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Lentiviral vectors have markedly enhanced gene therapy efficiency in treating congenital diseases, but their long-term safety remains controversial. Most gene therapies for congenital eye diseases need to be carried out at early ages, yet the assessment of related risks to ocular development posed by lentiviral vectors is challenging. Utilizing single-cell transcriptomic profiling on human retinal organoids, this study explored the impact of lentiviral vectors on the retinal development and found that lentiviral vectors can cause retinal precursor cells to shift toward photoreceptor fate through the up-regulation of key fate-determining genes such as PRDM1. Further investigation demonstrated that the intron and intergenic region of PRDM1 was bound by PHLDA1, which was also up-regulated by lentiviral vectors exposure. Importantly, knockdown of PHLDA1 successfully suppressed the lentivirus-induced differentiation bias of photoreceptor cells. The findings also suggest that while lentiviral vectors may disrupt the fate determination of retinal precursor cells, posing risks in early-stage retinal gene therapy, these risks could potentially be reduced by inhibiting the PHLDA1-PRDM1 axis.

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Hypoxia-ischemia (HI) is one of the main causes of neonatal brain injury. Mitophagy has been implicated in the degradation of damaged mitochondria and cell survival following neonatal brain HI injury. Pleckstrin homology-like domain family A member 1 (PHLDA1) plays vital roles in the progression of various disorders including the regulation of oxidative stress, the immune responses and apoptosis. In the present study we investigated the role of PHLDA1 in HI-induced neuronal injury and further explored the mechanisms underlying PHLDA1-regulated mitophagy in vivo and in vitro. HI model was established in newborn rats by ligation of the left common carotid artery plus exposure to an oxygen-deficient chamber with 8% O2 and 92% N2. In vitro studies were conducted in primary hippocampal neurons subjected to oxygen and glucose deprivation/-reoxygenation (OGD/R). We showed that the expression of PHLDA1 was significantly upregulated in the hippocampus of HI newborn rats and in OGD/R-treated primary neurons. Knockdown of PHLDA1 in neonatal rats via lentiviral vector not only significantly ameliorated HI-induced hippocampal neuronal injury but also markedly improved long-term cognitive function outcomes, whereas overexpression of PHLDA1 in neonatal rats via lentiviral vector aggravated these outcomes. PHLDA1 knockdown in primary neurons significantly reversed the reduction of cell viability and increase in intracellular reactive oxygen species (ROS) levels, and attenuated OGD-induced mitochondrial dysfunction, whereas overexpression of PHLDA1 decreased these parameters. In OGD/R-treated primary hippocampal neurons, we revealed that PHLDA1 knockdown enhanced mitophagy by activating FUNDC1, which was abolished by FUNDC1 knockdown or pretreatment with mitophagy inhibitor Mdivi-1 (25 µM). Notably, pretreatment with Mdivi-1 or the knockdown of FUNDC1 not only increased brain infarct volume, but also abolished the neuroprotective effect of PHLDA1 knockdown in HI newborn rats. Together, these results demonstrate that PHLDA1 contributes to neonatal HI-induced brain injury via inhibition of FUNDC1-mediated neuronal mitophagy.

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Neuroblastoma (NB) is the most common extracranial pediatric solid tumor originating from the abnormal development of cells of the sympathoadrenal lineage of the neural crest. Targeting GD2 ganglioside (GD2), a glycolipid expressed on neuroblastoma cells, with GD2 ganglioside-recognizing antibodies affects several pivotal signaling routes that drive or influence the malignant phenotype of the cells. Previously performed gene expression profiling helped us to identify the PHLDA1 (pleckstrin homology-like domain family A member 1) gene as the most upregulated gene in the IMR-32 human neuroblastoma cells treated with the mouse 14G2a monoclonal antibody. Mass spectrometry-based proteomic analyses were applied to better characterize a role of PHLDA1 protein in the response of neuroblastoma cells to chimeric ch14.18/CHO antibody. Additionally, global protein expression profile analysis in the IMR-32 cell line with PHLDA1 silencing revealed the increase in biological functions of mitochondria, accompanied by differentiation-like phenotype of the cells. Moreover, mass spectrometry analysis of the proteins co-immunoprecipitated using anti-PHLDA1-specific antibody, selected a group of possible PHLDA1 binding partners. Also, a more detailed analysis suggested that PHLDA1 interacts with the DCAF7/AUTS2 complex, a key component of neuronal differentiation in vitro. Importantly, our results indicate that PHLDA1 silencing enhances the EGF receptor signaling pathway and combinatory treatment of gefitinib and ch14.18/CHO antibodies might be beneficial for neuroblastoma patients. Data are available via ProteomeXchange with the identifier PXD044319.

