Reporter Assay Experiments Research Articles

N6-methyladenosine modification of circular RNA circASH2L suppresses growth and metastasis in hepatocellular carcinoma through regulating hsa-miR-525-3p/MTUS2 axis

BackgroundCircRNAs have been demonstrated to play a crucial role in regulating the growth and progression of various cancers, including hepatocellular carcinoma (HCC). Nevertheless, the circRNA's expression pattern and function in HCC need more investigation.MethodsBioinformatics techniques were used to identify differentially expressed circRNAs in HCC. CircASH2L expression in HCC tissues was assessed through qRT-PCR and ISH analysis. To assess circASH2L's impact on HCC progression, a variety of experiments were carried out both in vitro and in vivo, such as CCK8, colony formation, EdU assay, flow cytometry, transwell assay, and xenograft mouse model. Various experimental techniques including qRT-PCR, dual luciferase reporter assay, FISH, RNA pull-down, and RIP experiments were utilized to evaluate the relationship between circASH2L, miR-525-3p, and MTUS2. Additionally, experiments were conducted to explore the impact of m6A modification on circASH2L expression, including RNA stability assay, m6A RNA immunoprecipitation assay (MeRIP), and Co-IP experiments.ResultsWe found that circASH2L was downregulated in HCC tissues and the downregulation of circASH2L was significantly correlated with malignant characteristics as well as poor overall survival of patients with HCC. CircASH2L was found to inhibit cells growth, migration and invasion as well as tumorigenesis and metastasis in vivo. Mechanistically, we established that circASH2L directly interacted with miR-525-3p to enhance MTUS2 expression, subsequently leading to tumor suppression. Moreover, the influence of circASH2L on tumor suppression was attenuated by increasing miR-525-3p levels, and MTUS2 was recognized as an essential intermediary in circASH2L-induced tumor suppression. Additionally, N6−methyladenosine (m6A) modification was identified in circASH2L. Our data suggested that METTL3 was responsible for mediating m6A methylation of circASH2L, ultimately regulating circASH2L expression through the promotion of its degradation. These findings collectively highlight the role of circASH2L as a tumor suppressor through a unique circASH2L/miR-525-3p/MTUS2 axis, shedding light on the significance of m6A modification in regulating circASH2L function.ConclusionThe work emphasizes circASH2L as a promising therapeutic target for treating HCC, offering new insights into the role of circRNAs in HCC development.

Hypoxia-induced BAP1 enhances erastin-induced ferroptosis in nasopharyngeal carcinoma by stabilizing H2A

BackgroundHypoxia plays an important role in the chemotherapy resistance of nasopharyngeal carcinoma (NPC). Ferroptosis is a newly discovered form of programmed cell death and ferroptosis inducers showed promising therapeutic effects in some cancers. However, the sensibility of NPC cells to ferroptosis under the hypoxic microenvironment is still unclear, and this study was designed to clarify it.MethodsNPC cells, treated with erastin, were placed in a normoxia or hypoxic environment (5% CO2, 94% N2 and 1% O2) at 37℃for 24 h. After exposed to hypoxia, ferroptosis-associated phenotypes were detected by CCK8, MDA, GSH, lipid ROS and Fe. The gene expression profiles of head and neck squamous cell carcinoma (HNSCC) tissues were downloaded from the TCGA database to screen construction molecule. BAP1 was screened out and its functions on erastin-induced ferroptosis in NPC cells were detected by knockdown of BAP1. Luciferase reporter assay and co-IP experiment were performed to explore the molecular mechanism. Finally, the tumour xenograft model was applied to further verify these results in vivo.ResultsCCK8 assay showed that IC50 of NPC cells treated with erastin under hypoxia was significantly lower than that under normoxia. Hypoxia significantly increased the levels of lipid ROS and MDA, and decreased GSH content induced by erastin. A prognostic risk model for HNSCC with six ferroptosis-related genes was constructed and validated based on TCGA database. BAP1 was significantly up-regulated under hypoxia, and luciferase reporter assay showed that HIF-1α was an upstream transcription regulator of BAP1. Knockdown of BAP1 in NPC cells significantly increased the IC50 value of erastin under hypoxia and significantly ameliorated erastin-induced ferroptosis under hypoxia in aspect of lipid ROS, MDA content and GSH. Co-IP results showed that BAP1 mediated deubiquitination of H2A and decreased SLC7A11 expression. Finally, knockdown of BAP1 reduced sensitivity to erastin-induced ferroptosis in a tumour xenograft model. And the level of H2A was significantly decreased in xenograft tumors of BAP1 knockdown cells.ConclusionHypoxia-induced BAP1 enhances erastin-induced ferroptosis in NPC by stabilizing H2A. Ferroptosis inducers targeting BAP1 may be an effective way to improve chemotherapy resistance in NPC, especially in the hypoxic microenvironment.Graphical

