ABSTRACT
Lung cancer remains the leading cause of cancer-related deaths, with cigarette smoking being the most critical factor, linked to nearly 90% of lung cancer cases. NNK, a highly carcinogenic nitrosamine found in tobacco, is implicated in the lung cancer-causing effects of cigarette smoke. Although NNK is known to mutate or activate certain oncogenes, its potential interaction with p27 in modulating these carcinogenic effects is currently unexplored. Recent studies have identified specific downregulation of p27 in human squamous cell carcinoma, in contrast to adenocarcinoma. Additionally, exposure to NNK significantly suppresses p27 expression in human bronchial epithelial cells. Subsequent studies indicates that the downregulation of p27 is pivotal in NNK-induced cell transformation. Mechanistic investigations have shown that reduced p27 expression leads to increased level of ITCH, which facilitates the degradation of Jun B protein. This degradation in turn, augments miR-494 expression and its direct regulation of JAK1 mRNA stability and protein expression, ultimately activating STAT3 and driving cell transformation. In summary, our findings reveal that: (1) the downregulation of p27 increases Jun B expression by upregulating Jun B E3 ligase ITCH, which then boosts miR-494 transcription; (2) Elevated miR-494 directly binds to 3'-UTR of JAK1 mRNA, enhancing its stability and protein expression; and (3) The JAK1/STAT3 pathway is a downstream effector of p27, mediating the oncogenic effect of NNK in lung cancer. These findings provide significant insight into understanding the participation of mechanisms underlying p27 inhibition of NNK induced lung squamous cell carcinogenic effect.
Subject(s)
Bronchi , Carcinoma, Squamous Cell , Cell Transformation, Neoplastic , Cyclin-Dependent Kinase Inhibitor p27 , Epithelial Cells , Lung Neoplasms , Nitrosamines , Humans , Nitrosamines/toxicity , Bronchi/metabolism , Bronchi/pathology , Bronchi/drug effects , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Epithelial Cells/pathology , Carcinoma, Squamous Cell/chemically induced , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/chemically induced , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/drug effects , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cyclin-Dependent Kinase Inhibitor p27/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/chemically induced , Lung Neoplasms/genetics , Gene Expression Regulation, Neoplastic/drug effects , MicroRNAs/genetics , MicroRNAs/metabolism , Down-Regulation/drug effects , Carcinogens/toxicityABSTRACT
BACKGROUND: Gram-negative bacterial lipopolysaccharide (LPS) is a major component of inflammation and plays a key role in the pathogenesis of sepsis. According to our previous study, the expression of lipoprotein-associated phospholipase A2 (Lp-PLA2) is significantly upregulated in septic patients and is positively correlated with the severity of this disease. Herein, we investigated the potential roles of Lp-PLA2-targeting microRNAs (miRNAs) in LPS-induced inflammation in murine mononuclear macrophages (RAW264.7 cells). METHODS: In LPS-stimulated RAW264.7 cells, Lp-PLA2 was confirmed to be expressed during the inflammatory response. The function of microRNA-494-3p (miR-494-3p) in the LPS-induced inflammatory response of RAW264.7 cells was determined by the transfection of a miR-494-3p mimic or inhibitor in vitro. RESULTS: Compared to the control, LPS induced a significant increase in the Lp-PLA2 level, which was accompanied by the release of inflammatory mediators. The bioinformatics and qRTâPCR results indicated that the miR-494-3p level was associated with Lp-PLA2 expression in the LPS-induced inflammatory response of RAW264.7 cells. Dual-luciferase reporter assay results confirmed that the 3'-UTR of Lp-PLA2 was a functional target of microRNA-494-3p. During the LPS-induced inflammatory response of RAW264.7 cells, targeting Lp-PLA2 and transfecting miR-494-3p mimics significantly upregulated the expression of miR-494-3p, leading to a reduction in the release of inflammatory factors and conferring a protective effect on LPS-stimulated RAW264.7 cells. CONCLUSION: By targeting Lp-PLA2, miR-494-3p suppresses Lp-PLA2 secretion, thereby alleviating LPS-induced inflammation, which indicates that miR-494-3p may be a potential target for sepsis treatment.
