Lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells is inhibited by microRNA-494-3p via targeting lipoprotein-associated phospholipase A2.

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 qRT‒PCR 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.

Epithelium-derived exosomes promote silica nanoparticles-induced pulmonary fibroblast activation and collagen deposition via modulating fibrotic signaling pathways and their epigenetic regulations.

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.

Suppression of gastric cancer cell proliferation by miR-494-3p inhibitor-loaded engineered exosomes.

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.

Cadmium Exposure Induces Apoptosis and Necrosis of Thyroid Cells via the Regulation of miR-494-3p/PTEN Axis.

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.

Downregulation of circ_0035292 Alleviates LPS-Induced WI-38 Cell Injury via Targeting miR-494-3p/TLR4 Pathway in Infantile Pneumonia.

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.

Mechanism of KLF9 in airway inflammation in chronic obstructive pulmonary.

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.

Astaxanthin protects against pirarubicin-induced H9c2 cardiomyocytes by adjusting microRNA-494-3p-mediated MDM4/p53 signalling pathway.

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.

MiR-494-3p aggravates pirarubicin-induced cardiomyocyte injury by regulating MDM4/p53 signaling pathway.

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.

The mechanism by which miR-494-3p regulates PGC1-α-mediated inhibition of mitophagy in cardiomyocytes and alleviation of myocardial ischemia-reperfusion injury.

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 RT‒qPCR, 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.

LncRNA LINC00534 regulates cell proliferation and migration via the miR-494-3p/PTEN axis in HTR-8/SVneo cells.

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.

Cardiomyocyte-specific Peli1 contributes to the pressure overload-induced cardiac fibrosis through miR-494-3p-dependent exosomal communication.

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.

[LncRNA SNHG8 inhibits miR-494-3p expression to alleviate cerebral ischemia-reperfusion injury in mice].

OBJECTIVE: To explore the mechanism by which LncRNA SNHG8 regulates miR-494-3p expression to alleviate cerebral ischemia-reperfusion injury. METHODS: A mouse model of cerebral ischemia-reperfusion injury was established, and TTC staining was used to determine the infarct area; ELISA was used to detect the contents of the inflammatory factors IL-1ß, IL-6 and TNF-α in the brain tissue, and RT-qPCR was performed to detect the expression levels of LncRNA MALAT1 and miR-155-5p. A microglial cell model overexpressing LncRNA SNHG8 was exposed to oxygen-glucose deprivation/reoxygenation (OGD/R), and inflammatory reaction and apoptosis of the cells were detected using ELISA and flow cytometry. A luciferase reporter assay was used to detect the targeting relationship between LncRNA SNHG8 and miR-494-3p. We further constructed a microglial cell model overexpressing both LncRNA SNHG8 the miR-494-3p, and examined inflammatory reactions and apoptosis of the cells following OGD/R exposure. RESULTS: In the mouse model of cerebral ischemia-reperfusion injury, the contents of inflammatory factors IL-1ß, IL-6 and TNF-α increased significantly in the brain tissue (P < 0.001), where LncRNA SNHG8 expression was lowered (P < 0.01) and miR-494-3p expression increased significantly (P < 0.01). In the microglial cells, overexpression of LncRNA SNHG8 significantly inhibited the inflammatory reaction and apoptosis following OGD/R exposure (P < 0.01), and overexpression of LncRNA SNHG8 strongly inhibited the expression of miR-494-3p (P < 0.01). Overexpression of miR-494-3p in microglia overexpressing SNHG8 partially promoted inflammatory reaction and cell apoptosis in response to OGD/R (P < 0.05). CONCLUSION: LncRNA SNHG8 can improve cerebral ischemia-reperfusion injury in mice by inhibiting the expression of miR-494-3p and suppressing inflammatory reactions and apoptosis of the microglia.

MicroRNA-494-3p facilitates the progression of bladder cancer by mediating the KLF9/RGS2 axis.

Bladder cancer (BC) is a familiar malignancy with high morbidity and mortality. The effect of treatment is unsatisfactory after the metastasis and invasion of BC. Hence, more studies should be carried out to explore the metastasis of BC. RT-qPCR or/and western blot was conducted to evaluate miR-494-3p, KLF9, and RGS2 expression. Cell proliferation and invasion were estimated by MTT assay and transwell assay, respectively. Cell migration was tested by wound healing assay and transwell assay. Dual-luciferase reporter gene assay was employed to validate the interplay between miR-494-3p and KLF9 mRNA. The interaction between KLF9 and RGS2 promoter was verified using dual-luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay. miR-494-3p expression was upregulated, whereas KLF9 and RGS2 were downregulated in BC cells. miR-494-3p inhibition was competent to limit the growth of BC cells. KLF9 knockdown abolished the miR-494-3p depletion-mediated inhibitory growth of BC cells. Mechanistically, we found that KLF9 was a downstream gene of miR-494-3p and could bind to the promoter region of RGS2 to promote the expression of RGS2. Moreover, RGS2 knockdown abrogated the suppressive effects of miR-494-3p knockdown on the proliferation, migration, and invasion of BC cells. Notably, miR-494-3p inhibition obstructed the tumor growth in nude mice. miR-494-3p silencing inhibited the progression of BC by regulating the KLF9/RGS2 axis in vitro and in vivo, which laid the foundation for experiments of miR-494-3p in BC and provided therapeutic targets for BC.