Cuproptosis related ceRNA axis AC008083.2/miR-142-3p promotes the malignant progression of nasopharyngeal carcinoma through STRN3

Background CeRNA axis is an important way to regulate the occurrence and development of Nasopharyngeal carcinoma (NPC). Although the research on inducing cuproptosis of tumor cells is in the early stage of clinical practice, its mechanism of action is still of great significance for tumor treatment, including NPC. However, the regulation mechanism of cuproptosis in NPC by ceRNA network remains unclear. Methods The ceRNA network related to the survival of nasopharyngeal carcinoma related genes was constructed by bioinformatics. Dual-luciferase reporter assay and other experiments were used to prove the conclusion. Results Our findings indicate that the AC008083.2/miR-142-3p axis drives STRN3 to promote the malignant progression of NPC. By performing enrichment analysis and phenotypic assays, we demonstrated that the changes in the expressions of AC008083.2/miR-142-3p/NPC can affect the proliferation of NPC. Mechanistically, luciferase reporter gene assays suggested that AC008083.2 acts as a ceRNA of miR-142-3p to regulate the content of STRN3. Furthermore, the regulations of STRN3 and the malignant progression of NPC by AC008083.2 depends on miR-142-3p to some extent. Conclusions Our study reveals an innovative ceRNA regulatory network in NPC, which can be considered a new potential target for diagnosing and treating NPC.

Transplantation of miR-145a-5p modified M2 type microglia promotes the tissue repair of spinal cord injury in mice

BackgroundThe traumatic spinal cord injury (SCI) can cause immediate multi-faceted function loss or paralysis. Microglia, as one of tissue resident macrophages, has been reported to play a critical role in regulating inflammation response during SCI processes. And transplantation with M2 microglia into SCI mice promotes recovery of motor function. However, the M2 microglia can be easily re-educated and changed their phenotype due to the stimuli of tissue microenvironment. This study aimed to find a way to maintain the function of M2 microglia, which could exert an anti-inflammatory and pro-repair role, and further promote the repair of spinal cord injury.MethodsTo establish a standard murine spinal cord clip compression model using Dumont tying forceps. Using FACS, to sort microglia from C57BL/6 mice or CX3CR1GFP mice, and further culture them in vitro with different macrophage polarized medium. Also, to isolate primary microglia using density gradient centrifugation with the neonatal mice. To transfect miR-145a-5p into M2 microglia by Lipofectamine2000, and inject miR-145a-5p modified M2 microglia into the lesion sites of spinal cord for cell transplanted therapy. To evaluate the recovery of motor function in SCI mice through behavior analysis, immunofluorescence or histochemistry staining, Western blot and qRT-PCR detection. Application of reporter assay and molecular biology experiments to reveal the mechanism of miR-145a-5p modified M2 microglia therapy on SCI mice.ResultsWith in vitro experiments, we found that miR-145a-5p was highly expressed in M2 microglia, and miR-145a-5p overexpression could suppress M1 while promote M2 microglia polarization. And then delivery of miR-145a-5p overexpressed M2 microglia into the injured spinal cord area significantly accelerated locomotive recovery as well as prevented glia scar formation and neuron damage in mice, which was even better than M2 microglia transplantation. Further mechanisms showed that overexpressed miR-145a-5p in microglia inhibited the inflammatory response and maintained M2 macrophage phenotype by targeting TLR4/NF-κB signaling.ConclusionsThese findings indicate that transplantation of miR-145a-5p modified M2 microglia has more therapeutic potential for SCI than M2 microglia transplantation from epigenetic perspective.Graphical

High incidence and geographic distribution of cleft palate cases in Finland are associated with a regulatory variant in IRF6.

In Finland the frequency of isolated cleft palate (CP) is higher than that of isolated cleft lip with or without cleft palate (CL/P). This trend contrasts to that in other European countries but its genetic underpinnings are unknown. We performed a genome-wide association study for orofacial clefts, which include CL/P and CP, in the Finnish population. We identified rs570516915, a single nucleotide polymorphism that is highly enriched in Finns and Estonians, as being strongly associated with CP ( P = 5.25 × 10 -34 , OR = 8.65, 95% CI 6.11-12.25), but not with CL/P ( P = 7.2 × 10 -5 ), with genome-wide significance. The risk allele frequency of rs570516915 parallels the regional variation of CP prevalence in Finland, and the association was replicated in independent cohorts of CP cases from Finland ( P = 8.82 × 10 -28 ) and Estonia ( P = 1.25 × 10 -5 ). The risk allele of rs570516915 disrupts a conserved binding site for the transcription factor IRF6 within a previously characterized enhancer upstream of the IRF6 gene. Through reporter assay experiments we found that the risk allele of rs570516915 diminishes the enhancer activity. Oral epithelial cells derived from CRISPR-Cas9 edited induced pluripotent stem cells demonstrate that the CP-associated allele of rs570516915 concomitantly decreases the binding of IRF6 and the expression level of IRF6 , suggesting impaired IRF6 autoregulation as a molecular mechanism underlying the risk for CP.