ABSTRACT
BACKGROUND: In the context of increasing exposure to silica nanoparticles (SiNPs) and ensuing respiratory health risks, emerging evidence has suggested that SiNPs can cause a series of pathological lung injuries, including fibrotic lesions. However, the underlying mediators in the lung fibrogenesis caused by SiNPs have not yet been elucidated. RESULTS: The in vivo investigation verified that long-term inhalation exposure to SiNPs induced fibroblast activation and collagen deposition in the rat lungs. In vitro, the uptake of exosomes derived from SiNPs-stimulated lung epithelial cells (BEAS-2B) by fibroblasts (MRC-5) enhanced its proliferation, adhesion, and activation. In particular, the mechanistic investigation revealed SiNPs stimulated an increase of epithelium-secreted exosomal miR-494-3p and thereby disrupted the TGF-ß/BMPR2/Smad pathway in fibroblasts via targeting bone morphogenetic protein receptor 2 (BMPR2), ultimately resulting in fibroblast activation and collagen deposition. Conversely, the inhibitor of exosomes, GW4869, can abolish the induction of upregulated miR-494-3p and fibroblast activation in MRC-5 cells by the SiNPs-treated supernatants of BEAS-2B. Besides, inhibiting miR-494-3p or overexpression of BMPR2 could ameliorate fibroblast activation by interfering with the TGF-ß/BMPR2/Smad pathway. CONCLUSIONS: Our data suggested pulmonary epithelium-derived exosomes serve an essential role in fibroblast activation and collagen deposition in the lungs upon SiNPs stimuli, in particular, attributing to exosomal miR-494-3p targeting BMPR2 to modulate TGF-ß/BMPR2/Smad pathway. Hence, strategies targeting exosomes could be a new avenue in developing therapeutics against lung injury elicited by SiNPs.
Subject(s)
Collagen , Epigenesis, Genetic , Exosomes , Fibroblasts , Lung , MicroRNAs , Nanoparticles , Signal Transduction , Silicon Dioxide , Transforming Growth Factor beta , Exosomes/metabolism , Animals , Fibroblasts/metabolism , Fibroblasts/drug effects , Silicon Dioxide/chemistry , Signal Transduction/drug effects , Rats , Lung/metabolism , Lung/pathology , Collagen/metabolism , Humans , Nanoparticles/chemistry , MicroRNAs/metabolism , MicroRNAs/genetics , Cell Line , Transforming Growth Factor beta/metabolism , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/chemically induced , Male , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Rats, Sprague-Dawley , Epithelium/metabolism , Epithelium/drug effectsABSTRACT
Background: Gastric cancer necessitates novel treatments, and exosomes are promising therapeutic carriers. We created miR-494-3p inhibitor exosomes to assess their effects on gastric cancer cells. Methods: We conducted a comprehensive investigation into the expression of the oncogenic miR-494-3p in gastric cancer tissues from patients. Subsequently, we engineered miR-494-3p inhibitor-loaded exosomes and characterized their morphology and size through transmission electron microscopy and nanoparticle tracking analysis. We next determined the encapsulation efficiency of the miR-494-3p inhibitor within these exosomes and evaluated the exosomes' structural integrity by quantifying the presence of exosomal markers. Following these validations, we co-cultured miR-494-3p inhibitor exosomes with cancer cells and employed PKH26 staining to visualize the efficient endocytosis of engineered exosomes by gastric cancer cells and assess the impact of these modified exosomes on gastric cancer cell proliferation, apoptosis, migration, and invasion. Results: Increased expression of miR-494-3p was observed in gastric cancer tissues as compared to controls. Significant low miR-494-3p levels were found within miR-494-3p inhibitor exosomes, signifying effective encapsulation. The incorporation of miR-494-3p inhibitor into engineered exosomes did not alter exosome morphology or size. Finally, PKH26-stained exosomes clearly demonstrated efficient endocytosis by gastric cancer cells, leading to reduced proliferation, migration, invasion, and increased apoptosis. Conclusion: Our study identifies elevated miR-494-3p in gastric cancer tissues prompting the development of miR-494-3p inhibitor-loaded exosomes with efficient encapsulation. These engineered exosomes demonstrate successful endocytosis by cancer cells. This highlights their potential for therapeutic use in gastric cancer treatment by suppressing proliferation, migration, and invasion while enhancing apoptosis.