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Pleckstrin homeolike domain, family A, member 1 (PHLDA1) is a multifunctional protein that plays diverse roles in A variety of biological processes, including cell death, and hence its altered expression has been found in different types of cancer. Although studies have shown a regulatory relationship between p53 and PHLDA1, the molecular mechanism is still unclear. Especially, the role of PHLDA1 in the process of apoptosis is still controversial. In this study, we found that the expression of PHLDA1 in human cervical cancer cell lines was correlated with the up-expression of p53 after treatment with apoptosis-inducing factors. Subsequently, the binding site and the binding effect of p53 on the promoter region of PHLDA1 were verified by our bioinformatics data analysis and luciferase reporter assay. Indeed, we used CRISPR-Cas9 to knockout the p53 gene in HeLa cells and further confirmed that p53 can bind to the promoter region of PHLDA1 gene, and then directly regulate the expression of PHLDA1 by recruiting P300 and CBP to change the acetylation and methylation levels in the promoter region. Finally, a series of gain-of-function experiments further confirmed that p53 re-expression in HeLap53-/- cell can up-regulate the reduction of PHLDA1 caused by p53 knockout, and affect cell apoptosis and proliferation. Our study is the first to explore the regulatory mechanism of p53 on PHLDA1 by using the p53 gene knockout cell model, which further proves that PHLDA1 is a target-gene in p53-mediated apoptosis, and reveals the important role of PHLDA1 in cell fate determination.

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Objective: To investigate the regulatory mechanism of pleckstrin homology-like domain, family A, member 1 (PHLDA1) in sepsis-induced acute lung injury (ALI). Method: Mice model of sepsis were established by cecal ligation and puncture (CLP). The expression of PHLDA1 was reduced by injecting short hairpin RNA (shRNA)–PHLDA1 into the tail vein. The levels of PHLDA1, pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-1β, IL-18, super-oxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH), molecular mechanism related to pyroptosis, such as caspase 1, adaptor apoptosis-associated speck-like protein containing a CARD (ASC), and gasdermin D (GSDMD)-N, and nucleotide oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) were tested by Western blot analysis, quantitative real-time polymerase chain reaction, and enzyme-linked-immunosorbent serologic assay. Pathological changes in lung tissues were examined by hematoxylin and eosin staining. Wet–dry weight ratio of lung tissues was observed. Results: The expression of PHLDA1 was up-regulated in lung tissues from CLP-induced septic mice. Knockdown of PHLDA1 could reduce lung injury and wet–dry weight ratio in mice with sepsis-induced ALI. Moreover, silencing of PHLDA1 decreased the expressions of IL-1β, TNF-α, IL-18, IL-6, and MDA but increased SOD and GSH expressions in CLP-induced septic mice. The expressions of NLRP3, GSDMD-N, ASC, and caspase 1 were decreased by PHLDA1 silencing. Conclusion: Knockdown of PHLDA1 inhibited lung inflammation and pyroptosis in mice with sepsis-induced ALI by down-regulating NLRP3 (AU)

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OBJECTIVE: To investigate the regulatory mechanism of pleckstrin homology-like domain, family A, member 1 (PHLDA1) in sepsis-induced acute lung injury (ALI). METHOD: Mice model of sepsis were established by cecal ligation and puncture (CLP). The expression of PHLDA1 was reduced by injecting short hairpin RNA (shRNA)-PHLDA1 into the tail vein. The levels of PHLDA1, pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-1ß, IL-18, super-oxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH), molecular mechanism related to pyroptosis, such as caspase 1, adaptor apoptosis-associated speck-like protein containing a CARD (ASC), and gasdermin D (GSDMD)-N, and nucleotide oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) were tested by Western blot analysis, quantitative real-time polymerase chain reaction, and enzyme-linked-immunosorbent serologic assay. Pathological changes in lung tissues were examined by hematoxylin and eosin staining. Wet-dry weight ratio of lung tissues was observed. RESULTS: The expression of PHLDA1 was up-regulated in lung tissues from CLP-induced septic mice. Knockdown of PHLDA1 could reduce lung injury and wet-dry weight ratio in mice with sepsis-induced ALI. Moreover, silencing of PHLDA1 decreased the expressions of IL-1ß, TNF-α, IL-18, IL-6, and MDA but increased SOD and GSH expressions in CLP-induced septic mice. The expressions of NLRP3, GSDMD-N, ASC, and caspase 1 were decreased by PHLDA1 silencing. CONCLUSION: Knockdown of PHLDA1 inhibited lung inflammation and pyroptosis in mice with sepsis-induced ALI by down-regulating NLRP3.

