POLQ inhibition attenuates the stemness and ferroptosis resistance in gastric cancer cells via downregulation of dihydroorotate dehydrogenase.

Gastric cancer (GC) contains subpopulations of cancer stem cells (CSCs), which are described as the main contributors in tumor initiation and metastasis. It is necessary to clarify the molecular mechanism underlying CSCs phenotype and develop novel biomarkers and therapeutic targets for gastric cancer. Here, we show that POLQ positively regulates stem cell-like characteristics of gastric cancer cells, knockdown of POLQ suppressed the stemness of GC cells in vitro and in vivo. Further mechanistic studies revealed that POLQ knockdown could downregulate the expression of dihydroorotate dehydrogenase (DHODH). DHODH overexpression rescued the reduced stemness resulted by POLQ knockdown. Furthermore, we found that POLQ expression correlated with resistance to ferroptosis, and POLQ inhibition renders gastric cancer cells more vulnerable to ferroptosis. Further investigation revealed that POLQ regulated DHODH expression via the transcription factors E2F4, thereby regulating ferroptosis resistance and stemness of gastric cancer cells. Given the importance of POLQ in stemness and ferroptosis resistance of GC, we further evaluated the therapeutic potential of POLQ inhibitor novobiocin, the results show that novobiocin attenuates the stemness of GC cells and increased ferroptosis sensitivity. Moreover, the combination of POLQ inhibitor and ferroptosis inducer synergistically suppressed MGC-803 xenograft tumor growth and diminished metastasis. Our results identify a POLQ-mediated stemness and ferroptosis defense mechanism and provide a new therapeutic strategy for gastric cancer.

Kinetochore scaffold 1 downregulation suppressed the development of non-small cell lung cancer by inactivating the phosphatidylinositol 3 kinase/protein kinase B (AKT)/nuclear factor-kappa B pathway.

Kinetochore scaffold 1 (KNL1) is indispensable for generating motile micro-tubule attachments and isolating chromosomes. KNL1 is highly expressed in multiple middle-route tissues and promotes tumor development. However, how it functions in non-small cell lung cancer (NSCLC) is unclear. Real-time quantitative PCR (RT-qPCR) and Western blotting (WB) were used to determine KNL1 expression in NSCLC tissues and cells. The sh-KNL1 or oe-KNL1 was transfected into NSCLC cells. The colony formation assay, cell counting kit-8 (CCK-8) assay, and flow cytometry were used to evaluate cell proliferation and apoptosis. A transwell assay was used to monitor invasion and migration. The CCK-8 assay was used to measure NSCLC cell sensitivity to chemotherapy drugs. WB confirmed the protein levels of apoptosis-related proteins, cell cycle-associated proteins, and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappaB (NF-κB) pathway. A PI3K/AKT/NF-κB pathway inhibitor was used to intervene in NSCLC cell transfection along with oe-KNL1, thus revealing the function of the pathway in carcinogenicity mediated by KNL1. In result KNL1 expression was substantially increased in NSCLC tissues and cells. High-level KNL1 expression is related to the poor prognosis of NSCLC patients. KNL1 silencing bolstered promoted NSCLC cell apoptosis and inhibited proliferation, cell cycle progression, invasion, and EMT, whereas KNL1 silencing had the opposite effect. KNL1 knockdown increased NSCLC cell sensitivity to chemical drugs. KNL1 promoted PI3K/AKT/NF-κB pathway activation, while PI3K/AKT/NF-κB pathway inhibition weakened the procancer effect mediated by KNL1 overexpression but had little influence on KNL1 levels. We conclude that KNL1 activates the PI3K/AKT/NF-κB pathway to increase NSCLC progression and attenuate NSCLC sensitivity to chemotherapy drugs.

Meet the authors: Lori Emert-Sedlak and Tom Smithgall.

In an interview with Samantha Nelson, a scientific editor of Cell Chemical Biology, the first and corresponding authors of the research article entitled "PROTAC-mediated degradation of HIV-1 Nef efficiently restores cell-surface CD4 and MHC-I expression and blocks HIV-1 replication" share insights on their paper and life as scientists.

GGT5 facilitates migration and invasion through the induction of epithelial-mesenchymal transformation in gastric cancer.

