal microRNA-1246 (miR-1246) as a serum biomarker for breast cancer and understand the ...underlying mechanism in breast cancer progression. Methods: The expression levels of endogenous and exosomal miRNAs were examined by real time PCR, and the expression level of the target protein was detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study their uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-1246 was estimated by invasion assay and cell viability assay. Results: In this study, we demonstrate that exosomes carrying microRNA can be transferred among different cell lines through direct uptake. miR-1246 is highly expressed in metastatic breast cancer MDA-MB-231 cells compared to non-metastatic breast cancer cells or non-malignant breast cells. Moreover, miR-1246 can suppress the expression level of its target gene, Cyclin-G2 (CCNG2), indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could enhance the viability, migration and chemotherapy resistance of non-malignant HMLE cells. Conclusions: Together, our results support an important role of exosomes and exosomal miRNAs in regulating breast tumor progression, which highlights their potential for applications in miRNA-based therapeutics.
Gastric cancer is one of the most common malignant tumors. Cyclin G2 has been shown to be associated with the development of multiple types of tumors, but its underlying mechanisms in gastric tumors ...is not well-understood. The aim of this study is to investigate the role and the underlying mechanisms of cyclin G2 on Wnt/β-catenin signaling in gastric cancer.
Real-time PCR, immunohistochemistry and in silico assay were used to determine the expression of cyclin G2 in gastric cancer. TCGA datasets were used to evaluate the association between cyclin G2 expression and the prognostic landscape of gastric cancers. The effects of ectopic and endogenous cyclin G2 on the proliferation and migration of gastric cancer cells were assessed using the MTS assay, colony formation assay, cell cycle assay, wound healing assay and transwell assay. Moreover, a xenograft model and a metastasis model of nude mice was used to determine the influence of cyclin G2 on gastric tumor growth and migration in vivo. The effects of cyclin G2 expression on Wnt/β-catenin signaling were explored using a TOPFlash luciferase reporter assay, and the molecular mechanisms involved were investigated using immunoblots assay, yeast two-hybrid screening, immunoprecipitation and Duolink in situ PLA. Ccng2
mice were generated to further confirm the inhibitory effect of cyclin G2 on Wnt/β-catenin signaling in vivo. Furthermore, GSK-3β inhibitors were utilized to explore the role of Wnt/β-catenin signaling in the suppression effect of cyclin G2 on gastric cancer cell proliferation and migration.
We found that cyclin G2 levels were decreased in gastric cancer tissues and were associated with tumor size, migration and poor differentiation status. Moreover, overexpression of cyclin G2 attenuated tumor growth and metastasis both in vitro and in vivo. Dpr1 was identified as a cyclin G2-interacting protein which was required for the cyclin G2-mediated inhibition of β-catenin expression. Mechanically, cyclin G2 impacted the activity of CKI to phosphorylate Dpr1, which has been proved to be a protein that acts as a suppressor of Wnt/β-catenin signaling when unphosphorylated. Furthermore, GSK-3β inhibitors abolished the cyclin G2-induced suppression of cell proliferation and migration.
This study demonstrates that cyclin G2 suppresses Wnt/β-catenin signaling and inhibits gastric cancer cell growth and migration through Dapper1.
Impairment of ribosome biogenesis leads to p53 induction and cell cycle arrest, a checkpoint involved in human disease. Induction of p53 is attributed to the binding and inhibition of human double ...minute 2 (Hdm2) by a subset of ribosomal proteins (RPs): RPS7, RPL5, RPL11, and RPL23. However, we found that only RPL11 or RPL5, in a mutually dependent manner, elicit this response. We show that depletion of RPS7 or RPL23, like depletion of other RPs, except for RPL11 and RPL5, induces a p53 response and that the effects of RPS7 and RPL23 on p53 induction reported earlier may be ascribed to inhibition of global translation. Moreover, we made the surprising observation that codepletion of two essential RPs, one from each subunit, but not the same subunit, leads to suprainduction of p53. This led to the discovery that the previously proposed RPL11-dependent mechanism of p53 induction, thought to be caused by abrogation of 40S biogenesis and continued 60S biogenesis, is still operating, despite abrogation of 60S biogenesis. This response leads to both a G1 block and a novel G2/M block not observed when disrupting either subunit alone. Thus, induction of p53 is mediated by distinct mechanisms, with the data pointing to an essential role for ribosomal subunits beyond translation.
