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Lung Aug 2023Bronchopulmonary dysplasia (BPD) is associated with hyperoxia-induced oxidative stress-associated ferroptosis. This study examined the effect of E26 oncogene homolog 1...
PURPOSE
Bronchopulmonary dysplasia (BPD) is associated with hyperoxia-induced oxidative stress-associated ferroptosis. This study examined the effect of E26 oncogene homolog 1 (ETS1) on oxidative stress-associated ferroptosis in BPD.
METHODS
Hyperoxia-induced A549 cells and neonatal mice were used to establish BPD models. The effects of ETS1 on hyperoxia-induced ferroptosis-like changes in A549 cells were investigated by overexpression of ETS1 plasmid transfection and erastin treatment. Glucose consumption, lactate production, and NADPH levels were assessed by the glucose, lactate, and NADP/NADPH assay kits, respectively. The potential regulatory relationship between ETS1 and Nrf2/HO-1 was examined by treating hyperoxia-induced A549 cells with the Nrf2 inhibitor ML385. ETS1 effect on the Nrf2 promoter was explored by dual-luciferase reporter and chromatin immunoprecipitation assay. The effect of ETS1 on the symptoms of BPD mice was examined by injecting an adenovirus overexpressing ETS1.
RESULTS
ETS1 overexpression increased hyperoxia-induced cell viability, glucose consumption, lactate production, and NADPH levels and reduced inflammation and apoptosis in A549 cells. In animal experiments, ETS1 overexpression prevented weight loss, airway enlargement, and reductions in radial alveolar counts in BPD mice, while reducing the mean linear intercept, mean alveolar diameter and inflammation. ETS1 overexpression suppressed PTGS2 and CHAC1 expression, reduced ROS, MDA and ferrous iron (Fe) production and increased GSH levels in hyperoxia-induced A549 cells and BPD mice. In addition, ETS1 can bind to the Nrf2 promoter region and thus promote Nrf2 transcription. ETS1 overexpression increased the mRNA and protein levels of Nrf2, HO-1, xCT, and GPX4 in hyperoxia-induced A549 cells and BPD mice. In hyperoxia-induced A549 cells, erastin and ML385 treatment abolished the effect of ETS1 overexpression.
CONCLUSION
ETS1 is important in oxidative stress-related ferroptosis in a hyperoxia-induced BPD model, and the effect is partially mediated by the Nrf2/HO-1 axis.
Topics: Animals; Humans; Infant, Newborn; Mice; Animals, Newborn; Bronchopulmonary Dysplasia; Ferroptosis; Hyperoxia; Lung; NADP; NF-E2-Related Factor 2; Proto-Oncogene Protein c-ets-1
PubMed: 37490064
DOI: 10.1007/s00408-023-00639-1 -
Cancer Biology & Therapy Dec 2023Human epidermal growth factor receptor 2 positive (HER2) breast cancer (BC) is associated with poor prognosis. This study aimed to elucidate the role of miR-18a-5p in...
OBJECTIVE
Human epidermal growth factor receptor 2 positive (HER2) breast cancer (BC) is associated with poor prognosis. This study aimed to elucidate the role of miR-18a-5p in regulation of HER2-BC progression along with its mechanism of action.
METHODS
The expression of miR-18a-5p and HER2 in BC cells and tissues was analyzed using quantitative real-time PCR while protein level expression of AKT Serine/Threonine Kinase 1 (AKT), phosphorylated AKT (p-AKT), Phosphatidylinositol 3-kinase (PI3K), phosphorylated-PI3K (p-PI3K), and HER2 were assessed by western blotting. Cell Counting Kit-8, wound healing, and cell adhesion assays were used for in vitro analysis along with xenograft tumor model construction for in vivo analysis. Pearson correlation analysis and dual-luciferase reporter (DLR) assays were used to ascertain the targeting association between miR-18a-5p and HER2.
RESULTS
There was a downregulation of miR-18a-5p expression in the BC tissues and cells. Functionally, overexpression of miR-18a-5p prevented BC cells from proliferation, adherence, migration, and activation of the P-PI3K/P-AKT pathway. In vivo experiment revealed that tumor growth was suppressed when miR-18a-5p was overexpressed. In BC, HER2 overexpression increased cell proliferation, cell-cell adhesion, migration, and P-PI3K/P-AKT signaling, but overexpression of miR-18a-5p reversed this effect because of the target relationship between miR-18a-5p and HER2.