ABSTRACT
Therapy resistance and metastatic progression jointly determine the fatal outcome of cancer, therefore, elucidating their crosstalk may provide new opportunities to improve therapeutic efficacy and prevent recurrence and metastasis in esophageal squamous cell carcinoma (ESCC). Here, we have established radioresistant ESCC cells with the remarkable metastatic capacity, and identified miR-494-3p (miR494) as a radioresistant activator. Mechanistically, we demonstrated that cullin 3 (CUL3) is a direct target of miR494, which is transcriptionally regulated by JunD, and highlighted that JunD-miR494-CUL3 axis promotes radioresistance and metastasis by facilitating epithelial-mesenchymal transition (EMT) and restraining programmed cell death 1 ligand 1 (PD-L1) degradation. In clinical specimens, miR494 is significantly up-regulated and positively associated with T stage and lymph node metastasis in ESCC tissues and serum. Notably, patients with higher serum miR494 expression have poor prognosis, and patients with higher CUL3 expression have more conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs), less cancer-associated fibroblasts (CAF2/4), and tumor endothelial cells (TEC2/3) infiltration than patients with lower CUL3 expression, suggesting that CUL3 may be involved in tumor microenvironment (TME). Overall, miR494 may serve as a potential prognostic predictor and therapeutic target, providing a promising strategy for ESCC treatment.
Subject(s)
Carcinoma, Squamous Cell , Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , MicroRNAs , Humans , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/radiotherapy , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/radiotherapy , Carcinoma, Squamous Cell/metabolism , Esophageal Neoplasms/genetics , Esophageal Neoplasms/radiotherapy , Esophageal Neoplasms/metabolism , Endothelial Cells/metabolism , Prognosis , Epithelial-Mesenchymal Transition , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Cell Movement , Tumor Microenvironment , Proto-Oncogene Proteins c-jun/metabolism , Cullin Proteins/geneticsABSTRACT
Cadmium (Cd) exposure is a persistent pollution problem, necessitating caution in using cadmium-expelling complexing agents. Currently, there is no targeted therapy to treat Cd poisoning. The thyroid gland is a major endocrine organ that directly regulates thyroid hormones involved in various physiological processes and is a target organ for Cd accumulation. Herein, the effects of Cd exposure on swine thyroid glands were investigated. Six-week-old male pigs were randomly divided into the Cd and control groups. The control group was fed a normal diet containing 0 mg Cd/kg, while the Cd group was fed a diet containing 20 mg Cd/kg (CdCl2) for 40 days. The regulation mechanism of phosphatase and tensin homolog (PTEN) microRNA-494-3p (miR-494-3p) was evaluated to determine the toxic effects of Cd exposure on free radicals' cleaner. Notably, heat shock proteins (HSPs) were triggered as defense agents against Cd. Cd exposure increased the enzyme activity of superoxide dismutase1(SOD1) and SOD2, catalase (CAT), and glutathione (GSH), and the endoplasmic reticulum stress in thyroid cells. Histopathological staining, RT-qPCR, and Western Blot assays were further employed to detect possible apoptosis and necroptosis of thyroid cells induced by Cd exposure. The assays revealed increased thyroid inflammatory injury, fibrosis, and apoptosis caused by Cd exposure. This study demonstrates the role of microRNAs in regulating Cd toxicity in pig thyroid tissue and provides evidence of Cd's negative effects. It further provides an assessment of the toxicological impact of Cd as an environmental endocrine disruptor (ED) that threatens public health and safety, which forms a basis for the development of Cd poisoning treatment therapies.
ABSTRACT
BACKGROUND: With rapid progression, severe illness and high fatality rate, sepsis has become an acute and critical condition that seriously threatens human life and health. OBJECTIVE: To detect miR-210 and miR-494 expression in patients with sepsis and their relationship with severity and prognosis. METHODS: A total of 165 sepsis patients participated, including 105 patients with septic non-shock and 60 patients with septic shock. 53 sepsis patients died in 28 days, and 112 patients survived. The clinical information of all sepsis patients was retrospectively searched and reviewed. Based on the status of 28-day survival, they were categorized into survival group and death group. The expression levels in each group were compared on the first, third and seventh day. The ROC curve was applied to know the expression level of plasma miR-210 and miR-494 to predict the death. RESULTS: The two miRNAs expression of the septic shock group were significantly higher than that in sepsis non-shock group on the first, third and seventh day (all were P< 0.05). The ROC curve found that the AUC combined to predict the death on the third day was the largest, which was 0.925 (95%CI: 0.864-0.983). The sensitivity and specificity were 94.6% and 86.3%, respectively. CONCLUSION: The increased expression levels of plasma miR-210 and miR-494 are closely relevant to the severity and prognosis of sepsis patients. Combining the two items on the third day can predict the death of sepsis patients.