Transcription factor NFYA inhibits ferroptosis in lung adenocarcinoma cells by regulating PEBP1

BackgroundFerroptosis is an iron-dependent programmed cell death mediated by lipid peroxidation. The purpose was to explore the molecular mechanism by which phosphatidylethanolamine-binding protein 1 (PEBP1) regulates ferroptosis in lung adenocarcinoma (LUAD), hoping to identify novel therapeutic targets for LUAD. MethodsThe expression, enrichment pathways and upstream transcription factors of PEBP1 were analyzed using bioinformatics tools. Dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) experiments were conducted to validate the interaction and binding relationship between PEBP1 and the upstream transcription factor nuclear transcription factor Y subunit α (NFYA). Quantitative reverse transcription PCR (qRT-PCR) was conducted to measure the expression levels of PEBP1 and NFYA mRNA in LUAD cells. Cell viability was detected by cell counting kit-8 assay. In addition, levels of malondialdehyde (MDA), Fe2+, and lipid reactive oxygen species (ROS) were assessed to evaluate ferroptosis levels in LUAD cells. ResultsPEBP1 was downregulated and significantly enriched in the ferroptosis signaling pathway in LUAD. Overexpression of PEBP1 suppressed cell viability remarkably, while levels of MDA, Fe2+, and lipid ROS were increased. Conversely, knockdown of PEBP1 produced the opposite effects. The upstream transcription factor NFYA, predicted to be involved in the regulation of PEBP1, was also upregulated in LUAD. Dual-luciferase reporter assay, ChIP, and molecular experiments revealed that NFYA transcriptionally suppressed the expression of PEBP1, and overexpression of NFYA could reverse the effects caused by PEBP1 overexpression. ConclusionPEBP1 regulated ferroptosis in LUAD, and the transcription factor NFYA inhibited ferroptosis in LUAD cells by transcriptionally downregulating PEBP1 expression.

Upregulation of LncRNA UCA1 promotes cardiomyocyte proliferation by inhibiting the miR-128/SUZ12/P27 pathway

Enhancing cardiomyocyte proliferation is essential to reverse or slow down the heart failure progression in many cardiovascular diseases such as myocardial infarction (MI). Long non-coding RNAs (lncRNAs) have been reported to regulate cardiomyocyte proliferation. In particular, lncRNA urothelial carcinoma-associated 1 (lncUCA1) played multiple roles in regulating cell cycle progression and cardiovascular diseases, making lncUCA1 a potential target for promoting cardiomyocyte proliferation. However, the role of lncUCA1 in cardiomyocyte proliferation remains unknown. This study aimed at exploring the function and underlying molecular mechanism of lncUCA1 in cardiomyocyte proliferation. Quantitative RT-PCR showed that lncUCA1 expression decreased in postnatal hearts. Gain-and-loss-of-function experiments showed that lncUCA1 positively regulated cardiomyocyte proliferation in vitro and in vivo. The bioinformatics program identified miR-128 as a potential target of lncUCA1, and loss of miR-128 was reported to promote cardiomyocyte proliferation by inhibiting the SUZ12/P27 pathway. Luciferase reporter assay, qRT-PCR, western blotting, and immunostaining experiments further revealed that lncUCA1 acted as a ceRNA of miR-128 to upregulate its target SUZ12 and downregulate P27, thereby increasing cyclin B1, cyclin E, CDK1 and CDK2 expression to promote cardiomyocyte proliferation. In conclusion, upregulation of lncRNA UCA1 promoted cardiomyocyte proliferation by inhibiting the miR-128/SUZ12/P27 pathway. Our results indicated that lncUCA1 might be a new therapeutic target for stimulating cardiomyocyte proliferation.

LncRNA MIR181A2HG inhibits keratinocytes proliferation through miR-223-3p/SOX6 axis.