BACKGROUND: Gamma-glutamyltransferase 5 (GGT5), one of the two members in the GGT family (GGT1 and GGT5), plays a crucial role in oxidative regulation, inflammation promotion, and drug metabolism. Particularly in the tumorigenesis of various cancers, its significance has been recognized. Nevertheless, GGT5's role in gastric cancer (GC) remains ambiguous. This study delves into the function and prognostic significance of GGT5 in GC through a series of in vitro experiments. METHODS: Employing online bioinformatics analysis tools such as The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Kaplan-Meier plotter, and cBioPortal, we explored GGT5 characteristics and functions in GC. This encompassed aberrant expression, prognostic value, genomic alterations and mutations, immune cell infiltration, and associated signaling pathways. Immunohistochemistry was conducted to assess GGT5 expression in GC and adjacent normal tissues. Subsequently, univariate and multivariate logistic regression analyses were applied to investigate the associations between GGT5 and clinical characteristics. CCK8, wound healing, and migration assays were utilized to evaluate the impact of GGT5 on cell viability and migration. Additionally, Gene Set Enrichment Analysis (GSEA) and Western blot analysis were performed to scrutinize the activity of the epithelial-mesenchymal transformation (EMT) signaling pathway under GGT5 regulation. RESULTS: GGT5 exhibits upregulation in gastric cancer, with its overexpression significantly linked to histological differentiation in GC patients (P < 0.05). Multivariate analysis indicates that elevated GGT5 expression is an independent risk factor associated with poorer overall survival in gastric cancer patients (P < 0.05). In vitro experiments reveal that downregulation of GGT5 hampers the proliferation and migration of GC cell lines. Finally, GSEA using TCGA data highlights a significant correlation between GGT5 expression and genes associated with EMT, a finding further confirmed by Western blot analysis. CONCLUSIONS: GGT5 emerges as a promising prognostic biomarker and potential therapeutic target for GC.

BmSPP is a virus resistance gene in Bombyx mori.

Introduction: Signal peptide peptidase (SPP) is an intramembrane protease involved in a variety of biological processes, it participates in the processing of signal peptides after the release of the nascent protein to regulate the endoplasmic reticulum associated degradation (ERAD) pathway, binds misfolded membrane proteins, and aids in their clearance process. Additionally, it regulates normal immune surveillance and assists in the processing of viral proteins. Although SPP is essential for many viral infections, its role in silkworms remains unclear. Studying its role in the silkworm, Bombyx mori , may be helpful in breeding virus-resistant silkworms. Methods: First, we performed RT-qPCR to analyze the expression pattern of BmSPP. Subsequently, we inhibited BmSPP using the SPP inhibitor 1,3-di-(N-carboxybenzoyl-L-leucyl-L-leucylaminopropanone ((Z-LL)2-ketone) and downregulated the expression of BmSPP using CRISPR/Cas9 gene editing. Furthermore, we assessed the impact of these interventions on the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV). Results: We observed a decreased in the expression of BmSPP during viral proliferation. It was found that higher concentration of the inhibitor resulted in greater inhibition of BmNPV proliferation. The down-regulation of BmSPP in both in vivo and in vitro was found to affect the proliferation of BmNPV. In comparison to wild type silkworm, BmSPPKO silkworms exhibited a 12.4% reduction in mortality rate. Discussion: Collectively, this work demonstrates that BmSPP plays a negative regulatory role in silkworm resistance to BmNPV infection and is involved in virus proliferation and replication processes. This finding suggests that BmSPP servers as a target gene for BmNPV virus resistance in silkworms and can be utilized in resistance breeding programs.

[Downregulation of SETDB1 suppresses epithelial mesenchymal transition and invasion and metastasis in oral cancer via SOX7 methylation].

PURPOSE: To explore the mechanism of SETDB1 inhibiting epithelial mesenchymal transition (EMT),migration and invasion in oral cancer via SOX 7 methylation. METHODS: SETDB1 and SOX7 mRNA and protein expression levels in KB cells of oral cancer and oral mucosal epithelial ATCC cells were determined by qRT-PCR and Western blot (WB). SETDB1 si-RNA was structured, then transfect into KB cells of oral cancer by liposome-mediated method. siRNA-SETDB1 was the experimental group (si-S), siRNA empty vector was the negative control group (si-N), and untransfected KB cells were the blank control group(NC). SETDB1 mRNA and protein expression levels were detected by qRT-PCR and Western blot(WB), to verify the transfection effect. The methylation levels of SOX7 were determined by pyrosequencing. The expression of N-cadherin, Vimentin, ß-catenin, and Slug proteins was detected by WB. Cell viability was measured by MTT assay, migration ability was tested by scratch healing assay, and invasion ability was tested by Transwell chamber assay. Statistical analysis was performed with SPSS 21.0 software package. RESULTS: The results of Rt-qPCR and WB showed that the SETDB1 mRNA and protein expression decreased significantly in si-S group(P＜0.05). Pyrosequencing test results showed that the regulation of SETDB1 could significantly reduce the SOX7 methylation rate and increased the SOX7 protein expression. WB results showed that knockdown of SETDB1 significantly inhibited the expression of EMT-related proteins N-cadherin, Vimentin, ß-catenin and Slug in oral cancer KB cells (P＜0.05). The results of cell functology experiments showed that knockdown of SETDB1 could significantly inhibit survival, migration and invasion of KB cells. CONCLUSIONS: Downregulation of SETDB1 could suppress EMT, migration and invasion of oral cancer cells by regulating SOX7 methylation level, providing new ideas and targets for the diagnosis and treatment of oral cancer.