Subject(s)
MicroRNAs , Sepsis , Shock, Septic , Humans , Shock, Septic/genetics , Retrospective Studies , Sepsis/genetics , Sepsis/diagnosis , Prognosis , ROC CurveABSTRACT
Circular RNAs (circRNAs) have been confirmed to mediate infantile pneumonia development. In this, we investigated the role and new mechanism of circ_0035292 regulating infantile pneumonia progression. Lipopolysaccharide (LPS)-treated WI-38 cells were used to mimic infantile pneumonia cell injury models. Quantitative real-time PCR was used to measure circ_0035292, microRNA (miR)-494-3p and toll-like receptor 4 (TLR4). Cell proliferation and apoptosis were assessed by MTT assay, EdU assay, and flow cytometry. Protein expression was tested using western blot analysis. Inflammation and oxidative stress were evaluated by measuring IL-6, IL-1ß, MDA and SOD levels using ELISA assay and corresponding kits. RNA interaction was confirmed by dual-luciferase reporter assay and RIP assay. Circ_0035292 had elevated expression in infantile pneumonia patients and LPS-induced WI-38 cells. Silenced circ_0035292 could enhance WI-38 cell proliferation, while suppress apoptosis, inflammation and oxidative stress under LPS treatment. Mechanically, circ_0035292 targeted miR-494-3p to positively regulate TLR4. The rescue experiments indicated that miR-494-3p inhibitor abolished the function of circ_0035292 knockdown, and TLR4 overexpression reversed the inhibitory effect of miR-494-3p on LPS-induced WI-38 cell injury. Circ_0035292 might be a potential target for infantile pneumonia treatment, which knockdown could relieve LPS-induced cell injury via the regulation of miR-494-3p/TLR4 axis.
ABSTRACT
PURPOSE: Pirarubicin (THP) is an antitumour drug widely used in clinical practice, but its cardiotoxicity limits its application. THP cardiotoxicity must be treated as soon as possible. There is an urgent need to find drugs that alleviate THP cardiotoxicity. The purpose of this study was to investigate the effects and mechanisms of Astaxanthin (AST) on THP-induced cardiomyocytes. METHODS: Rat cardiomyocytes H9c2 were induced with THP. The effects of AST on THP-induced H9c2 and its mechanism were investigated by CCK8, reactive oxygen species assay, tunnel assay, flow cytometry, RT-qPCR, and Western blot. RESULTS: AST increased cell viability, inhibited apoptosis and accelerated cell cycle progression, reduced oxidative damage and inflammatory response in THP-induced H9c2; down-regulated miR-494-3p expression, promoted MDM4 expression, inhibited p53 activation, and suppressed apoptosis-related protein expression. Overexpression of MiR-494-3p reversed the above effects of AST. CONCLUSIONS: AST can inhibit H9c2 apoptosis induced by THP and attenuate H9c2 damage by THP, which may be achieved by downregulating miR-494-3p, upregulating MDM4, and inhibiting p53.
Subject(s)
MicroRNAs , Tumor Suppressor Protein p53 , Rats , Animals , Tumor Suppressor Protein p53/metabolism , Cell Line , MicroRNAs/metabolism , Myocytes, Cardiac , Cardiotoxicity/prevention & control , ApoptosisABSTRACT
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an airway-associated lung disorder, resulting in airway inflammation. This article aimed to explore the role of the krüppel-like factor 9 (KLF9)/microRNA (miR)-494-3p/phosphatase and tensin homolog (PTEN) axis in airway inflammation and pave a theoretical foundation for the treatment of COPD. METHODS: The COPD mouse model was established by exposure to cigarette smoke, followed by measurements of total cells, neutrophils, macrophages, and hematoxylin and eosin staining. The COPD cell model was established on human lung epithelial cells BEAS-2B using cigarette smoke extract. Cell viability was assessed by cell counting kit-8 assay. miR-494-3p, KLF9, PTEN, and NLR family, pyrin domain containing 3 (NLRP3) levels in tissues and cells were measured by quantitative real-time polymerase chain reaction or Western blot assay. Inflammatory factors (TNF-α/IL-6/IL-8/IFN-γ) were measured by enzyme-linked immunosorbent assay. Interactions among KLF9, miR-494-3p, and PTEN 3'UTR were verified by chromatin immunoprecipitation and dual-luciferase assays. RESULTS: KLF9 was upregulated in lung tissues of COPD mice. Inhibition of KLF9 alleviated airway inflammation, reduced intrapulmonary inflammatory cell infiltration, and repressed NLRP3 expression. KLF9 bound to the miR-494-3p promoter and increased miR-494-3p expression, and miR-494-3p negatively regulated PTEN expression. miR-494-3p overexpression or Nigericin treatment reversed KLF9 knockdown-driven repression of NLRP3 inflammasome and inflammation. CONCLUSION: KLF9 bound to the miR-494-3p promoter and repressed PTEN expression, thereby facilitating NLRP3 inflammasome-mediated inflammation.