CONCLUSION
miR-18a-5p inhibits HER2 BC progression by targeting HER2 to inhibit PI3K/AKT pathway activation. A theoretical foundation for the identification of new therapeutic targets for HER2 BC may be provided by the miR-18a-5p - HER2 axis.
Topics: Humans; Female; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; MicroRNAs; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Movement
PubMed: 37394766
DOI: 10.1080/15384047.2023.2224512 -
Cell Death & Disease Oct 2023RNA polymerase mitochondria (POLRMT) is essential for mitochondrial transcription machinery and other mitochondrial functions. Its expression and potential functions in...
RNA polymerase mitochondria (POLRMT) is essential for mitochondrial transcription machinery and other mitochondrial functions. Its expression and potential functions in prostate cancer were explored here. The Cancer Genome Atlas prostate cancer cohort (TCGA PRAD) shows that POLRMT mRNA expression is upregulated in prostate cancer tissues and POLRMT upregulation is correlated with poor patients' survival. POLRMT mRNA and protein levels were upregulated in local prostate cancer tissues and different primary/immortalized prostate cancer cells. Genetic depletion of POLRMT, using viral shRNA or CRISPR/Cas9 gene editing methods, impaired mitochondrial functions in prostate cancer cells, leading to mitochondrial depolarization, oxidative stress, mitochondria complex I inhibition, and ATP depletion. Moreover, POLRMT depletion resulted in robust inhibition of prostate cancer cell viability, proliferation, and migration, and provoked apoptosis. Conversely, prostate cancer cell proliferation, migration, and ATP contents were strengthened following ectopic POLRMT overexpression. In vivo, intratumoral injection of POLRMT shRNA adeno-associated virus impeded prostate cancer xenograft growth in nude mice. POLRMT silencing, oxidative stress, and ATP depletion were detected in POLRMT shRNA-treated prostate cancer xenograft tissues. IMT1 (inhibitor of mitochondrial transcription 1), the first-in-class POLRMT inhibitor, inhibited prostate cancer cell growth in vitro and in vivo. Together, overexpressed POLRMT is an important mitochondrial protein for prostate cancer cell growth, representing a novel and promising diagnostic and therapeutic oncotarget.
Topics: Male; Animals; Mice; Humans; Mitochondrial Proteins; Mice, Nude; Mitochondria; Prostatic Neoplasms; RNA, Small Interfering; RNA, Messenger; Adenosine Triphosphate; Cell Proliferation; Cell Line, Tumor; DNA-Directed RNA Polymerases
PubMed: 37816734
DOI: 10.1038/s41419-023-06203-2 -
Molecular Medicine (Cambridge, Mass.) Oct 2023Rab-interacting lysosomal protein (RILP) contains an alpha-helical coil with an unexplored biological function in osteosarcoma. This study investigated the expression of...
BACKGROUND
Rab-interacting lysosomal protein (RILP) contains an alpha-helical coil with an unexplored biological function in osteosarcoma. This study investigated the expression of RILP in osteosarcoma cells and tissues to determine the effect of RILP on the biological behaviors of osteosarcoma cells and the underlying mechanism.
METHODS
Tumor Immune Estimation Resource (TIMER) database, The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were used for bioinformatic analysis. Co-immunoprecipitation experiment was used to determine whether the two proteins were interacting. In functional tests, cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, transwell invasion assay, Immunofluorescence (IF) assay and immunohistochemical (IHC) assay were performed.
RESULTS
Overexpression of RILP significantly inhibited proliferation and impaired metastasis ability of osteosarcoma cells, while silencing of RILP showed the opposite trend. RNA-seq data analysis was applied in 143B cells and pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in the PI3K/AKT pathway. We further verified that overexpression of RILP restrained the PI3K/AKT/mTOR signaling pathway and induced autophagy in osteosarcoma cells, while the opposite trend was observed when PI3K pathway activator 740Y-P was used. 3-Methyladenine (3-MA), a selective autophagy inhibitor, partially attenuated the inhibitory effect of RILP on the migration and invasion ability of osteosarcoma cells, suggesting the involvement of autophagy in epithelial-mesenchymal transition regulation in osteosarcoma cells. Growth factor receptor binding protein-10 (Grb10), an adaptor protein, was confirmed as a potential target of RILP to restrain the PI3K/AKT signaling pathway. We subcutaneously injected stably overexpressing 143B osteosarcoma cells into nude mice and observed that overexpression of RILP inhibited tumor growth by inhibiting the PI3K/AKT/mTOR pathway.