Psoriasis is a complex and recurrent chronic inflammatory skin disease, and the abnormal proliferation of keratinocytes plays a crucial role in the pathogenesis of psoriasis. Long non-coding RNAs (lncRNAs) play an indispensable role in regulating cellular functions. This research aims to explore the potential impact of lncRNA MIR181A2HG on the regulation of keratinocyte proliferation. The expression level of MIR181A2HG and the mRNA level of KRT6, KRT16, and SOX6 were assessed using qRT-PCR. The viability and proliferation of keratinocytes were evaluated using CCK-8 and EdU assays. Cell cycle analysis was performed using flow cytometry. Dual-luciferase reporter assays were applied to test the interaction among MIR181A2HG/miR-223-3p/SOX6. Protein level was detected by Western blotting analysis. The findings indicated that psoriasis lesions tissue exhibited lower levels of MIR181A2HG expression compared to normal tissue. The overexpression of MIR181A2HG resulted in the inhibition of HaCaT keratinocytes proliferation. The knockdown of MIR181A2HG promoted cell proliferation. The dual-luciferase reporter assay and rescue experiments provided evidence of the interaction among MIR181A2HG, SOX6, and miR-223-3p. The lncRNA MIR181A2HG functions as a miR-223-3p sponge targeting SOX6 to regulate the proliferation of keratinocytes, which suggested that MIR181A2HG/miR-223-3p/SOX6 might be potential diagnostic and therapeutic targets for psoriasis.

NEAT1_1 confers gefitinib resistance in lung adenocarcinoma through promoting AKR1C1-mediated ferroptosis defence

Gefitinib is one of the most extensively utilized epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) for treating advanced lung adenocarcinoma (LUAD) patients harboring EGFR mutation. However, the emergence of drug resistance significantly compromised the clinical efficacy of EGFR-TKIs. Gaining further insights into the molecular mechanisms underlying gefitinib resistance holds promise for developing novel strategies to overcome the resistance and improve the prognosis in LUAD patients. Here, we identified that the inhibitory efficacy of gefitinib on EGFR-mutated LUAD cells was partially dependent on the induction of ferroptosis, and ferroptosis protection resulted in gefitinib resistance. Among the ferroptosis suppressors, aldo-keto reductase family 1 member C1 (AKR1C1) exhibited significant upregulation in gefitinib-resistant strains of LUAD cells and predicted poor progression-free survival (PFS) and overall survival (OS) of LUAD patients who received first-generation EGFR-TKI treatment. Knockdown of AKR1C1 partially reversed drug resistance by re-sensitizing the LUAD cells to gefitinib-mediated ferroptosis. The decreased expression of miR-338-3p contributed to the aberrant upregulation of AKR1C1 in gefitinib-resistant LUAD cells. Furthermore, upregulated long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1_1 (NEAT1_1) sponged miR-338-3p to neutralize its suppression on AKR1C1. Dual-luciferase reporter assay and miRNA rescue experiment confirmed the NEAT1_1/miR-338-3p/AKR1C1 axis in EGFR-mutated LUAD cells. Gain- and loss-of-function assays demonstrated that the NEAT1_1/miR-338-3p/AKR1C1 axis promoted gefitinib resistance, proliferation, migration, and invasion in LUAD cells. This study reveals the effects of NEAT1_1/miR-338-3p/AKR1C1 axis-mediated ferroptosis defence in gefitinib resistance in LUAD. Thus, targeting NEAT1_1/miR-338-3p/AKR1C1 axis might be a novel strategy for overcoming gefitinib resistance in LUAD harboring EGFR mutation.

The SOX2/PDIA6 axis mediates aerobic glycolysis to promote stemness in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is an aggressive and rapidly expanding lung cancer. Abnormal upregulation or knockdown of PDIA6 expression can predict poor prognosis in various cancers. This study aimed to investigate the biological function of PDIA6 in NSCLC. SOX2 and PDIA6 expression in NSCLC tissues and regulatory relationship between them were analyzed using bioinformatics. GSEA was performed on the enrichment pathway of PDIA6. qRT-PCR was utilized to examine expression of SOX2 and PDIA6 in NSCLC tissues and cells, and dual-luciferase reporter assay and ChIP experiments were performed to validate their regulatory relationship. CCK-8 experiment was conducted to assess cell viability, western blot was to examine levels of stem cell markers and proteins related to aerobic glycolysis pathway in cells. Cell sphere formation assay was used to evaluate efficiency of cell sphere formation. Reagent kits were used to measure glycolysis levels and glycolysis products. High expression of PDIA6 in NSCLC was linked to aerobic glycolysis. Knockdown of PDIA6 reduced cell viability, expression of stem cell surface markers, and cell sphere formation efficiency in NSCLC. Overexpression of PDIA6 could enhance cell viability and promote aerobic glycolysis, but the addition of 2-DG could reverse this result. Bioinformatics predicted the existence of upstream transcription factor SOX2 for PDIA6, and SOX2 was significantly upregulated in NSCLC, and they had a binding relationship. Further experiments revealed that PDIA6 overexpression restored repressive effect of knocking down SOX2 on aerobic glycolysis and cell stemness. This work revealed that the SOX2/PDIA6 axis mediated aerobic glycolysis to promote NSCLC cell stemness, providing new therapeutic strategies for NSCLC.