Subject(s)
MicroRNAs , Pulmonary Disease, Chronic Obstructive , Animals , Humans , Mice , Inflammasomes/metabolism , Inflammation/metabolism , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Lung , MicroRNAs/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/metabolismABSTRACT
Umbilical cord-mesenchymal stem cells (UC-MSCs)-derived exosomes have been considered as an effective treatment for ischemic stroke. CircRNA BBS2 (circBBS2) was demonstrated to be down-regulated in patients with ischemic stroke. However, the role of UC-MSCs-derived exosomal circBBS2 in ischemic stroke and potential mechanisms remain unclear. Hypoxia/reperfusion (H/R)-exposed SH-SY5Y cells and middle cerebral artery occlusion (MCAO)-treated rats were served as in vitro and in vivo models of ischemic stroke. Target gene expression was detected by qRT-PCR. Cell viability was assessed by MTT assay. Ferroptosis was determined by iron, MDA, GSH, and lipid ROS levels. Protein levels were measured by Western blotting. The target relationships among circBBS2, miR-494, and SLC7A11 were validated by RNA-pull down, RIP, and dual-luciferase reporter assays. TTC and HE staining were performed to evaluate cerebral infarction volume and neuropathological changes. circBBS2 was lowly expressed and ferroptosis was triggered in MCAO rats and H/R-stimulated SH-SY5Y cells. UC-MSCs-derived exosomes enhanced cell viability and restrained ferroptosis via increasing circBBS2 expression in SH-SY5Y cells. Mechanistically, circBBS2 sponged miR-494 to enhance the SLC7A11 level. Knockdown of miR-494 or SLC7A11 reversed the effects of silencing circBBS2 or miR-494 on ferroptosis of SH-SY5Y cells, respectively. Furthermore, UC-MSCs-derived exosomes attenuated ischemic stroke in rats via delivering circBBS2 to inhibit ferroptosis. UC-MSCs-derived exosomal circBBS2 enhanced SLC7A11 expression via sponging miR-494, therefore repressing ferroptosis and relieving ischemic stroke. Our findings shed light on a novel mechanism for UC-MSCs-derived exosomes in the treatment of ischemic stroke.
Subject(s)
Ferroptosis , Ischemic Stroke , MicroRNAs , Neuroblastoma , Animals , Humans , Rats , Amino Acid Transport System y+/genetics , Ferroptosis/genetics , Hypoxia , Ischemic Stroke/genetics , MicroRNAs/genetics , RNA, Circular/geneticsABSTRACT
Lung cancer primarily arises from exposure to various environmental factors, particularly airborne pollutants. Among the various lung carcinogens, benzo(a)pyrene and its metabolite B[a]PDE are the strongest ones that actively contribute to lung cancer development. ATG7 is an E1-like activating enzyme and contributes to activating autophagic responses in mammal cells. However, the potential alterations of ATG7 and its role in B[a]PDE-caused lung carcinogenesis remain unknown. Here, we found that B[a]PDE exposure promoted ATG7 expression in mouse lung tissues, while B[a]PDE exposure resulted in ATG7 induction in human normal bronchial epithelial cells. Our studies also demonstrated a significant correlation between high ATG7 expression levels and poor overall survival in lung cancer patients. ATG7 knockdown significantly repressed Beas-2B cell transformation upon B[a]PDE exposure, and such promotive effect of ATG7 on cell transformation mediated the p27 translation inhibition. Further studies revealed that miR-373 inhibition was required to stabilize ATG7 mRNA, therefore increasing ATG7 expression following B[a]PDE exposure, while ATG7 induction led to the autophagic degradation of the DNA methyltransferase 3 Beta (DNMT3B) protein, in turn promoted miR-494 transcription via its promoter region methylation status suppression. We also found that the miR-494 upregulation inhibited p27 protein translation and promoted bronchial epithelial cell transformation via its directly targeting p27 mRNA 3'-UTR region. Current studies, to the best of our knowledge, are for the first time to identify that ATG7 induction and its mediated autophagy is critical for B[a]PDE-induced transformation of human normal epithelial cells.