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Doxorubicin is a common chemotherapeutic agent in clinic, but myocardial toxicity limits its use. Fibroblast growth factor (FGF) 10, a multifunctional paracrine growth factor, plays diverse roles in embryonic and postnatal heart development as well as in cardiac regeneration and repair. In this study we investigated the role of FGF10 as a potential modulator of doxorubicin-induced cardiac cytotoxicity and the underlying molecular mechanisms. Fgf10+/- mice and an inducible dominant negative FGFR2b transgenic mouse model (Rosa26rtTA; tet(O)sFgfr2b) were used to determine the effect of Fgf10 hypomorph or blocking of endogenous FGFR2b ligands activity on doxorubicin-induced myocardial injury. Acute myocardial injury was induced by a single injection of doxorubicin (25 mg/kg, i.p.). Then cardiac function was evaluated using echocardiography, and DNA damage, oxidative stress and apoptosis in cardiac tissue were assessed. We showed that doxorubicin treatment markedly decreased the expression of FGFR2b ligands including FGF10 in cardiac tissue of wild type mice, whereas Fgf10+/- mice exhibited a greater degree of oxidative stress, DNA damage and apoptosis as compared with the Fgf10+/+ control. Pre-treatment with recombinant FGF10 protein significantly attenuated doxorubicin-induced oxidative stress, DNA damage and apoptosis both in doxorubicin-treated mice and in doxorubicin-treated HL-1 cells and NRCMs. We demonstrated that FGF10 protected against doxorubicin-induced myocardial toxicity via activation of FGFR2/Pleckstrin homology-like domain family A member 1 (PHLDA1)/Akt axis. Overall, our results unveil a potent protective effect of FGF10 against doxorubicin-induced myocardial injury and identify FGFR2b/PHLDA1/Akt axis as a potential therapeutic target for patients receiving doxorubicin treatment.

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Tight regulation of Toll-like receptor (TLR)-mediated inflammatory responses is important for innate immunity. Here, we show that T-cell death-associated gene 51 (TDAG51/PHLDA1) is a novel regulator of the transcription factor FoxO1, regulating inflammatory mediator production in the lipopolysaccharide (LPS)-induced inflammatory response. TDAG51 induction by LPS stimulation was mediated by the TLR2/4 signaling pathway in bone marrow-derived macrophages (BMMs). LPS-induced inflammatory mediator production was significantly decreased in TDAG51-deficient BMMs. In TDAG51-deficient mice, LPS- or pathogenic Escherichia coli infection-induced lethal shock was reduced by decreasing serum proinflammatory cytokine levels. The recruitment of 14-3-3ζ to FoxO1 was competitively inhibited by the TDAG51-FoxO1 interaction, leading to blockade of FoxO1 cytoplasmic translocation and thereby strengthening FoxO1 nuclear accumulation. TDAG51/FoxO1 double-deficient BMMs showed significantly reduced inflammatory mediator production compared with TDAG51- or FoxO1-deficient BMMs. TDAG51/FoxO1 double deficiency protected mice against LPS- or pathogenic E. coli infection-induced lethal shock by weakening the systemic inflammatory response. Thus, these results indicate that TDAG51 acts as a regulator of the transcription factor FoxO1, leading to strengthened FoxO1 activity in the LPS-induced inflammatory response.

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The downregulation of Pleckstrin Homology-Like Domain family A member 1 (PHLDA1) expression mediates resistance to targeted therapies in receptor tyrosine kinase-driven cancers. The restoration and maintenance of PHLDA1 levels in cancer cells thus constitutes a potential strategy to circumvent resistance to inhibitors of receptor tyrosine kinases. Through a pharmacological approach, we identify the inhibition of MAPK signalling as a crucial step in PHLDA1 downregulation. Further ChIP-qPCR analysis revealed that MEK1/2 inhibition produces significant epigenetic changes at the PHLDA1 locus, specifically a decrease in the activatory marks H3Kme3 and H3K27ac. In line with this, we show that treatment with the clinically relevant class I histone deacetylase (HDAC) inhibitor 4SC-202 restores PHLDA1 expression in lapatinib-resistant human epidermal growth factor receptor-2 (HER2)+ breast cancer cells. Critically, we show that when given in combination, 4SC-202 and lapatinib exert synergistic effects on 2D cell proliferation and colony formation capacity. We therefore propose that co-treatment with 4SC-202 may prolong the clinical efficacy of lapatinib in HER2+ breast cancer patients.