Hepatitis C virus (HCV) exposure leads to persistent life‐long infections characterized by chronic inflammation often developing into cirrhosis and hepatocellular carcinoma. The mechanism by which ...HCV remains in the liver while inducing an inflammatory and antiviral response remains unclear. Though the innate immune response to HCV in patients seems to be quite active, HCV has been shown in cell culture to employ a diverse array of innate immune antagonists, which suggests that current model systems to study interactions between HCV and the innate immune system are not representative of what happens in vivo. We recently showed that hepatoma‐derived HepG2 cells support the entire HCV life cycle if the liver‐specific microRNA, miR‐122, is expressed along with the entry factor, CD81 (termed HepG2‐HFL cells). We found that there was a striking difference in these cells' ability to sustain HCV infection and spread when compared with Huh‐7 and Huh‐7.5 cells. Additionally, HepG2‐HFL cells exhibited a more robust antiviral response when challenged with other RNA viruses and viral mimetics than Huh‐7 and Huh‐7.5 cells. HCV infection elicited a potent interferon‐lambda (IFN‐λ), IFN‐stimulated gene, and cytokine response in HepG2‐HFL cells, but not in Huh‐7 cells, suggesting that HepG2‐HFL cells more faithfully recapitulate the innate immune response to HCV infection in vivo. Using this model, we found that blocking the retinoic acid‐inducible gene I (RIG‐I)‐like receptor pathway or the IFN‐λ‐signaling pathway promoted HCV infection and spread in HepG2‐HFL cells. Conclusion: HepG2‐HFL cells represent a new system to study the interaction between HCV and the innate immune system, solidifying the importance of IFN‐λ in hepatic response to HCV infection and revealing non‐redundant roles of RIG‐I and melanoma differentiation‐associated protein 5 in HCV recognition and repression of infection. (Hepatology 2014;60:1170–1179)
In vitro preclinical models for the assessment of drug-induced liver injury (DILI) are usually based on cryopreserved primary human hepatocytes (cPHH) or human hepatic tumor-derived cell lines; ...however, it is unclear how well such cell models reflect the normal function of liver cells. The physiological, pharmacological, and toxicological phenotyping of available cell-based systems is necessary in order to decide the testing purpose for which they are fit. We have therefore undertaken a global proteomic analysis of 3 human-derived hepatic cell lines (HepG2, Upcyte, and HepaRG) in comparison with cPHH with a focus on drug metabolizing enzymes and transport proteins (DMETs), as well as Nrf2-regulated proteins. In total, 4946 proteins were identified, of which 2722 proteins were common across all cell models, including 128 DMETs. Approximately 90% reduction in expression of cytochromes P450 was observed in HepG2 and Upcyte cells, and approximately 60% in HepaRG cells relative to cPHH. Drug transporter expression was also lower compared with cPHH with the exception of MRP3 and P-gp (MDR1) which appeared to be significantly expressed in HepaRG cells. In contrast, a high proportion of Nrf2-regulated proteins were more highly expressed in the cell lines compared with cPHH. The proteomic database derived here will provide a rational basis for the context-specific selection of the most appropriate 'hepatocyte-like' cell for the evaluation of particular cellular functions associated with DILI and, at the same time, assist in the construction of a testing paradigm which takes into account the in vivo disposition of a new drug.
To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin ...homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G1/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G2/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.
Background: DNA damage triggers cell cycle checkpoints to halt cell division ahead of DNA repair.