Subject(s)
Lung Neoplasms , MicroRNAs , Humans , Animals , Mice , Proteolysis , Methylation , Up-Regulation , Epithelial Cells , Promoter Regions, Genetic , MicroRNAs/genetics , MammalsABSTRACT
OBJECTIVE: Pirarubicin (THP) is a widely used antitumor drug in clinical practice, but its cardiotoxicity limits its use. There is an urgent need to find drugs to alleviate the cardiotoxicity of THP. This study aimed to investigate the effect and mechanism of miR-494-3p on THP-induced cardiomyocytes. METHODS: THP induced immortalized mouse cardiomyocytes HL-1, silenced or overexpressed miR-494-3p. The effects of miR-494-3p on HL-1 contained in THP were investigated by CCK8, flow cytometry, ROS detection, JC-1 mitochondrial membrane potential detection, TUNEL cell apoptosis detection, RT-qPCR, and Western blot. RESULTS: miR-494-3p could reduce cell viability, increase oxidative damage, and promote cell apoptosis; at the same time, it inhibited the expression of MDM4, promoted the activation of p53, and promoted the expression of apoptosis-related proteins. MiR-494-3p inhibitors have the opposite effect. CONCLUSION: miR-494-3p can aggravate THP damage to HL-1, which may be achieved by downregulating MDM4 and promoting p53. miR-494-3p is one of the important miRNAs in THP-induced cardiotoxicity, which provides theoretical support for its possible use as a therapeutic target for THP-induced cardiovascular disease.
Subject(s)
MicroRNAs , Signal Transduction , Mice , Animals , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Myocytes, Cardiac , Cardiotoxicity/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , ApoptosisABSTRACT
BACKGROUND: Metabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker. METHODS: Bioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats. RESULTS: MiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells. CONCLUSIONS: MiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , MicroRNAs , Rats , Animals , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Sorafenib/pharmacology , Sorafenib/therapeutic use , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Drug Resistance, Neoplasm/genetics , MicroRNAs/metabolismABSTRACT
OBJECTIVE: The purpose of this study was to explore whether miR-494-3p inhibits the occurrence of mitochondrial autophagy in cardiomyocytes by inhibiting the expression of PGC1-α and to supplement the theoretical basis for the role of autophagy in cardiac injury induced by hypoxia/reperfusion (H/R). METHODS: The expression of miR-494-3p was detected by RTâqPCR, and the expression of PGC1-α, autophagy-related proteins (LC3, Beclin 1), apoptosis-related proteins (Bax and Bcl-2), PINK1/Parkin signaling pathway-related proteins (PINK1, Parkin) and mitochondrial change-related proteins (Mfn1, Mfn2, OPA1) was detected by Western blotting. The changes in mitochondrial membrane potential were detected by JC-1 staining (ΔΨm). The formation of autophagosomes was observed by transmission electron microscopy. Cell proliferation activity was detected by CCK-8, and cell apoptosis was detected by flow cytometry. A dual-luciferase gene reporter assay identified a targeted binding site between miR-494-3p and PGC1-α. RESULTS: The results showed that miR-494-3p and PGC1-α were differentially expressed in H/R cardiomyocytes; that is, the expression of miR-494-3p was downregulated, and the expression of PGC1-α was upregulated. In addition, mitochondrial autophagy occurred in H/R cardiomyocytes. That is, LC3-II/LC3-I, Beclin 1, PINK1, and Parkin expression was upregulated, Mfn1, Mfn2, and OPA1 expression was downregulated, and the mitochondrial membrane potential was decreased. The transfection of miR-494-3p mimic can significantly improve the cell proliferation activity of cardiomyocytes and inhibit the occurrence of cardiomyocyte apoptosis and autophagy, while the transfection of miR-494-3p inhibitor has the opposite result. After transfection of the miR-494-3p mimic, treatment with autophagy inhibitors and activators changed the effects of miR-494-3p on cardiomyocyte proliferation and apoptosis. At the same time, the overexpression of PGC1-α reversed the promoting effect of miR-494-3p on cardiomyocyte proliferation and the inhibitory effect on apoptosis and autophagy. CONCLUSION: MiR-494-3p can target and negatively regulate the expression of PGC1-α to inhibit mitophagy in cardiomyocytes.