N6-methyladenosine-modified MIB1 promotes stemness properties and peritoneal metastasis of gastric cancer cells by ubiquitinating DDX3X.

Peritoneal metastasis (PM), one of the most typical forms of metastasis in advanced gastric cancer (GC), indicates a poor prognosis. Exploring the potential molecular mechanism of PM is urgently necessary, as it has not been well studied. E3 ubiquitin ligase has been widely established to exert a biological function in various cancers, but its mechanism of action in GC with PM remains unknown. The effect of MIB1 on PM of GC was confirmed in vitro and in vivo. Co-immunoprecipitation (Co-IP) and mass spectrometry demonstrated the association between MIB1 and DDX3X. Western blot, flow cytometry and immunofluorescence determined that DDX3X was ubiquitylated by MIB1 and promoted stemness. We further confirmed that METTL3 promoted the up-regulation of MIB1 by RNA immunoprecipitation (RIP), luciferase reporter assay and other experiments. We observed that the E3 ubiquitin ligase Mind bomb 1 (MIB1) was highly expressed in PMs, and patients with PM with high MIB1 expression showed a worse prognosis than those with low MIB1 expression. Mechanistically, our study demonstrated that the E3 ubiquitin ligase MIB1 promoted epithelial-mesenchymal transition (EMT) progression and stemness in GC cells by degrading DDX3X. In addition, METTL3 mediated m6A modification to stabilize MIB1, which required the m6A reader IGF2BP2. Our study elucidated the specific molecular mechanism by which MIB1 promotes PM of GC, and suggested that targeting the METTL3-MIB1-DDX3X axis may be a promising therapeutic strategy for GC with PM.

METTL4 mediated-N6-methyladenosine promotes acute lung injury by activating ferroptosis in alveolar epithelial cells

Sepsis-induced acute lung injury has been deemed to be an life-threatening pulmonary dysfunction caused by a dysregulated host response to infection. The modification of N6-Methyladenosine (m6A) is implicated in several biological processes, including mitochondrial transcription and ferroptosis. Ferroptosis is an iron-dependent type of programed cell death, which plays a role in sepsis-induced acute lung injury (ALI). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial regulator of intracellular oxidative homeostasis, linked to ferroptosis resistance. This research aims to explore the effect of m6A in ferroptosis in sepsis-induced ALI. First, we found a time-dependent dynamic alteration on pulmonary methylation level during sepsis-induced ALI. We identified METTL4 as a differentially expressed gene in ALI mice using m6A sequencing and RNA-sequencing, and revealed the methylation of several ferroptosis related genes (Nrf2). Thus, we generated METTL4 deficiency mice and found that METTL4 knockdown alleviated ferroptosis, as evidenced by lipid ROS, MDA, Fe2+, as well as alterations in GPX4 and SLC7A11 protein expression. Consistently, we found that METTL4 silencing could decrease ferroptosis sensitivity in LPS-induced TC-1 cells. Furthermore, both the dual-luciferase reporter assay and rescue experiments indicated that METTL4 mediated the N6-methyladenosine of Nrf2 3′UTR, then YTHDF2 binded with the m6A site, promoting the degradation of Nrf2. In conclusion, we revealed that METTL4 promoted alveolar epithelial cells ferroptosis in sepsis-induced lung injury via N6-methyladenosine of Nrf2, which might provide a novel approach to therapeutic strategies for sepsis-induced ALI.

Circ_MAPK9 promotes STAT3 and LDHA expression by silencing miR-642b-3p and affects the progression of hepatocellular carcinoma