CONCLUSION
Our study revealed that the expression of RILP was associated with favorable prognosis of osteosarcoma and RILP inhibits proliferation, migration, and invasion and promotes autophagy in osteosarcoma cells via Grb10-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. In the future, targeting RILP may be a potential strategy for osteosarcoma treatment.
Topics: Animals; Mice; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; GRB10 Adaptor Protein; Mice, Nude; Osteosarcoma; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Humans
PubMed: 37789274
DOI: 10.1186/s10020-023-00722-6 -
Biogerontology Oct 2023SARS-Cov-2 infection, which has caused the COVID-19 global pandemic, triggers cellular senescence. In this study, we investigate the role of the SARS-COV-2 spike protein...
SARS-Cov-2 infection, which has caused the COVID-19 global pandemic, triggers cellular senescence. In this study, we investigate the role of the SARS-COV-2 spike protein (S-protein) in regulating the senescence of RPE cells. The results showed that administration or overexpression of S-protein in ARPE-19 decreased cell proliferation with cell cycle arrest at the G1 phase. S-protein increased SA-β-Gal positive ARPE-19 cells with high expression of P53 and P21, senescence-associated inflammatory factors (e.g., IL-1β, IL-6, IL-8, ICAM, and VEGF), and ROS. Elimination of ROS by N-acetyl cysteine (NAC) or knocking down p21 by siRNA diminished S-protein-induced ARPE cell senescence. Both administrated and overexpressed S-protein colocalize with the ER and upregulate ER-stress-associated BIP, CHOP, ATF3, and ATF6 expression. S-protein induced P65 protein nuclear translocation. Inhibition of NF-κB by bay-11-7082 reduced S-protein-mediated expression of senescence-associated factors. Moreover, the intravitreal injection of S-protein upregulates senescence-associated inflammatory factors in the zebrafish retina. In conclusions, the S-protein of SARS-Cov-2 induces cellular senescence of ARPE-19 cells in vitro and the expression of senescence-associated cytokines in zebrafish retina in vivo likely by activating ER stress, ROS, and NF-κb. These results may uncover a potential association between SARS-cov-2 infection and development of AMD.
Topics: Animals; Humans; Spike Glycoprotein, Coronavirus; Reactive Oxygen Species; NF-kappa B; Tumor Suppressor Protein p53; Zebrafish; COVID-19; SARS-CoV-2; Cellular Senescence
PubMed: 36738354
DOI: 10.1007/s10522-023-10019-0 -
Cell Death and Differentiation Aug 2023MAD2 is a spindle assembly checkpoint protein that participates in the formation of mitotic checkpoint complex, which blocks mitotic progression. RNF8, an established...
MAD2 is a spindle assembly checkpoint protein that participates in the formation of mitotic checkpoint complex, which blocks mitotic progression. RNF8, an established DNA damage response protein, has been implicated in mitotic checkpoint regulation but its exact role remains poorly understood. Here, RNF8 proximity proteomics uncovered a role of RNF8-MAD2 in generating the mitotic checkpoint signal. Specifically, RNF8 competes with a small pool of p31 for binding to the closed conformer of MAD2 via its RING domain, while CAMK2D serves as a molecular scaffold to concentrate the RNF8-MAD2 complex via transient/weak interactions between its p-Thr287 and RNF8's FHA domain. Accordingly, RNF8 overexpression impairs glioma stem cell (GSC) mitotic progression in a FHA- and RING-dependent manner. Importantly, low RNF8 expression correlates with inferior glioma outcome and RNF8 overexpression impedes GSC tumorigenicity. Last, we identify PLK1 inhibitor that mimics RNF8 overexpression using a chemical biology approach, and demonstrate a PLK1/HSP90 inhibitor combination that synergistically reduces GSC proliferation and stemness. Thus, our study has unveiled a previously unrecognized CAMK2D-RNF8-MAD2 complex in regulating mitotic checkpoint with relevance to gliomas, which is therapeutically targetable.
Topics: Humans; Adaptor Proteins, Signal Transducing; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Cycle Proteins; DNA-Binding Proteins; Glioma; M Phase Cell Cycle Checkpoints; Mad2 Proteins; Mitosis; Nuclear Proteins; Spindle Apparatus; Ubiquitin-Protein Ligases
PubMed: 37468549
DOI: 10.1038/s41418-023-01192-3 -
International Journal of Biological... 2023Gastric cancer (GC) is a common malignancy and remains the fourth-leading cause of cancer-related deaths worldwide. Oncogenic potential of SDC2 has been implicated in...