Multi-Omics Analysis Reveals the Distinct Features of Metabolism Pathways Supporting the Fruit Size and Color Variation of Giant Pumpkin.

Pumpkin (Cucurbita maxima) is an important vegetable crop of the Cucurbitaceae plant family. The fruits of pumpkin are often used as directly edible food or raw material for a number of processed foods. In nature, mature pumpkin fruits differ in size, shape, and color. The Atlantic Giant (AG) cultivar has the world's largest fruits and is described as the giant pumpkin. AG is well-known for its large and bright-colored fruits with high ornamental and economic value. At present, there are insufficient studies that have focused on the formation factors of the AG cultivar. To address these knowledge gaps, we performed comparative transcriptome, proteome, and metabolome analysis of fruits from the AG cultivar and a pumpkin with relatively small fruit (Hubbard). The results indicate that up-regulation of gene-encoded expansins contributed to fruit cell expansion, and the increased presence of photoassimilates (stachyose and D-glucose) and jasmonic acid (JA) accumulation worked together in terms of the formation of large fruit in the AG cultivar. Notably, perhaps due to the rapid transport of photoassimilates, abundant stachyose that was not converted into glucose in time was detected in giant pumpkin fruits, implying that a unique mode of assimilate unloading is in existence in the AG cultivar. The potential molecular regulatory network of photoassimilate metabolism closely related to pumpkin fruit expansion was also investigated, finding that three MYB transcription factors, namely CmaCh02G015900, CmaCh01G018100, and CmaCh06G011110, may be involved in metabolic regulation. In addition, neoxanthin (a type of carotenoid) exhibited decreased accumulation that was attributed to the down-regulation of carotenoid biosynthesis genes in AG fruits, which may lead to pigmentation differences between the two pumpkin cultivars. Our current work will provide new insights into the potential formation factors of giant pumpkins for further systematic elucidation.

Overexpression of miR-25 Downregulates the Expression of ROBO2 in Idiopathic Intellectual Disability.

Idiopathic intellectual disability (IID) encompasses the cases of intellectual disability (ID) without a known cause and represents approximately 50% of all cases. Neural progenitor cells (NPCs) from the olfactory neuroepithelium (NEO) contain the same information as the cells found in the brain, but they are more accessible. Some miRNAs have been identified and associated with ID of known etiology. However, in idiopathic ID, the effect of miRNAs is poorly understood. The aim of this study was to determine the miRNAs regulating the expression of mRNAs that may be involved in development of IID. Expression profiles were obtained using NPC-NEO cells from IID patients and healthy controls by microarray. A total of 796 miRNAs and 28,869 mRNAs were analyzed. Several miRNAs were overexpressed in the IID patients compared to controls. miR-25 had the greatest expression. In silico analysis showed that ROBO2 was the target for miR-25, with the highest specificity and being the most down-regulated. In vitro assay showed an increase of miR-25 expression induced a decrease in ROBO2 expression. In neurodevelopment, ROBO2 plays a crucial role in episodic learning and memory, so its down-regulation, caused by miR-25, could have a fundamental role in the intellectual disability that, until now, has been considered idiopathic.

MiR-21-5p alleviates trigeminal neuralgia in rats through down-regulation of voltage-gated potassium channel Kv1.1. / MiR-21-5p通过下调电压门控钾离子通道Kv1.1è¡¨è¾¾å‡è½»å¤§é¼ ä¸‰å‰ç¥žç»ç—›.