Extracellular Polysaccharide from Rhizopus nigricans Inhibits Hepatocellular Carcinoma via miR-494-3p/TRIM36 Axis and Cyclin E Ubiquitination.

Background and Aims: This study was designed to uncover the mechanism for extracellular polysaccharide (EPS1-1)-mediated effects on hepatocellular carcinoma (HCC) development. Methods: HCC cells were treated with EPS1-1, miR-494-3p mimic, sh-TRIM36, and pcDNA3.1-TRIM36. The levels of miR-494-3p and TRIM36 were measured in normal hepatocytes, THLE-2, and HepG2 and HuH7HCC cell lines, along with the protein expression of cyclin D/E and p21. The proliferation, cell cycle, and apoptosis of HCC cells were assayed. The interactions between miR-494-3p and TRIM36, and between TRIM36 and cyclin E were assessed. Finally, the expression and localization of TRIM36 and cyclin E were monitored, and tumor apoptosis was detected, in tumor xenograft model. Results: EPS1-1 suppressed HCC cell proliferation and cyclin D/E expression and promoted apoptosis and p21 expression. miR-494-3p was upregulated and TRIM36 was downregulated in HCC cells. Transfection with miR-494-3p mimic or sh-TRIM36 facilitated HCC cell proliferation and the expression of cyclin D/E protein but they inhibited apoptosis and p21 expression in the presence of EPS1-1. Overexpression of TRIM36 further consolidated EPS1-1-mediated inhibition of HCC proliferation, cyclin D/E, and the promotion of apoptosis and p21 expression. Those effects were reversed by miR-494-3p overexpression. TRIM36 was a target gene of miR-494-3p, and TRIM36 induced cyclin E ubiquitination. EPS1-1 suppressed cyclin E expression, promoted TRIM36 expression and tumor apoptosis, all of which were abrogated by increasing the expression of miR-494-3p in vivo. Conclusions: EPS1-1 protected against HCC by limiting its proliferation and survival through the miR-494-3p/TRIM36 axis and by inducing cyclin E ubiquitination.

Circular RNA hsa_circ_0000317 inhibits non-small cell lung cancer progression through regulating microRNA-494-3p/phosphatase and tensin homolog deleted on chromosome 10 axis.

BACKGROUND: Circular RNA (circRNA), a group of non-coding RNA, is pivotal in the progression of various cancers, including Non-Small Cell Lung Cancer (NSCLC). Some circRNAs have been reported to be implicated in the progression of NSCLC, however, the function and molecular mechanism of hsa_circ_0000317 (circ_0000317) in NSCLC have not been fully understood. METHODS: The significantly differentially expressed circRNA in NSCLC tissues, circ_0000317, was screened out by microarray. Circ_0000317, microRNA(miR)-494-3p and Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) expressions in NSCLC tissues were respectively probed by quantitative real-time polymerase chain reaction and western blot assay. MTT and Transwell assays were adopted to examine the growth, migration, and invasion of NSCLC cells. Bioinformatics, luciferase reporter gene assay, RNA immunoprecipitation, and RNA pull-down assay were conducted to probe the relationships among circ_0000317, miR-494-3p, and PTEN. RESULTS: Circ_0000317 expression level was reduced in NSCLC tissues and cell lines. Circ_0000317 expression in NSCLC patients was associated with TNM stage and lymphatic metastasis. Circ_0000317 overexpression restrained the proliferation, migration, and invasion of NSCLC cells, but co-transfection of miR-494-3p mimics partially reversed this effect. In addition, circ_0000317, was identified as a competitive endogenous RNA, which could sponge miR-494-3p to increase PTEN expression and activate PI3K/AKT pathway. CONCLUSION: Circ_0000317, inhibits NSCLC progression via modulating miR-494-3p/PTEN/PI3K/AKT pathway.

Specific miRNAs Change After 3 Months of GH treatment and Contribute to Explain the Growth Response After 12 Months.