Subject(s)
MicroRNAs , Mitophagy , Myocardial Reperfusion Injury , Rats , Apoptosis , Beclin-1/metabolism , Hypoxia/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Mitophagy/genetics , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/metabolism , Myocytes, Cardiac/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Protein Kinases/metabolism , AnimalsABSTRACT
BACKGROUND: Increasing evidence suggests that mitophagy is responsible for the pathogenesis of intervertebral disk (IVD) degeneration. Previous studies have shown that Duhuo Jisheng Decoction (DHJSD), a classic Fangji of traditional Chinese medicine, can delay IVD degeneration; however, its specific mechanism of action is unknown. In this study, we investigated the mechanism by which DHJSD treatment prevented IVD degeneration in IL-1ß-treated human nucleus pulposus (NP) cells in vitro. METHODS: Cell Counting Kit-8 was performed to explore the effects of DHJSD on the viability of NP cells exposed to IL-1ß. The mechanism by which DHJSD delays IVD degeneration was explored using luciferase reporter assay, RT-qPCR, western blotting, TUNEL assay, mitophagy detection assay, Mito-SOX, Mitotracker and in situ hybridization. RESULTS: We observed that DHJSD enhanced the viability of NP cells treated with IL-1ß in a concentration-time dependent approach. Moreover, DHJSD lessened IL-1ß-induced NP apoptosis and mitochondrial dysfunction and activated mitophagy in NP cells treated with IL-1ß. Mitophagy suppressor cyclosporin A reversed the beneficial impacts of DHJSD in NP cells. In addition, the differential expression of miR-494 regulated IL-1ß-induced NP apoptosis and mitochondrial dysfunction, and the protective impact of miR-494 on NP cells treated with IL-1ß was achieved by mitophagy activation, which was regulated by its target gene, sirtuin 3 (SIRT3). Finally, we observed that DHJSD treatment could effectively delay IL-1ß-induced NP apoptosis by affecting the miR-494/SIRT3/mitophagy signal axis. CONCLUSIONS: These results show that the miR-494/SIRT3/mitophagy signaling pathway is responsible for the apoptosis and mitochondrial dysfunction of NP cells and that DHJSD may exert protective effects against IVD degeneration by regulating the miR-494/SIRT3/mitophagy signal axis.
Subject(s)
Intervertebral Disc Degeneration , MicroRNAs , Nucleus Pulposus , Sirtuin 3 , Humans , Sirtuin 3/genetics , Sirtuin 3/metabolism , Mitophagy , Cells, Cultured , Intervertebral Disc Degeneration/pathology , Apoptosis , MicroRNAs/metabolism , Mitochondria/geneticsABSTRACT
Aberrant expression of circular RNA (circRNA) is closely linked to the progression of various human cancers, including prostate cancer (PCa). In this research, we aimed to investigate the biological role of and mechanisms of circ_0085494 in PCa. The quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to detect circ_0085494, miR-497-5p, and nuclear receptor binding protein 1 (NRBP1) mRNA expression in PCa tissues and cell lines. Subsequently, colony formation, cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and transwell assays were performed to evaluate PCa cell proliferation, migration, and invasion. Western blot assay was applied for assessing the protein levels. Dual-luciferase reporter and RNA pull-down assays were implemented for verifying the association between miR-497-5p and circ_0085494 or NRBP1. The role of circ_0085494 in vivo was measured by establishing a mice xenograft model. Circ_0085494 was highly expressed in PCa tissues and cells, and its absence suppressed PCa cell proliferation, migration, and invasion. Circ_0085494 impacts NRBP1 content by adsorbing miR-497-5p. Meanwhile, the repression of circ_0085494 absence on tumor growth in vivo was validated. Our finding revealed that circ_0085494 downregulation might repress PCa tumor progression through in part regulating the miR-497-5p/NRBP1 pathway.
Subject(s)
MicroRNAs , Prostatic Neoplasms , Male , Humans , Animals , Mice , RNA, Circular/genetics , Prostatic Neoplasms/genetics , Cell Line , Cell Proliferation , MicroRNAs/genetics , Receptors, Cytoplasmic and Nuclear , Vesicular Transport ProteinsABSTRACT
BACKGROUND: LncRNA LINC00534 has been found to be differentially expressed in placental tissue samples of preeclampsia (PE), but the exact mechanism is still unclear. METHODS: In vitro assays were carried out in HTR-8/SVneo cells using various methods, including cell counting kit-8 (CCK-8), transwells, flow cytometry, and Western blotting (WB) and quantitative polymerase chain reaction. RNA pull-down and bioinformatics analysis were applied to examine other potential underlying mechanisms involved. RESULTS: We found that there was a high expression of LINC00534 in the placental tissues of patients with PE. LINC00534 overexpression (OE) significantly inhibited cell proliferation and migration as well as accelerated cell apoptosis in HTR8/SVneo cells. The knockdown of LINC00534 produced an opposite trend. Mechanistically, LINC00534 promoted the expressions of PTEN (Phosphatase and tensin homolog) through decreasing miR-494-3p. Further rescue studies showed that LINC00534 played a role by targeting mir-494-3p, which controlled the growth and migration of HTR-8/SVneo trophoblast cells via regulating PTEN/PI3K/AKT (Phosphatidylinositol3-kinase/protein kinase B). Moreover, lncRNA pull-down assay identified 198 potential bound proteins for LINC00534. Those proteins were mostly involved in RNA processing and modification, posttranslational modification, protein turnover, and chaperones. CONCLUSION: Overall, by suppressing HTR8/SVneo cell growth and migration via the miR-494-3p/PTEN axis and other mechanisms, LINC00534 offers new insight into PE pathogenesis.