OBJECTIVES: Trigeminal neuralgia (TN) is a common neuropathic pain. Voltage-gated potassium channel (Kv) has been confirmed to be involved in the occurrence and development of TN, but the specific mechanism is still unclear. MicroRNA may be involved in neuropathic pain by regulating the expression of Kv channels and neuronal excitability in trigeminal ganglion (TG). This study aims to explore the relationship between Kv1.1 and miR-21-5p in TG with a TN model, evaluate whether miR-21-5p has a regulatory effect on Kv1.1, and to provide a new target and experimental basis for the treatment of TN. METHODS: A total of 48 SD rats were randomly divided into 6 groups: 1) a sham group (n=12), the rats were only sutured at the surgical incision without nerve ligation; 2) a sham+agomir NC group (n=6), the sham rats were microinjected with agomir NC through stereotactic brain injection in the surgical side of TG; 3) a sham+miR-21-5p agomir group (n=6), the sham rats were microinjected with miR-21-5p agomir via stereotactic brain injection in the surgical side of TG; 4) a TN group (n=12), a TN rat model was constructed using the chronic constriction injury of the distal infraorbital nerve (dIoN-CCI) method with chromium intestinal thread; 5) a TN+antagonist NC group (n=6), TN rats were microinjected with antagonist NC through stereotactic brain injection method in the surgical side of TG; 6) a TN+miR-21-5p antagonist group (n=6), TN rats were microinjected with miR-21-5p antagonist through stereotactic brain injection in the surgical side of TG. The change of mechanical pain threshold in rats of each group after surgery was detected. The expressions of Kv1.1 and miR-21-5p in the operative TG of rats were detected by Western blotting and real-time reverse transcription polymerase chain reaction. Dual luciferase reporter genes were used to determine whether there was a target relationship between Kv1.1 and miR-21-5p and whether miR-21-5p directly affected the 3'-UTR terminal of KCNA1. The effect of brain stereotaxic injection was evaluated by immunofluorescence assay, and then the analogue of miR-21-5p (agomir) and agomir NC were injected into the TG of rats in the sham group by brain stereotaxic apparatus to overexpress miR-21-5p. The miR-21-5p inhibitor (antagomir) and antagomir NC were injected into TG of rats in the TN group to inhibit the expression of miR-21-5p. The behavioral changes of rats before and after administration were observed, and the expression changes of miR-21-5p and Kv1.1 in TG of rats after intervention were detected. RESULTS: Compared with the baseline pain threshold, the facial mechanical pain threshold of rats in the TN group was significantly decreased from the 5th to 15th day after the surgery (P<0.05), and the facial mechanical pain threshold of rats in the sham group was stable at the normal level, which proved that the dIoN-CCI model was successfully constructed. Compared with the sham group, the expression of Kv1.1 mRNA and protein in TG of the TN group was down-regulated (both P<0.05), and the expression of miR-21-5p was up-regulated (P<0.05). The results of dual luciferase report showed that the luciferase activity of rno-miR-21-5p mimics and KCNA1 WT transfected with 6 nmol/L or 20 nmol/L were significantly decreased compared with those transfected with mimic NC and wild-type KCNA1 WT, respectively (P<0.001). Compared with low dose rno-miR-21-5p mimics (6 nmol/L) co-transfection group, the relative activity of luciferase in the high dose rno-miR-21-5p mimics (20 nmol/L) cotransfection group was significantly decreased (P<0.001). The results of immunofluorescence showed that drugs were accurately injected into TG through stereotaxic brain. After the expression of miR-21-5p in the TN group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were increased. After overexpression of miR-21-5p in the sham group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were decreased. CONCLUSIONS: Both Kv1.1 and miR-21-5p are involved in TN and miR-21-5p can regulate Kv1.1 expression by binding to the 3'-UTR of KCNA1.

HERC5 downregulation in non-small cell lung cancer is associated with altered energy metabolism and metastasis.

BACKGROUND: Metastasis is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC) patients. We previously showed that low HERC5 expression predicts early tumor dissemination and a dismal prognosis in NSCLC patients. Here, we performed functional studies to unravel the mechanism underlying the "metastasis-suppressor" effect of HERC5, with a focus on mitochondrial metabolism pathways. METHODS: We assessed cell proliferation, colony formation potential, anchorage-independent growth, migration, and wound healing in NSCLC cell line models with HERC5 overexpression (OE) or knockout (KO). To study early tumor cell dissemination, we used these cell line models in zebrafish experiments and performed intracardial injections in nude mice. Mass spectrometry (MS) was used to analyze protein changes in whole-cell extracts. Furthermore, electron microscopy (EM) imaging, cellular respiration, glycolytic activity, and lactate production were used to investigate the relationships with mitochondrial energy metabolism pathways. RESULTS: Using different in vitro NSCLC cell line models, we showed that NSCLC cells with low HERC5 expression had increased malignant and invasive properties. Furthermore, two different in vivo models in zebrafish and a xenograft mouse model showed increased dissemination and metastasis formation (in particular in the brain). Functional enrichment clustering of MS data revealed an increase in mitochondrial proteins in vitro when HERC5 levels were high. Loss of HERC5 leads to an increased Warburg effect, leading to improved adaptation and survival under prolonged inhibition of oxidative phosphorylation. CONCLUSIONS: Taken together, these results indicate that low HERC5 expression increases the metastatic potential of NSCLC in vitro and in vivo. Furthermore, HERC5-induced proteomic changes influence mitochondrial pathways, ultimately leading to alterations in energy metabolism and demonstrating its role as a new potential metastasis suppressor gene.