Gastric cancer (GC) is a common malignancy and remains the fourth-leading cause of cancer-related deaths worldwide. Oncogenic potential of SDC2 has been implicated in multiple types of cancers, yet its role and underlying molecular mechanisms in GC remain unknown. Here, we found that SDC2 was highly expressed in GC and its upregulation correlated with poor prognosis in GC patients. Depletion of SDC2 significantly suppressed the growth and invasive capability of GC cells, while overexpressing SDC2 exerts opposite effects. Combined bioinformatics and experimental analyses substantiated that overexpression of SDC2 activated the AKT signaling pathway in GC, mechanistically through the interaction between SDC2 and PDK1-PH domain, thereby facilitating PDK1 membrane translocation to promote AKT activation. Moreover, SDC2 could also function as a co-receptor for FGF2 and was profoundly involved in the FGF2-AKT signaling axis in GC. Lastly, we revealed a mechanism on the USP14-mediated stabilization of SDC2 that is likely to contribute to SDC2 upregulation in GC tissues. Furthermore, we showed that IU1, a potent USP14 inhibitor, decreased the abundance of SDC2 in GC cells. Our findings indicate that SDC2 functions as a novel GC oncogene and has potential utility as a diagnostic marker and therapeutic target for GC.
Topics: Humans; Cell Line, Tumor; Cell Proliferation; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Proto-Oncogene Proteins c-akt; Signal Transduction; Stomach Neoplasms; Syndecan-2; Ubiquitin Thiolesterase
PubMed: 37496999
DOI: 10.7150/ijbs.84331 -
Proceedings of the National Academy of... Dec 2023Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells...
Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.
Topics: Mice; Animals; Circadian Clocks; CLOCK Proteins; Phase Separation; Period Circadian Proteins; Circadian Rhythm; Microbodies; ARNTL Transcription Factors; Mammals
PubMed: 38127982
DOI: 10.1073/pnas.2318274120 -
Cell Death & Disease Dec 2023The current study tested the expression and potential functions of Gαi1 in nasopharyngeal carcinoma (NPC). The Cancer Genome Atlas (TCGA) database results demonstrate...
The current study tested the expression and potential functions of Gαi1 in nasopharyngeal carcinoma (NPC). The Cancer Genome Atlas (TCGA) database results demonstrate that Gαi1 transcripts' number in NPC tissues is significantly higher than that in the normal nasal epithelial tissues. Its overexpression correlates with poor survival in certain NPC patients. Moreover, Gαi1 is significantly upregulated in NPC tissues of local primary patients and in different primary human NPC cells. Whereas its expression is relatively low in cancer-surrounding normal tissues and in primary nasal epithelial cells. Genetic silencing (via shRNA strategy) or knockout (via CRISPR-sgRNA method) of Gαi1 substantially suppressed viability, proliferation, cell cycle progression, and migration in primary NPC cells, causing significant caspase-apoptosis activation. Contrarily, ectopic Gαi1 expression exerted pro-tumorigenic activity and strengthened cell proliferation and migration in primary NPC cells. Gαi1 is important for Akt-mTOR activation in NPC cells. Akt-S6K phosphorylation was downregulated after Gαi1 shRNA or KO in primary NPC cells, but strengthened following Gαi1 overexpression. In Gαi1-silenced primary NPC cells, a S473D constitutively-active mutant Akt1 (caAkt1) restored Akt-S6K phosphorylation and ameliorated Gαi1 shRNA-induced proliferation inhibition, migration reduction and apoptosis. Bioinformatics analyses proposed zinc finger protein 384 (ZNF384) as a potential transcription factor of Gαi1. In primary NPC cells, ZNF384 shRNA or knockout (via CRISPR-sgRNA method) decreased Gαi1 mRNA and protein expression, whereas ZNF384 overexpression upregulated it. Importantly, there was an increased binding between ZNF384 protein and the Gαi1 promoter in human NPC tissues and different NPC cells. In vivo studies showed that intratumoral injection of Gαi1-shRNA-expressing adeno-associated virus (AAV) impeded subcutaneous NPC xenograft growth in nude mice. Gαi1 downregulation, Akt-mTOR inactivation, and apoptosis induction were detected in Gαi1-silenced NPC xenograft tissues. Gαi1 KO also effectively inhibited the growth of NPC xenografts in nude mice. Together, overexpressed Gαi1 exerts pro-tumorigenic activity in NPC possibly by promoting Akt-mTOR activation.