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BACKGROUND: During ischemic stroke treatment, cerebral ischemia/reperfusion (I/R) injury results in neuronal cell death and neurological dysfunctions in brain. Previous studies indicate that basic helix-loop-helix family member e40 (BHLHE40) exerts protective effects on the pathology of neurogenic diseases. However, the protective function of BHLHE40 in I/R is unclear. OBJECTIVES: This study aimed to explore the expression, role and potential mechanism of BHLHE40 after ischemia. MATERIAL AND METHODS: We established models of I/R injury in rats and of oxygen-glucose deprivation/reoxygenation (OGD/R) in primary hippocampal neurons. Nissl and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to detect neuronal injury and apoptosis. Immunofluorescence was used to detect BHLHE40 expression. Cell viability and cell damage measurements were conducted using Cell Counting Kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) assay. The regulation of BHLHE40 to pleckstrin homology-like domain family A, member 1 (PHLDA1) was assessed using the dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay. RESULTS: Cerebral I/R rats exhibited severe neuronal loss and apoptosis in hippocampal cornu Ammonis 1 (CA1) region, accompanied by downregulated BHLHE40 expression at both mRNA and protein levels, indicating that BHLHE40 may regulate the apoptosis of hippocampal neurons. The function of BHLHE40 in neuronal apoptosis during cerebral I/R was further explored by establishing an OGD/R model in vitro. Low expression of BHLHE40 was also observed in neurons treated with OGD/R. The OGD/R administration inhibited cell viability and enhanced cell apoptosis in hippocampal neurons, whereas BHLHE40 overexpression reversed those changes. Mechanistically, we demonstrated that BHLHE40 could repress PHLDA1 transcription by binding to PHLDA1 promoter. The PHLDA1 is a facilitator of neuronal damage in brain I/R injury and its upregulation reversed the effects caused by BHLHE40 overexpression in vitro. CONCLUSIONS: The transcription factor BHLHE40 may protect against brain I/R injury through repressing cell damage via regulating PHLDA1 transcription. Thus, BHLHE40 may be a candidate gene for further study of molecular or therapeutic targets for I/R.

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Mitochondrial dysfunction and endoplasmic reticulum (ER) stress occur in ischemic stroke. The disruption of these two organelles can directly lead to cell death through various signaling pathways. Thus, investigation of the associated molecular mechanisms in cerebral ischemia is a prerequisite for stroke treatment. Pleckstrin homology-like domain family A member 1 (PHLDA1) is a multifunctional protein that can modulate mitochondrial function and ER stress in cardiomyocyte and cancer cells. This work studied the role of PHLDA1 in cerebral ischemic/reperfusion (I/R) injury and explored the underlying mechanisms associated with mitochondrial functions and ER stress. Middle cerebral artery occlusion/reperfusion (MCAO/R)-treated mice and oxygen-glucose deprivation/reoxygenation (OGD/R)-stimulated neurons were used as I/R models in vivo and in vitro, respectively. PHLDA1 was upregulated in ischemic penumbra of MCAO/R-induced mice and OGD/R-exposed neurons. In vitro, PHLDA1 knockdown protected neurons from OGD/R-induced apoptosis. In vivo, PHLDA1 silencing facilitated functional recovery and reduced cerebral infarct volume. Mechanistically, PHLDA1 knockdown promoted PPARγ nuclear translocation, which may mediate the effects on reversion of mitochondrial functions and alleviation of ER stress. In summary, PHLDA1 knockdown alleviates neuronal ischemic injuries in mice. PPARγ activation and mitochondrial dysfunction and endoplasmic reticulum stress attenuation are involved in the underlying mechanisms.