METTL14 downregulation drives S100A4+ monocyte-derived macrophages via MyD88/NF-κB pathway to promote MAFLD progression.

Without intervention, a considerable proportion of patients with metabolism-associated fatty liver disease (MAFLD) will progress from simple steatosis to metabolism-associated steatohepatitis (MASH), liver fibrosis, and even hepatocellular carcinoma. However, the molecular mechanisms that control progressive MAFLD have yet to be fully determined. Here, we unraveled that the expression of the N6-methyladenosine (m6A) methyltransferase METTL14 is remarkably downregulated in the livers of both patients and several murine models of MAFLD, whereas hepatocyte-specific depletion of this methyltransferase aggravated lipid accumulation, liver injury, and fibrosis. Conversely, hepatic Mettl14 overexpression alleviated the above pathophysiological changes in mice fed on a high-fat diet (HFD). Notably, in vivo and in vitro mechanistic studies indicated that METTL14 downregulation decreased the level of GLS2 by affecting the translation efficiency mediated by YTHDF1 in an m6A-depedent manner, which might help to form an oxidative stress microenvironment and accordingly recruit Cx3cr1+Ccr2+ monocyte-derived macrophages (Mo-macs). In detail, Cx3cr1+Ccr2+ Mo-macs can be categorized into M1-like macrophages and S100A4-positive macrophages and then further activate hepatic stellate cells (HSCs) to promote liver fibrosis. Further experiments revealed that CX3CR1 can activate the transcription of S100A4 via CX3CR1/MyD88/NF-κB signaling pathway in Cx3cr1+Ccr2+ Mo-macs. Restoration of METTL14 or GLS2, or interfering with this signal transduction pathway such as inhibiting MyD88 could ameliorate liver injuries and fibrosis. Taken together, these findings indicate potential therapies for the treatment of MAFLD progression.

Downregulation of lncRNA EPB41L4A-AS1 promotes gastric cancer cell proliferation, migration and invasion.

BACKGROUND: The incidence of gastric cancer ranks the first among digestive tract tumors in China. However, there are no specific symptoms in the early stage of the tumor and the diagnosis process is complex, so more effective detection methods are very needed. In this study, a novel long noncoding RNA (lncRNA) was introduced as a diagnostic biomarker for gastric cancer, which brought new thinking to the exploration of its pathological mechanism and clinical prediction. METHODS: The level of lncRNA EPB41L4A-AS1 (EPB41L4A-AS1) in gastric cancer serum and cells was verified via real-time quantitative polymerase chain reaction (RT-qPCR). Receiver operating characteristic (ROC) curve was performed based on the EPB41L4A-AS1 level, and the diagnostic possibility of EPB41L4A-AS was analyzed. The chi-square test evaluated the correlation between EPB41L4A-AS expression and clinical information. The cells were cultured and transfected in vitro, and the mediations of abnormal EPB41L4A-AS level on the viability and motility of gastric cancer cells were verified through cell counting kit-8 (CCK-8) and Transwell assay. Furthermore, luciferase activity assay was performed to confirm the sponge molecule microRNA-17-5p (miR-17-5p) of EPB41L4A-AS1. RESULTS: EPB41L4A-AS1 was decreased in gastric cancer, and low EPB41L4A-AS1 level indicated resultful diagnostic value. Overexpression of EPB41L4A-AS1 inhibited the activity of gastric cancer cells, while knockdown of EPB41L4A-AS1 promoted tumor deterioration. EPB41L4A-AS1 directly targeted and regulated the expression ofmiR-17-5p. CONCLUSION: This study elaborated that EPB41L4A-AS1 is lowly expressed in gastric cancer. Silencing EPB41L4A-AS1 was beneficial to cell proliferation, migration, and invasion. EPB41L4A-AS1 provides a new possibility for the diagnosis of gastric cancer patients by targeting miR-17-5p.

PD-1 downregulation enhances CAR-T cell antitumor efficiency by preserving a cell memory phenotype and reducing exhaustion.