BackgroundAberrant expression and activation of circular RNAs (circRNAs) are closely associated with various cancers. The role of circ_MAPK9 (hsa_circ_0001566) in cancer progression remains unknown. This study aims to investigate the function, mechanism and clinical significance of circ_MAPK9 in hepatocellular carcinoma (HCC).MethodsCirc_MAPK9 expression on the microarray of tumor from clinical HCC patients was detected by in situ hybridization (ISH). Circ_MAPK9 knockdown was achieved with siRNAs in SMMC-7721 and SK-Hep1 HCC cell lines. The biological function of circ_MAPK9 was verified in vitro by CCK8 test, colony formation assay, transwell assay, PI-Annexin V staining, and in vivo by xenograft tumor in nude mice. Fluorescent in situ hybridization (FISH), subcellular fractionation assay, a dual-luciferase reporter assay and rescue experiments were employed for further mechanistic investigation.ResultsThe expression of circ_MAPK9 was significantly up-regulated in HCC tissues and cells, which was found to be associated with poor prognosis. Patients with high expression of circ_MAPK9 had a shorter overall survival and disease-free survival in comparison to those with low circ_MAPK9 expression. Functional assays showed that circ_MAPK9 knockdown suppressed cellular proliferation, migration, invasion and tumor growth in vivo, and promoted apoptosis in HCC cells. Moreover, we found that circ_MAPK9 knockdown could inhibit aerobic glycolysis by decreasing the production of adenosine triphosphate (ATP) and lactic acid, which was mediated by lactate dehydrogenase (LDHA). Mechanistically, circ_MAPK9 functioned as ceRNA via sponging miR-642b-3p and alleviated the inhibitory effect of miR-642b-3p on its target signal transducer and activator of transcription 3 (STAT3) and LDHA, thereby leading to STAT3 activation and LDHA expression.ConclusionsCirc_MAPK9, as an oncogene, promotes HCC growth and metastasis through miR-642b-3p/STAT3-LDHA axis. Circ_MAPK9 could serve as a potential biomarker for HCC poor prognosis and diagnosis.

NOTCH3 promotes docetaxel resistance of prostate cancer cells through regulating TUBB3 and MAPK signaling pathway.

Docetaxel is the preferred chemotherapeutic agent in patients with castrate-resistant prostate cancer (CRPC). However, patients eventually develop docetaxel resistance and in the absence of effective treatment options. Consequently, it is essential to investigate the mechanisms generating docetaxel resistance and develop novel alternative therapeutic targets. RNA sequencing was undertaken on docetaxel-sensitive and docetaxel-resistant prostate cancer (PCa) cells. Subsequently, chemoresistance, cancer stemness, and lipid metabolism were investigated. To obtain insight into the precise activities and action mechanisms of NOTCH3 in docetaxel-resistant PCa, immunoprecipitation, mass spectrometry, ChIP, luciferase reporter assay, cell metabolism, and animal experiments were performed. Through RNA sequencing analysis, we found that NOTCH3 expression was markedly higher in docetaxel-resistant cells relative to parental cells, and that this trend was continued in docetaxel-resistant PCa tissues. Experiments invitro and invivo revealed that NOTCH3 enhanced stemness, lipid metabolism, and docetaxel resistance in PCa. Mechanistically, NOTCH3 is bound to TUBB3 and activates the MAPK signaling pathway. Moreover, NOTCH3 was directly regulated by MEF2A in docetaxel-resistant cells. Notably, targeting NOTCH3 and the MEF2A/TUBB3 signaling axis was related to docetaxel chemoresistance in PCa. Overall, these results demonstrated that NOTCH3 fostered stemness, lipid metabolism, and docetaxel resistance in PCa via the TUBB3 and MAPK signaling pathways. Therefore, NOTCH3 may be employed as a prognostic biomarker in PCa patients. NOTCH3 could be a therapeutic target for PCa patients, particularly those who have developed docetaxel resistance.

Naringenin regulates cigarette smoke extract-induced extracellular vesicles from alveolar macrophage to attenuate the mouse lung epithelial ferroptosis through activating EV miR-23a-3p/ACSL4 axis

BackgroundAlveolar macrophages are one of the momentous regulators in pulmonary inflammatory responses, which can secrete extracellular vesicles (EVs) packing miRNAs. Ferroptosis, an iron-dependent cell death, is associated with cigarette smoke-induced lung injury, and EVs have been reported to regulate ferroptosis by transporting intracellular iron. However, the regulatory mechanism of alveolar macrophage-derived EVs has not been clearly illuminated in smoking-related pulmonary ferroptosis. Despite the known anti-ferroptosis effects of naringenin in lung injury, whether naringenin controls EVs-mediated ferroptosis has not yet been explored. PurposeWe explore the effects of EVs from cigarette smoke-stimulated alveolar macrophages in lung epithelial ferroptosis, and elucidate the EV miRNA-mediated pharmacological mechanism of naringenin. Study design and methodsDifferential and ultracentrifugation were conducted to extract EVs from different alveolar macrophages treatment groups in vitro. Both intratracheal instilled mice and treated epithelial cells were used to investigate the roles of EVs from alveolar macrophages involved in ferroptosis. Small RNA sequencing analysis was performed to distinguish altered miRNAs in EVs. The ferroptotic effects of EV miRNAs were examined by applying dual-Luciferase reporter assay and miRNA inhibitor transfection experiment. ResultsHere, we firstly reported that EVs from cigarette smoke extract-induced alveolar macrophages (CSE-EVs) provoked pulmonary epithelial ferroptosis. The ferroptosis inhibitor ferrostatin-1 treatment reversed these changes in vitro. Moreover, EVs from naringenin and CSE co-treated alveolar macrophages (CSE+Naringenin-EVs) markedly attenuated the lung epithelial ferroptosis compared with CSE-EVs. Notably, we identified miR-23a-3p as the most dramatically changed miRNA among Normal-EVs, CSE-EVs, and CSE+Naringenin-EVs. Further experimental investigation showed that ACSL4, a pro-ferroptotic gene leading to lipid peroxidation, was negatively regulated by miR-23a-3p. The inhibition of miR-23a-3p diminished the efficacy of CSE+Naringenin-EVs. ConclusionOur findings firstly provided evidence that naringenin elevated the EV miR-23a-3p level from CSE-induced alveolar macrophages, thereby inhibiting the mouse lung epithelial ferroptosis via targeting ACSL4, and further complemented the mechanism of cigarette-induced lung injury and the protection of naringenin in a paracrine manner. The administration of miR-23a-3p-enriched EVs has the potential to ameliorate pulmonary ferroptosis.