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OBJECTIVE: Pleckstrin homology-like domain family A member 1 (PHLDA1) is involved in the progression of intestine-related diseases, but its role and related mechanisms in Necrotizing enterocolitis (NEC) are unclear. The aim of this study was to better understand the function of PHLDA1 in NEC and the underlying mechanisms. METHODS: A neonatal mouse model of NEC was established by hypoxic hypothermia, and sh-PHLDA1 was transfected into mice to observe the mortality of each group within 4 days. The levels of IL-1ß, IL-6, IL-18 and TNF-α were measured by PCR and ELISA. ROS, MDA, SOD, and GSH-Px levels were detected by Dihydroethidium (DHE) method and kit; expression of pyroptosis-related factors including NLRP3, ASC, cleaved-caspase1, GSDMD-N, IL-1ß, IL-18, and Nrf2 were detected by western-blot; mechanistically, the effects of transfection of sh-PHLDA1 and ML385 (Nrf2 inhibitor) were investigated, and the expression of pyroptosis-related factors was detected again. RESULTS: PHLDA1 was highly expressed in the intestinal tissues of NEC mice, and transfection of sh-PHLDA1 improved the survival rate, alleviated intestinal lesions, improved intestinal inflammation, oxidative stress and cellular scorching in NEC. In addition, sh-PHLDA1 was able to inhibit NLRP3 activation and pyroptosis by activating Nrf2. CONCLUSION: Knockdown of PHLDA1 attenuated necrotizing small intestinal colitis by enhancing Nrf2 expression to inhibit NLRP3 inflammasome activation and pyroptosis.

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Background Acute kidney injury (AKI), a prevalent complication of sepsis, causes substantial burden on patients’ families as well as the society. More reliable markers are urgently required for the prevention and treatment of AKI. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) was implicated in various diseases, but its involvement in sepsis-induced AKI remains to be explored. The JNK/ERK pathway has been revealed as being involved in progression of sepsis. One previous study demonstrated that PHLDA1 could activate the JNK/ERK pathway in hepatic ischemia/reperfusion injury. Nevertheless, involvement of PHLDA1 in sepsis-triggered AKI through the JNK/ERK pathway has not been probed. Methods A cecal ligation and punctured (CLP) mice model of sepsis-induced AKI was established. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining were applied to evaluate the expression of PHLDA1. Concentration of blood urea nitrogen (BUN) and serum creatinine (Scr), inflammation markers, including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α, as well as oxidative stress-associated proteins (catalase, malondialdehyde, superoxide dismutase, and glutathione), in the kidney tissues of mice were evaluated by enzyme-linked-immunosorbent serologic assay. Western blot analysis was applied for measuring protein expression levels. Results The BUN and SCr levels in mice were obviously elevated in the CLP group compared to the sham group. Moreover, the expression of PHLDA1 was also elevated in the CLP group in comparison to the sham group. Down-regulation of PHLDA1 alleviated renal injury, inflammation, and oxidative stress in AKI model. Mechanistic study showed that PHLDA1 knockdown suppressed the activation of c-JUN N-terminal kinase/p38 and extracellular signal-regulated kinase (JNK/ERK) pathway (AU)

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BACKGROUND: Acute kidney injury (AKI), a prevalent complication of sepsis, causes substantial burden on patients' families as well as the society. More reliable markers are urgently required for the prevention and treatment of AKI. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) was implicated in various diseases, but its involvement in sepsis-induced AKI remains to be explored. The JNK/ERK pathway has been revealed as being involved in progression of sepsis. One previous study demonstrated that PHLDA1 could activate the JNK/ERK pathway in hepatic ischemia/reperfusion injury. Nevertheless, involvement of PHLDA1 in sepsis-triggered AKI through the JNK/ERK pathway has not been probed. METHODS: A cecal ligation and punctured (CLP) mice model of sepsis-induced AKI was established. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining were applied to evaluate the expression of PHLDA1. Concentration of blood urea nitrogen (BUN) and serum creatinine (Scr), inflammation markers, including interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α, as well as oxidative stress-associated proteins (catalase, malondialdehyde, superoxide dismutase, and glutathione), in the kidney tissues of mice were evaluated by enzyme-linked-immunosorbent serologic assay. Western blot analysis was applied for measuring protein expression levels. RESULTS: The BUN and SCr levels in mice were obviously elevated in the CLP group compared to the sham group. Moreover, the expression of PHLDA1 was also elevated in the CLP group in comparison to the sham group. Down-regulation of PHLDA1 alleviated renal injury, inflammation, and oxidative stress in AKI model. Mechanistic study showed that PHLDA1 knockdown suppressed the activation of c-JUN N-terminal kinase/p38 and extracellular signal-regulated kinase (JNK/ERK) pathway. CONCLUSION: Down-regulation of PHLDA1 suppressed inflammation and oxidative stress through the modulation of JNK/ERK pathway in sepsis-induced AKI. The results could offer a novel insight into the treatment of patients with sepsis-induced AKI.