Context: There is growing evidence of the role of epigenetic regulation of growth, and miRNAs potentially play a role. Objective: The aim of this study is to identify changes in circulating miRNAs following GH treatment in subjects with isolated idiopathic GH deficiency (IIGHD) after the first 3 months of treatment, and verify whether these early changes can predict growth response. Design and Methods: The expression profiles of 384 miRNAs were analyzed in serum in 10 prepubertal patients with IIGHD (5 M, 5 F) at two time points before starting GH treatment (t-3, t0), and at 3 months on treatment (t+3). MiRNAs with a fold change (FC) >+1.5 or <-1.5 at t+3 were considered as differentially expressed. In silico analysis of target genes and pathways led to a validation step on 8 miRNAs in 25 patients. Clinical and biochemical parameters were collected at baseline, and at 6 and 12 months. Simple linear regression analysis and multiple stepwise linear regression models were used to explain the growth response. Results: Sixteen miRNAs were upregulated and 2 were downregulated at t+3 months. MiR-199a-5p (p = 0.020), miR-335-5p (p = 0.001), and miR-494-3p (p = 0.026) were confirmed to be upregulated at t+3. Changes were independent of GH peak values at testing, and levels stabilized after 12 months. The predicted growth response at 12 months was considerably improved compared with models using the common clinical and biochemical parameters. Conclusions: MiR-199a-5p, miR-335-5p, and miR-494-3p changed after 3 months of GH treatment and likely reflected both the degree of GH deficiency and the sensitivity to treatment. Furthermore, they were of considerable importance to predict growth response.

CircVAPA promotes small cell lung cancer progression by modulating the miR-377-3p and miR-494-3p/IGF1R/AKT axis.

BACKGROUND: Multiple lines of evidence have demonstrated that circular RNAs (circRNAs) play oncogenic or tumor-suppressive roles in various human cancers. Nevertheless, the biological functions of circRNAs in small cell lung cancer (SCLC) are still elusive. METHODS: CircVAPA (annotated as hsa_circ_0006990) was identified by mining the circRNA profiling dataset of six paired SCLC tissues and the RNA-seq data of serum samples from 36 SCLC patients and 118 healthy controls. The circVAPA expression level was evaluated using quantitative real-time PCR in SCLC cells and tissues. Cell viability, colony formation, cell cycle and apoptosis analysis assays and in vivo tumorigenesis were used to reveal the biological roles of circVAPA. The underlying mechanism of circVAPA was investigated by Western blot, RNA pulldown, RNA immunoprecipitation, dual-luciferase reporter assay and rescue experiments. RESULTS: We revealed that circVAPA, derived from exons 2-4 of the vesicle-associated membrane protein-associated protein A (VAPA) gene, exhibited higher expression levels in SCLC cell lines, clinical tissues, and serum from SCLC patients than the controls, and facilitated SCLC progression in vitro and in vivo. Mechanistically, circVAPA activated the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway by modulating the miR-377-3p and miR-494-3p/insulin-like growth factor 1 receptor (IGF1R) axis to accelerate SCLC progression. Furthermore, circVAPA depletion markedly enhanced the inhibitory effects of BMS-536924, an IGF1R kinase inhibitor in cellular and xenograft mouse models. CONCLUSIONS: CircVAPA promotes SCLC progression via the miR-377-3p and miR-494-3p/IGF1R/AKT axis. We hope to develop clinical protocols of combinations of circVAPA inhibition and BMS-536924 addition for treating SCLC with circVAPA upregulation.

MiR-494-3p Upregulation Exacerbates Cerebral Ischemia Injury by Targeting Bhlhe40.

PURPOSE: Cerebral ischemia is related to insufficient blood supply and is characterized by abnormal reactive oxygen species (ROS) production and cell apoptosis. Previous studies have revealed a key role for basic helix-loop-helix family member e40 (Bhlhe40) in oxidative stress and cell apoptosis. This study aimed to investigate the roles of miR-494-3p in cerebral ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: A mouse middle cerebral artery occlusion (MCAO/R) model was established to mimic cerebral ischemia in vivo. Brain infarct area was assessed using triphenyl tetrazolium chloride staining. Oxygen-glucose deprivation/reoxygenation (OGD/R) operation was adopted to mimic neuronal injury in vitro. Cell apoptosis was analyzed by flow cytometry. The relationship between miR-494-3p and Bhlhe40 was validated by luciferase reporter and RNA immunoprecipitation assays. RESULTS: Bhlhe40 expression was downregulated both in MCAO/R animal models and OGD/R-induced SH-SY5Y cells. Bhlhe40 overexpression inhibited cell apoptosis and reduced ROS production in SH-SY5Y cells after OGD/R treatment. MiR-494-3p was verified to bind to Bhlhe40 and negatively regulate Bhlhe40 expression. Additionally, cell apoptosis and ROS production in OGD/R-treated SH-SY5Y cells were accelerated by miR-494-3p overexpression. Rescue experiments suggested that Bhlhe40 could reverse the effects of miR-494-3p overexpression on ROS production and cell apoptosis. CONCLUSION: MiR-494-3p exacerbates brain injury and neuronal injury by regulating Bhlhe40 after I/R.