TTC13 expression and STAT3 activation may form a positive feedback loop to promote ccRCC progression

Background Renal cell carcinoma (RCC) originates from renal tubular epithelial cells and is mainly classified into three histological types, including clear cell renal cell carcinoma (ccRCC) which accounts for about 75% of all kidney cancers and is characterized by its strong invasiveness and poor prognosis. Hence, it is imperative to understand the mechanisms underlying the occurrence and progression of ccRCC to identify effective biomarkers for the early diagnosis and the prognosis prediction. Methods The mRNA level of TTC13 was quantified by RT-PCR, while the protein level was determined by western blot and immunohistochemistry (IHC) staining. Cell proliferation was measured by cck-8, and cell apoptosis was detected by flow cytometry. The binding of STAT3 to the promoter region of TTC13 was determined by the luciferase reporter assay and chip experiments. STAT3 nuclear translocation was assessed by immunofluorescence staining. Results We found that TTC13 was up-regulated in ccRCC, and TTC13 promoted cell proliferation as well as inhibited cell apoptosis and autophagy of ccRCC through wnt/β-catenin and IL6-JAK-STAT3 signaling pathways. Furthermore, TTC13 might play a role in the immune infiltration and immunotherapy of ccRCC. Mechanistically, STAT3 activated the transcription of TTC13 gene. Conclusions STAT3 directly regulated TTC13 expression through a positive feedback loop mechanism to promote ccRCC cell proliferation as well as reduce cell apoptosis and autophagy. These findings suggested new and effective therapeutic targets for more accurate and personalized treatment strategies.

EBV-Encoded MicroRNA-BART17-3p Targets DDX3X and Promotes EBV Infection in EBV-Associated T/Natural Killer-Cell Lymphoproliferative Diseases.

Epstein-Barr virus (EBV) persistently infects T/natural killer (NK) cells causing an array of refractory EBV-associated T/NK-cell lymphoproliferative disorders. EBV-encoded microRNAs are important regulators for EBV latent infection and tumorigenesis. However, the roles of most EBV microRNAs in EBV-infected T/NK cells remain poorly understood. On the basis of a search of the doRiNA database and the BiBiServ2-RNAhybrid website, we predicted that EBV-miR-BART17-3p targeted DDX3X, and we verified the hypothesis by dual-luciferase reporter assay and cell function experiments. In addition, we collected 50 EBV-positive T-, B-, and NK-cell samples from the peripheral blood of EBV-positive cases to examine the role of EBV-miR-BART17-3p in the disease. We found that EBV-miR-BART17-3p directly targeted DDX3X and downregulated DDX3X expression. By analyzing EBV-positive cell samples from cell lines and patients, we found that EBV-miR-BART17-3p was highly expressed only in EBV-positive NK cells and that the overexpression was significantly related to high EBV loads in EBV-infected NK cells. Furthermore, we found that EBV-miR-BART17-3p downregulated the RIG-I-like receptor antiviral pathway and promoted the expression of EBV-encoded proteins in EBV-infected NK cells by targeting DDX3X. Our study showed that EBV-miR-BART17-3p was abundantly expressed in EBV-infected NK cells and inhibited the important antivirus immune responses of hosts by targeting DDX3X of the RIG-I-like receptor pathway. These findings could help us gain insights into the pathogenic mechanisms underlying EBV-associated T/NK-cell lymphoproliferative disorders and find the potential therapeutic target.