BACKGROUND: Despite the encouraging outcome of chimeric antigen receptor T cell (CAR-T) targeting B cell maturation antigen (BCMA) in managing relapsed or refractory multiple myeloma (RRMM) patients, the therapeutic side effects and dysfunctions of CAR-T cells have limited the efficacy and clinical application of this promising approach. METHODS: In this study, we incorporated a short hairpin RNA cassette targeting PD-1 into a BCMA-CAR with an OX-40 costimulatory domain. The transduced PD-1KD BCMA CAR-T cells were evaluated for surface CAR expression, T-cell proliferation, cytotoxicity, cytokine production, and subsets when they were exposed to a single or repetitive antigen stimulation. Safety and efficacy were initially observed in a phase I clinical trial for RRMM patients. RESULTS: Compared with parental BCMA CAR-T cells, PD-1KD BCMA CAR-T cell therapy showed reduced T-cell exhaustion and increased percentage of memory T cells in vitro. Better antitumor activity in vivo was also observed in PD-1KD BCMA CAR-T group. In the phase I clinical trial of the CAR-T cell therapy for seven RRMM patients, safety and efficacy were initially observed in all seven patients, including four patients (4/7, 57.1%) with at least one extramedullary site and four patients (4/7, 57.1%) with high-risk cytogenetics. The overall response rate was 85.7% (6/7). Four patients had a stringent complete response (sCR), one patient had a CR, one patient had a partial response, and one patient had stable disease. Safety profile was also observed in these patients, with an incidence of manageable mild to moderate cytokine release syndrome and without the occurrence of neurological toxicity. CONCLUSIONS: Our study demonstrates a design concept of CAR-T cells independent of antigen specificity and provides an alternative approach for improving the efficacy of CAR-T cell therapy.

Forskolin-mediated cAMP activation upregulates TNF-α expression despite NF-κB downregulation in LPS-treated Schwann cells.

Although Schwann cells have been found to play a key role in inflammation and repair following nerve injury, the exact pathway is still unknown. To explore the mechanism by which Schwann cells exert their effects in the neuron microenvironment, we investigated two main inflammatory pathways: the NF-κB and cAMP pathways, and their downstream signaling molecules. In this study, lipopolysaccharide (LPS), a bacterial endotoxin, was used to activate the NF-κB pathway, and forskolin, a plant extract, was used to activate the cAMP pathway. The rat RT4-D6P2T Schwann cell line was treated with 0.1, 1, or 10 µg/mL of LPS, with or without 2 µM of forskolin, for 1, 3, 12, and 24 hours to determine the effects of elevated cAMP levels on LPS-treated cell viability. To investigate the effects of elevated cAMP levels on the expression of downstream signaling effector proteins, specifically NF-κB, TNF-α, AKAP95, and cyclin D3, as well as TNF-α secretion, RT4-D6P2T cells were incubated in the various treatment combinations for a 3-hour time period. Overall, results from the CellTiter-Glo viability assay revealed that forskolin increased viability in cells treated with smaller doses of LPS for 1 and 24 hours. For all time points, 10 µg/mL of LPS noticeably reduced viability regardless of forskolin treatment. Results from the Western blot analysis revealed that, at 10 µg/mL of LPS, forskolin upregulated the expression of TNF-α despite a downregulation of NF-κB, which was also accompanied by a decrease in TNF-α secretion. These results provide evidence that cAMP might regulate TNF-α expression through alternate pathways. Furthermore, although cAMP activation altered AKAP95 and cyclin D3 expression at different doses of LPS, there does not appear to be an association between the expression of AKAP95 or cyclin D3 and the expression of TNF-α. Exploring the possible interactions between cAMP, NF-κB, and other key inflammatory signaling pathways might reveal a potential therapeutic target for the treatment of nerve injury and inflammation.

Trametinib sensitizes KRAS-mutant lung adenocarcinoma tumors to PD-1/PD-L1 axis blockade via Id1 downregulation.

BACKGROUND: The identification of novel therapeutic strategies to overcome resistance to the MEK inhibitor trametinib in mutant KRAS lung adenocarcinoma (LUAD) is a challenge. This study analyzes the effects of trametinib on Id1 protein, a key factor involved in the KRAS oncogenic pathway, and investigates the role of Id1 in the acquired resistance to trametinib as well as the synergistic anticancer effect of trametinib combined with immunotherapy in KRAS-mutant LUAD. METHODS: We evaluated the effects of trametinib on KRAS-mutant LUAD by Western blot, RNA-seq and different syngeneic mouse models. Genetic modulation of Id1 expression was performed in KRAS-mutant LUAD cells by lentiviral or retroviral transductions of specific vectors. Cell viability was assessed by cell proliferation and colony formation assays. PD-L1 expression and apoptosis were measured by flow cytometry. The anti-tumor efficacy of the combined treatment with trametinib and PD-1 blockade was investigated in KRAS-mutant LUAD mouse models, and the effects on the tumor immune infiltrate were analyzed by flow cytometry and immunohistochemistry. RESULTS: We found that trametinib activates the proteasome-ubiquitin system to downregulate Id1 in KRAS-mutant LUAD tumors. Moreover, we found that Id1 plays a major role in the acquired resistance to trametinib treatment in KRAS-mutant LUAD cells. Using two preclinical syngeneic KRAS-mutant LUAD mouse models, we found that trametinib synergizes with PD-1/PD-L1 blockade to hamper lung cancer progression and increase survival. This anti-tumor activity depended on trametinib-mediated Id1 reduction and was associated with a less immunosuppressive tumor microenvironment and increased PD-L1 expression on tumor cells. CONCLUSIONS: Our data demonstrate that Id1 expression is involved in the resistance to trametinib and in the synergistic effect of trametinib with anti-PD-1 therapy in KRAS-mutant LUAD tumors. These findings suggest a potential therapeutic approach for immunotherapy-refractory KRAS-mutant lung cancers.