Topics: Animals; Humans; Mice; Cell Line, Tumor; Cell Proliferation; Mice, Nude; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Proto-Oncogene Proteins c-akt; RNA, Guide, CRISPR-Cas Systems; RNA, Small Interfering; Signal Transduction; TOR Serine-Threonine Kinases; Transcription Factors; GTP-Binding Protein alpha Subunits, Gi-Go
PubMed: 38049415
DOI: 10.1038/s41419-023-06308-8 -
Theranostics 2023In the bone marrow microenvironment (BMME), mesenchymal stem/stromal cells (MSCs) control the self-renewal of both healthy and cancerous hematopoietic stem/progenitor...
In the bone marrow microenvironment (BMME), mesenchymal stem/stromal cells (MSCs) control the self-renewal of both healthy and cancerous hematopoietic stem/progenitor cells (HSPCs). We previously showed that leukemia-derived MSCs change neighbor MSCs into leukemia-permissive states and boost leukemia cell proliferation, survival, and chemotherapy resistance. But the mechanisms behind how the state changes are still not fully understood. Here, we took a reverse engineering approach to determine BCR-ABL1+ leukemia cells activated transcriptional factor C/EBPβ, resulting in miR130a/b-3p production. Then, we back-tracked from clinical specimen transcriptome sequencing to cell co-culture, molecular and cellular assays, flow cytometry, single-cell transcriptome, and transcriptional regulation to determine the molecular mechanisms of BCR-ABL1-driven exosome-miR130b-3p-mediated gap-junction Cx43 MSC intercellular communications. BCR-ABL1-driven exosome-miR130a/b-3p mediated gap-junction Cx43 (a.k.a., GJA1) BMSC intercellular communications for subclonal evolution in leukemic microenvironment by targeting BMSCs-expressed HLAs, thereby potentially maintaining BMSCs with self-renewal properties and reduced BMSC immunogenicity. The Cx43 and miR-130a/b subclonal MSCs subsets of differentiation state could be reversed to Cx43 and miR-130a/b subclones of the higher stemness state in Cx43-overexpressed subclonal MSCs. Both miR-130a and miR-130b might only inhibit Cx43 translation or degrade Cx43 proteins and did not affect Cx43 mRNA stability. The subclonal evolution was further confirmed by single-cell transcriptome profiling of MSCs, which suggested that Cx43 regulated their stemness and played normal roles in immunomodulation antigen processing. Thus, upregulated miR-130a/b promoted osteogenesis and adipogenesis from BMSCs, thereby decreasing cancer progression. Our clinical data validated that the expression of many genes in human major histocompatibility was negatively associated with the stemness of MSCs, and several immune checkpoint proteins contributing to immune escape in tumors were overexpressed after either miR-130a or miR-130b overexpression, such as CD274, LAG3, PDCD1, and TNFRSF4. Not only did immune response-related cytokine-cytokine receptor interactions and PI3K-AKT pathways, including EGR3, TNFRSF1B, but also NDRG2 leukemic-associated inflammatory factors, such as IFNB1, CXCL1, CXCL10, and CCL7 manifest upon miR-130a/b overexpression. Either BCR siRNAs or ABL1 siRNAs assay showed significantly decreased miR-130a and miR-130b expression, and chromatin immunoprecipitation sequencing confirmed that the regulation of miR-130a and miR-130b expression is BCR-ABL1-dependent. BCR-ABL1 induces miR-130a/b expression through the upregulation of transcriptional factor C/EBPβ. C/EBPβ could bind directly to the promoter region of miR-130b-3p, not miR-130a-3p. BCR-ABL1-driven exosome-miR130a-3p could interact with Cx43, and further impact GJIC in TME. Our findings shed light on how leukemia BCR-ABL1-driven exosome-miR130b-3p could interact with gap-junction Cx43, and further impact GJIC in TME, implications for leukemic therapies of subclonal evolution.
Topics: Humans; Cell Communication; Connexin 43; Exosomes; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MicroRNAs; Phosphatidylinositol 3-Kinases; Tumor Microenvironment; Tumor Suppressor Proteins
PubMed: 37554265
DOI: 10.7150/thno.83178