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Background: The molecular mechanisms of EWS-FLI-mediating target genes and downstream pathways may provide a new way in the targeted therapy of Ewing sarcoma. Meanwhile, enhancers transcript non-coding RNAs, known as enhancer RNAs (eRNAs), which may serve as potential diagnosis markers and therapeutic targets in Ewing sarcoma. Materials and methods: Differentially expressed genes (DEGs) were identified between 85 Ewing sarcoma samples downloaded from the Treehouse database and 3 normal bone samples downloaded from the Sequence Read Archive database. Included in DEGs, differentially expressed eRNAs (DEeRNAs) and target genes corresponding to DEeRNAs (DETGs), as well as the differentially expressed TFs, were annotated. Then, cell type identification by estimating relative subsets of known RNA transcripts (CIBERSORT) was used to infer portions of infiltrating immune cells in Ewing sarcoma and normal bone samples. To evaluate the prognostic value of DEeRNAs and immune function, cross validation, independent prognosis analysis, and Kaplan-Meier survival analysis were implemented using sarcoma samples from the Cancer Genome Atlas database. Next, hallmarks of cancer by gene set variation analysis (GSVA) and immune gene sets by single-sample gene set enrichment analysis (ssGSEA) were identified to be significantly associated with Ewing sarcoma. After screening by co-expression analysis, most significant DEeRNAs, DETGs and DETFs, immune cells, immune gene sets, and hallmarks of cancer were merged to construct a co-expression regulatory network to eventually identify the key DEeRNAs in tumorigenesis of Ewing sarcoma. Moreover, Connectivity Map Analysis was utilized to identify small molecules targeting Ewing sarcoma. External validation based on multidimensional online databases and scRNA-seq analysis were used to verify our key findings. Results: A six-different-dimension regulatory network was constructed based on 17 DEeRNAs, 29 DETFs, 9 DETGs, 5 immune cells, 24 immune gene sets, and 8 hallmarks of cancer. Four key DEeRNAs (CCR1, CD3D, PHLDA1, and RASD1) showed significant co-expression relationships in the network. Connectivity Map Analysis screened two candidate compounds, MS-275 and pyrvinium, that might target Ewing sarcoma. PHLDA1 (key DEeRNA) was extensively expressed in cancer stem cells of Ewing sarcoma, which might play a critical role in the tumorigenesis of Ewing sarcoma. Conclusion: PHLDA1 is a key regulator in the tumorigenesis and progression of Ewing sarcoma. PHLDA1 is directly repressed by EWS/FLI1 protein and low expression of FOSL2, resulting in the deregulation of FOX proteins and CC chemokine receptors. The decrease of infiltrating T-lymphocytes and TNFA signaling may promote tumorigenesis and progression of Ewing sarcoma.

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Sevoflurane anesthesia induces neurocognitive impairment and pyroptosis in the developing brain. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) was involved in neuronal apoptosis, oxidative stress and inflammation during ischemic stroke. The role of PHLDA1 in sevoflurane-induced pyroptosis in developing rats was investigated. Firstly, neonatal rats at day 7 was exposed to 2.0% sevoflurane for 6 h to induce neurotoxicity. Pathological analysis showed that sevoflurane anesthesia induced hippocampal injury and reduced the number of neurons. The expression of PHLDA1 was elevated in hippocampus of sevoflurane-treated rats. Secondly, sevoflurane anesthesia-treated neonatal rats were injected with adeno-associated virus serotype (AAV) to mediate knockdown of PHLDA1. Injection with AAV-shPHLDA1 ameliorated sevoflurane-induced hippocampal injury and neurocognitive impairment in rats. Moreover, knockdown of PHLDA1 increased the number of neurons in sevoflurane-treated rats. Silence of PHLDA1 suppressed neuronal apoptosis, and inhibited pyroptosis in sevoflurane-treated rats. Thirdly, PHLDA1 was also elevated in sevoflurane-treated primary neuronal cells. Loss of PHLDA1 also enhanced cell viability and suppressed pyroptosis of sevoflurane-treated primary neuronal cells. Lastly, silence of PHLDA1 reduced protein expression of TRAF6 and p-Rac1 in sevoflurane-treated rats and neuronal cells. Over-expression of TRAF6 attenuated PHLDA1 silence-induced increase of cell viability and decreased pyroptosis in neuronal cells. In conclusion, loss of PHLDA1 protected against sevoflurane-induced pyroptosis in developing rats through inhibition of TRAF6-mediated activation of Rac1.