Molecular interplay between ecdysone receptor and retinoid X receptor in regulating the molting of the Chinese mitten crab, Eriocheir sinensis.

Molting is a pivotal biological process regulated by the ecdysteroid signaling pathway that requires molecular coordination of two transcription factors, Ecdysone receptor (EcR) and ultraspiracle (USP) in arthropods. However, the molecular interplay of EcR and Retinoid X receptor (RXR), the crustacean homolog of USP in the ecdysteroid signaling pathway, is not well understood. In this study, we conducted temporal and spatial expression, co-immunoprecipitation (CO-IP), and luciferase reporter assay experiments to investigate the molecular function and interplay of EcR and RXR during the molting process of the Chinese mitten crab, Eriocheir sinensis. The results showed that the expression level of RXR was more stable and significantly higher than EcR during the entire molting process. However, the expression level of EcR fluctuated dynamically and increased sharply at the premolt stage. The CO-IP and luciferase reporter assay results confirmed the molecular interplay of EcR and RXR. The heterodimer complex formed by the two transcription factors significantly induced the transcription of E75, an essential gene in the ecdysteroid signaling pathway. Our study unveiled the diverse molecular function and molecular interplay of EcR and RXR; RXR is possibly a "constitutive-type" gene, and EcR is possibly a vital speed-limiting gene while both EcR and RXR are required to initiate the ecdysteroid signaling cascade, which may be indispensable for molting regulation in E. sinensis. The results provide a theoretical basis for the endocrine control of molting in E. sinensis and novel insights into the molecular mechanism of molting mediated by the ecdysteroid signaling pathway in crustaceans.

Circ_0114581 promotes osteogenic differentiation of BMSCs via the MiR-155-5p/HNRNPA3 axis

Osteoporosis (OP) is a common metabolic bone disease characterized by deterioration of bone tissue structure, reduction of bone mass, and susceptibility to fracture. More and new suitable therapeutic targets need to be discovered. The purpose of this study was to explore the ceRNA mechanisms of circRNAs involved in osteoporosis. In this study, a competing endogenous RNA (ceRNA) regulatory network was obtained through the application of OP-related high throughput data sets. Our results provided evidence that HNRNPA3 was involved in the regulation of osteogenic differentiation in BMSCs. Testing of human bone tissues and ovariectomized mice bones proved that its expression level was negatively correlated with OP. The utilization of miRNA mimic or inhibitor proved that miR-155-5p could negatively regulate the expression of HNRNPA3, while overexpression of hsa_circ_0114581 with a circRNA overexpression vector proved that hsa_circ_0114581 could indirectly promoted HNRNPA3 expression and osteogenic differentiation by sponging hsa-miR-155-5p. A serious of luciferase reporter assay experiments further verified the binding site between miR-155-5p and HNRNPA3 and the binding site between miR-155-5p and hsa_circ_0114581. This study proved that the hsa_circ_0114581/hsa-miR-155-5p/HNRNPA3 axis was related with OP. The results reveal valuable insights into the pathogenesis of OP and noncoding RNA markers that may have a treatment role and will help to provide hypotheses for future studies.

EIF4A3-regulated hsa_circ_0001445 can inhibit the progression of laryngeal squamous cell carcinoma via hsa-miR-432-5p-dependent up-regulation of RGMA expression

ABSTRACT Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor in the head and neck, the 5-year relative survival rate of patients diagnosed with laryngeal cancer was estimated to be 61% from 2012 to 2018. An increasing number of studies have shown that circular RNAs (circRNAs) play a key role in the occurrence and development of cancer and may function as cancer biomarkers and new therapeutic targets. At present, the research on the relationship between circRNAs and LSCC is still in its infancy and needs further exploration. In this study, we found a circRNA (hsa_circ_0001445) associated with LSCC based on bioinformatics analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) assay indicated that the expression of hsa_circ_0001445 was down-regulated in LSCC tissues and cell lines. Notably, the expression of hsa_circ_0001445 was negatively correlated with aggressive clinicopathological features and poor prognosis. Then, functional experiments found that overexpression of hsa_circ_0001445 inhibited the proliferation, migration and invasion of LSCC cells and tumor growth in vivo. Mechanistically, RNA immunoprecipitation (RIP), biotin-labeled probe pull-down, luciferase reporter assay and western blot experiments were employed and found that EIF4A3 reduced the expression of hsa_circ_0001445, and the direct binding of hsa_circ_0001445 to hsa-miR-432-5p attenuated the inhibitory effect of hsa-miR-432-5p on RGMA. In summary, our research suggests that hsa_circ_0001445 may be used as a potential prognostic biomarker and therapeutic target for LSCC.