Subject(s)
MicroRNAs , Pre-Eclampsia , RNA, Long Noncoding , Humans , Pregnancy , Female , MicroRNAs/genetics , MicroRNAs/metabolism , Placenta/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Trophoblasts/metabolism , Cell Proliferation/genetics , Pre-Eclampsia/genetics , Pre-Eclampsia/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolismABSTRACT
Cardiac fibrosis is an essential pathological process in pressure overload (PO)-induced heart failure. Recently, myocyte-fibroblast communication is proven to be critical in heart failure, in which, pathological growth of cardiomyocytes (CMs) may promote fibrosis via miRNAs-containing exosomes (Exos). Peli1 regulates the activation of NF-κB and AP-1, which has been demonstrated to engage in miRNA transcription in cardiomyocytes. Therefore, we hypothesized that Peli1 in CMs regulates the activation of cardiac fibroblasts (CFs) through an exosomal miRNA-mediated paracrine mechanism, thereby promoting cardiac fibrosis. We found that CM-conditional deletion of Peli1 improved PO-induced cardiac fibrosis. Moreover, Exos from mechanical stretch (MS)-induced WT CMs (WT MS-Exos) promote activation of CFs, Peli1-/- MS-Exos reversed it. Furthermore, miRNA microarray and qPCR analysis showed that miR-494-3p was increased in WT MS-Exos while being down regulated in Peli1-/- MS-Exos. Mechanistically, Peli1 promoted miR-494-3p expression via NF-κB/AP-1 in CMs, and then miR-494-3p induced CFs activation by inhibiting PTEN and amplifying the phosphorylation of AKT, SMAD2/3, and ERK. Collectively, our study suggests that CMs Peli1 contributes to myocardial fibrosis via CMs-derived miR-494-3p-enriched exosomes under PO, and provides a potential exosomal miRNA-based therapy for cardiac fibrosis.
Subject(s)
Cell Communication , Exosomes , Heart Failure , Myocytes, Cardiac , Humans , Exosomes/genetics , Exosomes/metabolism , Fibrosis/etiology , Fibrosis/genetics , Fibrosis/metabolism , Fibrosis/pathology , Heart Failure/genetics , Heart Failure/metabolism , Heart Failure/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , NF-kappa B/genetics , NF-kappa B/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factor AP-1/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Heart Diseases/etiology , Heart Diseases/genetics , Heart Diseases/metabolism , Heart Diseases/pathology , Fibroblasts/metabolism , Fibroblasts/pathology , Cell Communication/genetics , Cell Communication/physiologyABSTRACT
Epiretinal membranes (ERMs) are the result of fibro-cellular proliferation that cause distortion and impairment of central vision. We hypothesized that select microRNAs (miRs) regulate retinal fibro-proliferation and ERM formation. Following IRB approval, a pilot study was performed in patients presenting for retina surgery with and without clinical ERMs. Total RNA was isolated from ERM tissue and controls from non-ERM vitreous and subjected to miR profiling via microarray analysis. MiR-494 was identified as the only miR selectively expressed at significantly greater levels, and in silico analysis identified p27 as a putative fibroproliferative gene target of miR-494. In vitro testing of miR-494 and p27 in fibrotic transformation was assessed in spontaneously immortalized human retinal pigment epithelial (RPE) and human Müller cell lines, stimulated to transform into a fibroproliferative state via transforming growth factor beta (TGFß). Fibroproliferative transformation was characterized by de novo cellular expression of alpha smooth muscle actin (αSMA). In both RPE and Müller cells, both TGFß and miR-494 mimic decreased p27 expression. In parallel experiments, transfection with p27 siRNA augmented TGFß-induced αSMA expression, while only in RPE cells did co-transfection with miR-494 inhibitor decrease αSMA levels. These results demonstrate that miR-494 augments fibrotic transformation in both Müller cells and RPEs, however only in RPEs does miR-494 mediate fibrotic transformation via p27. As p27 is known to regulate cellular proliferation and differentiation, future studies should extend clinical testing of miR-494 and/or p27 as a potential novel non-surgical therapy for ERMs, as well as identify relevant miR-494 targets in Müller cells.