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Activation of the Ras signaling pathway promotes the growth of malignant human glioblastoma multiforme (GBM). Mutations in Ras are rare in GBM, elevated levels of activated Ras are prevalently observed in GBM. However, the potential mechanism of how Ras is activated in GBM remains unclear. In this study, we screened a new interacted protein of Ras, PHLDA1. Our findings confirmed that PHLDA1 acted as an oncogene and promoted glioma progression and recurrence. We demonstrated that PHLDA1 was upregulated in GBM tissues and cells. PHLDA1 overexpression promoted cell proliferation and tumor growth. In terms of mechanism, PHLDA1 promoted cell proliferation by regulating Ras/Raf/Mek/Erk signaling pathway. Moreover, Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation. PHLDA1 and Src competed for binding with Ras, inhibiting Ras phosphorylation by Src and rescuing Ras activity. This study may provide a new idea of the molecular mechanism underlying glioma progression and a novel potential therapeutic target for comprehensive glioblastoma treatment.

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Emphysematous phenotype is the most important phenotypic component of chronic obstructive pulmonary disease and is associated with substantial morbidity and mortality. The current pharmaceutical treatments and therapeutic procedures do not reduce pulmonary damage in patients with emphysematous phenotype. Therefore, it is important to identify effector molecules that can be used as interfering targets in such patients. Apoptosis of type II alveolar epithelial cells plays a key role in the phenotypic formation. This study aimed to further explore the molecular mechanisms involved in this process. The number of type II alveolar epithelial cells was significantly reduced due to increased apoptosis in patients with emphysematous phenotype compared to those with non-emphysematous phenotype. Pleckstrin homology like domain, family A, member 1 (PHLDA1) was mainly distributed in type II alveolar epithelial cells in both groups but was markedly reduced in patients with emphysematous phenotype. Overexpression of PHLDA1 prevented cigarette smoke extract-stimulated apoptosis of type II alveolar epithelial cells, whereas its knockdown worsened the apoptosis. PHLDA1 binding ability to tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) was weakened after exposure to cigarette smoke extract, with decreased PHLDA1 level lowering the abundance of YWHAE and attenuating the binding ability of YWHAE to p-Bad. These results demonstrate that considerable apoptosis of type II alveolar epithelial cells occurs in patients with emphysematous phenotype, and PHLDA1 may act as an effective antiapoptotic factor via YWHAE. Moreover, PHLDA1 may serve as a potential interfering target, providing insights into therapeutic strategies for emphysematous phenotype.

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[This corrects the article DOI: 10.3389/fimmu.2022.731500.].

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Pleckstrin homology-like domain, family A, member 1 (PHLDA1) has been reported to be expressed in many mammalian tissues and cells. However, the functions and exact mechanisms of PHLDA1 remain unclear. In this study, we found that PHLDA1 expression was significantly altered in macrophages after exposure to lipopolysaccharide (LPS) in vitro, suggesting that PHLDA1 may be involved in the regulation of TLR4 signaling pathway activated by LPS. PHLDA1 attenuated the production of LPS-stimulated proinflammatory cytokines (TNF-α, IL-6, and IL-1ß). Further research showed that the phosphorylation levels of some important signal molecules in TLR4/MyD88-mediated MAPK and NF-κB signaling pathways were reduced by PHLDA1, which in turn impaired the transcription factors NF-κB and AP1 nuclear translocation and their responsive element activities. Furthermore, we found that PHLDA1 repressed LPS-induced proinflammatory cytokine production via binding to Tollip which restrained TLR4 signaling pathway. A mouse model of endotoxemia was established to confirm the above similar results. In brief, our findings demonstrate that PHLDA1 is a negative regulator of LPS-induced proinflammatory cytokine production by Tollip, suggesting that PHLDA1 plays an anti-inflammatory role through inhibiting the TLR4/MyD88 signaling pathway with the help of Tollip. PHLDA1 may be a novel therapeutic target in treating endotoxemia.

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