Results: Ectopic cyclin G2 (CycG2) induces a Chk2-dependent cell cycle arrest, and depletion of endogenous CycG2 attenuates doxorubicin-induced G2/M-phase cell cycle arrest.
Conclusion: CycG2 influences checkpoint signaling and is required for G2/M arrest responses to genotoxic stress.
Significance: Proper checkpoint function is important for genomic integrity and tumor suppression.
Hydroquinone (HQ), a major metabolite of benzene and known hematotoxic carcinogen. MicroRNA 1246 (miR-1246), an oncogene, regulates target genes in carcinogenesis including leukemia. This study ...investigates the impact of exosomal derived miR-1246 from HQ-transformed (HQ19) cells on cell-to-cell communication in recipient TK6 cells.
RNA sequencing was used to identify differentially expressed exosomal miRNAs in HQ19 cells and its phosphate buffered solution control cells (PBS19), which were then confirmed using qRT-PCR. The impact of exosomal miR-1246 derived from HQ-transformed cells on cell cycle distribution was investigated in recipient TK6 cells.
RNA sequencing analysis revealed that 34 exosomal miRNAs were upregulated and 158 miRNAs were downregulated in HQ19 cells compared with PBS19 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses predicted that their targets are enriched in cancer development-related pathways, such as MAPK signaling, microRNAs in cancer, apoptosis, PI3K-Akt signaling, cell cycle, Ras signaling, and Chronic myeloid leukemia. Eleven miRNAs were confirmed to have differential expression through qRT-PCR, with 6 upregulated (miR-140-3p, miR-551b-3p, miR-7-5p, miR-1290, miR-92a-3p, and miR-1246) and 5 downregulated (miR-183-5p, miR-26a-5p, miR-30c-5p, miR-205-5p, and miR-99b-3p). Among these, miR-1246 exhibited the highest expression level. HQ exposure resulted in a concentration-dependent increase in miR-1246 levels and decrease Cyclin G2 (CCNG2) levels in TK6 cells. Similarly, exosomes from HQ19 exhibited similar effects as HQ exposure. Dual luciferase reporter gene assays indicated that miR-1246 could band to CCNG2. After HQ exposure, exosomal miR-1246 induced cell cycle arrest at the S phase, elevating the expression of genes like pRb, E2F1, and Cyclin D1 associated with S phase checkpoint. However, silencing miR-1246 caused G2/M-phase arrest.
HQ-transformed cells' exosomal miR-1246 targets CCNG2, regulating TK6 cell cycle arrest, highlighting its potential as a biomarker for HQ-induced malignant transformation.
We construct a compact manifold with a closed G2 structure not admitting any torsion‐free G2 structure, which is non‐formal and has first Betti number b1=1$b_1=1$. We develop a method of resolution ...for orbifolds that arise as a quotient M/Z2$M/{{\mathbb {Z}}_2}$ with M a closed G2 manifold under the assumption that the singular locus carries a nowhere‐vanishing closed 1‐form.
In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining ...(D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors.
In favorable conditions, eukaryotic cells proceed irreversibly through the cell division cycle (G1-S-G2-M) in order to produce two daughter cells with the same number and identity of chromosomes of ...their progenitor. The integrity of this process is maintained by "checkpoints" that hold a cell at particular transition points of the cycle until all requisite events are completed. The crucial functions of these checkpoints seem to depend on irreversible bistability of the underlying checkpoint control systems. Bistability of cell cycle transitions has been confirmed experimentally in frog egg extracts, budding yeast cells and mammalian cells. For fission yeast cells, a recent paper by Patterson
(2021) provides experimental evidence for an abrupt transition from G2 phase into mitosis, and we show that these data are consistent with a stochastic model of a bistable switch governing the G2/M checkpoint. Interestingly, our model suggests that their experimental data could also be explained by a reversible/sigmoidal switch, and stochastic simulations confirm this supposition. We propose a simple modification of their experimental protocol that could provide convincing evidence for (or against) bistability of the G2/M transition in fission yeast.
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