SLC7A11 inhibits ferroptosis and downregulates PD-L1 levels in lung adenocarcinoma.

Introduction: Lung adenocarcinoma (LUAD) is a prevalent form of lung cancer originating from lung glandular cells with low survival rates despite recent therapeutic advances due to its diverse and complex nature. Recent evidence suggests a link between ferroptosis and the effectiveness of anti-PD-L1 therapy, with potential synergistic effects. Methods: Our study comprehensively analyzed the expression patterns of ferroptosis regulators in LUAD and their association with prognosis and PD-L1 expression. Furthermore, we identified two distinct subtypes of LUAD through consensus clustering of ferroptosis regulators, revealing significant tumor heterogeneity, divergent PD-L1 expression, and varying prognoses between the subtypes. Results: Among the selected ferroptosis regulators, SLC7A11 emerged as an independent prognostic marker for LUAD patients and exhibited a negative correlation with PD-L1 expression. Subsequent investigations revealed high expression of SLC7A11 in the LUAD population. In vitro experiments demonstrated that overexpression of SLC7A11 led to reduced PD-L1 expression and inhibited ferroptosis in A549 cells, underscoring the significant role of SLC7A11 in LUAD. Additionally, pan-cancer analyses indicated an association between SLC7A11 and the expression of immune checkpoint genes across multiple cancer types with poor prognoses. Discussion: From a clinical standpoint, these findings offer a foundation for identifying and optimizing potential combination strategies to enhance the therapeutic effectiveness of immune checkpoint inhibitors and improve the prognosis of patients with LUAD.

Downregulation of HMGCS2 mediated AECIIs lipid metabolic alteration promotes pulmonary fibrosis by activating fibroblasts.

BACKGROUND: Abnormal lipid metabolism has recently been reported as a crucial signature of idiopathic pulmonary fibrosis (IPF). However, the origin and biological function of the lipid and possible mechanisms of increased lipid content in the pathogenesis of IPF remains undetermined. METHODS: Oil-red staining and immunofluorescence analysis were used to detect lipid accumulation in mouse lung fibrosis frozen sections, Bleomycin-treated human type II alveolar epithelial cells (AECIIs) and lung fibroblast. Untargeted Lipid omics analysis was applied to investigate differential lipid species and identified LysoPC was utilized to treat human lung fibroblasts and mice. Microarray and single-cell RNA expression data sets identified lipid metabolism-related differentially expressed genes. Gain of function experiment was used to study the function of 3-hydroxy-3-methylglutaryl-Coa Synthase 2 (HMGCS2) in regulating AECIIs lipid metabolism. Mice with AECII-HMGCS2 high were established by intratracheally delivering HBAAV2/6-SFTPC- HMGCS2 adeno-associated virus. Western blot, Co-immunoprecipitation, immunofluorescence, site-directed mutation and flow cytometry were utilized to investigate the mechanisms of HMGCS2-mediated lipid metabolism in AECIIs. RESULTS: Injured AECIIs were the primary source of accumulated lipids in response to Bleomycin stimulation. LysoPCs released by injured AECIIs could activate lung fibroblasts, thus promoting the progression of pulmonary fibrosis. Mechanistically, HMGCS2 was decreased explicitly in AECIIs and ectopic expression of HMGCS2 in AECIIs using the AAV system significantly alleviated experimental mouse lung fibrosis progression via modulating lipid degradation in AECIIs through promoting CPT1A and CPT2 expression by interacting with PPARα. CONCLUSIONS: These data unveiled a novel etiological mechanism of HMGCS2-mediated AECII lipid metabolism in the genesis and development of pulmonary fibrosis and provided a novel target for clinical intervention.