Hsa_circ_0007142 contributes to cisplatin resistance in esophageal squamous cell carcinoma via miR-494-3p/LASP1 axis.

BACKGROUND: Chemoresistance is one of the major obstacles for tumor treatment. Circular RNAs (circRNAs) have been confirmed to play vital roles in chemoresistance of cancer, including esophageal squamous cell carcinoma (ESCC). We investigated the roles and mechanisms of circ_0007142 in cisplatin (DDP) resistance of ESCC. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to determine the levels of circ_0007142, DOCK1 mRNA, microRNA-494-3p (miR-494-3p) and LIM And SH3 Protein 1 (LASP1) mRNA. RNase R assay was conducted to analyze the characteristic of circ_0007142. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate IC50 of DDP. Flow cytometry analysis, 5-ethynyl-2'-deoxyuridine (EdU) assay and transwell assay were carried out to examine cell apoptosis, proliferation and invasion, respectively. Dual-luciferase reporter assay was employed to verify the association between miR-494-3p and circ_0007142 or LASP1. Murine xenograft assay was conducted to investigate the role of circ_0007142 in DDP resistant in vivo. The protein level of LASP1 in tumors was measured by Immunohistochemistry (IHC) analysis. RESULTS: Circ_0007142 was upregulated in DDP-resistant ESCC tissues and cells. Circ_0007142 knockdown improved DDP sensitivity, induced cell apoptosis and hampered cell proliferation and invasion in DDP-resistant ESCC cells. Circ_0007142 functioned as the sponge for miR-494-3p and miR-494-3p inhibition reversed the impacts of circ_0007142 knockdown on DDP resistance, cell apoptosis, proliferation, and invasion. LASP1 was a target of miR-494-3p, and the effects on DDP resistance, cell apoptosis, growth, and invasion mediated by LASP1 downregulation were rescued by miR-494-3p inhibition. Moreover, circ_0007142 knockdown enhanced DDP sensitivity in vivo. CONCLUSION: Circ_0007142 improved DDP resistance of ESCC by upregulating LASP1 via sponging miR-494-3p.

Golgi phosphoprotein 3 promotes angiogenesis and sorafenib resistance in hepatocellular carcinoma via upregulating exosomal miR-494-3p.

BACKGROUND: Golgi phosphoprotein 3 (GOLPH3) has been frequently reported as an oncoprotein in a variety of tumors. However, its role in the cancer-associated intercellular signaling communication has not yet been explored. This study aimed at exploring whether GOLPH3 regulates angiogenesis and sorafenib resistance via exosomal mechanisms in hepatocellular carcinoma (HCC). METHODS: In vivo assays were performed to elucidate the function of GOLPH3 in HCC. Exosomes of HCC cells were isolated by differential centrifugation, and then measured and quantified using nanoparticle tracking analysis (NTA), BCA assay, western blot (WB), and transmission electron microscopy (TEM). Differentially expressed miRNAs in exosome were analyzed and verified through small RNA sequencing (sRNA-seq) and reverse-transcription polymerase chain reaction (RT-PCR). In addition, a series of in vitro assays were performed to determine the function of exosomes and miR-494-3p in HCC. The candidate target gene of miR-494-3p was identified by bioinformatics prediction and dual-luciferase reporter assay. RESULTS: Downregulation of GOLPH3 expression could suppress angiogenesis and enhance sorafenib sensitivity in HCC. Exosomes derived from GOLPH3 overexpression HCC cells promoted the angiogenesis ability of HUVECs and induced sorafenib resistance in HCC cells. A total of 13 differentially expressed miRNAs between negative control and GOLPH3 knockdown group were found in exosomes. However, GOLPH3 was only associated with miR-494-3p expression level in exosomes derived from HCC cells without affecting total cellular miR-494-3p content. Results confirmed that exosomal miR-494-3p promotes angiogenesis of HUVECs and sorafenib resistance in HCC cells through directly targeting PTEN. CONCLUSIONS: HCC cells with high expression levels of GOLPH3 could promote angiogenesis and sorafenib resistance by enhancing exosomal miR-494-3p secretion to recipient HUVECs and HCC cells, respectively.