MicroRNA-503-5p protects streptozotocin-induced erectile dysfunction in diabetic rats by downregulating SYDE2.

Plentiful studies have clarified miRNAs take on a key role in the sexual dysfunction of diabetic rats. This study aimed to figure out microRNA (miR)-503-5p/SYDE2 axis' latent mechanisms in streptozotocin-induced diabetic rat sexual dysfunction. A model of erectile dysfunction (ED) in diabetic rats was established by injecting streptozotocin. MiR-503-5p and SYDE2 in ED rats were altered by injection of miR-503-5p mimic or si/oe-SYDE2. The targeting link between miR-503-5p and SYDE2 was testified. ICP/MAP value was tested by pressure sensor; Penile capillary abundance was assessed; Penile cGMP and AGEs were detected; penile smooth muscle cell apoptosis was assessed; MiR-503-5p and SYDE2 were tested. In streptozotocin-induced ED rats, miR-503-5p was reduced and SYDE2 was elevated. Elevating miR-503-5p or silencing of SYDE2 can enhance penile erection rate, ICP/MAP value, capillary abundance, and cGMP but reduce AGEs and penile smooth muscle cell apoptosis rate in ED rats. Strengthening SYDE2 with elevating miR-503-5p turned around the accelerating effect of elevated miR-503-5p on penile erection in ED rats. SYDE2 was a downstream target gene of miR-503-5p. MiR-503-5p protects streptozotocin-induced sexual dysfunction in diabetic rats by targeting SYDE2.

Downregulation of Glis3 in INS1 cells exposed to chronically elevated glucose contributes to glucotoxicity-associated ß cell dysfunction.

Chronically elevated levels of glucose are deleterious to pancreatic ß cells and contribute to ß cell dysfunction, which is characterized by decreased insulin production and a loss of ß cell identity. The Krüppel-like transcription factor, Glis3 has previously been shown to positively regulate insulin transcription and mutations within the Glis3 locus have been associated with the development of several pathologies including type 2 diabetes mellitus. In this report, we show that Glis3 is significantly downregulated at the transcriptional level in INS1 832/13 cells within hours of being subjected to high glucose concentrations and that diminished expression of Glis3 is at least partly attributable to increased oxidative stress. CRISPR/Cas9-mediated knockdown of Glis3 indicated that the transcription factor was required to maintain normal levels of both insulin and MafA expression and reduced Glis3 expression was concomitant with an upregulation of ß cell disallowed genes. We provide evidence that Glis3 acts similarly to a pioneer factor at the insulin promoter where it permissively remodels the chromatin to allow access to a transcriptional regulatory complex including Pdx1 and MafA. Finally, evidence is presented that Glis3 can positively regulate MafA transcription through its pancreas-specific promoter and that MafA reciprocally regulates Glis3 expression. Collectively, these results suggest that decreased Glis3 expression in ß cells exposed to chronic hyperglycemia may contribute significantly to reduced insulin transcription and a loss of ß cell identity.

CircPHGDH downregulation decreases papillary thyroid cancer progression through miR-122-5p/PKM2 axis.

BACKGROUND: Although papillary thyroid carcinoma (PTC) has a favorable prognosis, it could affect patient life quality and become a serious threat because of invasion and metastasis. Many investigations have suggested that circular RNAs (circRNAs) are involved in different cancer regulations. Nevertheless, circRNAs role in invasive PTC remains unclear. METHODS: In the present investigation, next-generation sequencing was applied to explore abnormal circRNA expression. The expression of circRNA phosphoglycerate dehydrogenase (circPHGDH) in PTC cell lines and tissues were examined. Then, we investigated regulatory mechanism and circPHGDH downstream targets using bioinformatics analysis and luciferase reporting analysis. Then transwell migration, Cell Counting Kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used for cells migration and proliferation analysis. In vivo metastasis and tumorigenesis assays were also employed to evaluate the circPHGDH role in PTC. RESULTS: The data showcased that circPHGDH expression increased in both PTC cell lines and tissues, which suggested that circPHGDH functions in PTC progression. circPHGDH downregulation suppressed PTC invasion and proliferation in both in vivo and in vitro experiments. Bioinformatics and luciferase reporter results confirmed that both microRNA (miR)-122-5p and pyruvate kinase M2 subtype (PKM2) were downstream targets of circPHGDH. PKM2 overexpression or miR-122-5p suppression reversed PTC cell invasion and proliferation post silencing circPHGDH by restoring aerobic glycolysis. CONCLUSION: Taken together, our research found that circPHGDH downregulation reduced PTC progression via miR-122-5p/PKM2 axis regulation mediated by aerobic glycolysis.