Circ_0027791 contributes to the growth and immune evasion of hepatocellular carcinoma via the miR-496/programmed cell death ligand 1 axis in an m6A-dependent manner.

Emerging evidence indicates the critical roles of circular RNAs in the development of multiple cancers, containing hepatocellular carcinoma (HCC). Herein, our present research reported the biological function and mechanism of circ_0027791 in HCC progression. Circ_0027791, microRNA-496 (miR-496), programmed cell death ligand 1 (PDL1), and methyltransferase-like 3 (METTL3) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, proliferation, invasion, and sphere formation ability were detected using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, 5-ethynyl-2'-deoxyuridine, transwell, and sphere formation assays. Macrophage polarization was detected using flow cytometry assay. To understand the role of circ_0027791 during the immune escape, HCC cells were cocultured with peripheral blood mononuclear cells or cytokine-induced killer (CIK) cells in vitro. A xenograft mouse model was applied to assess the function of circ_0027791 in vivo. After prediction using circinteractome and miRDB, the binding between miR-496 and circ_0027791 or PDL1 was validated based on a dual-luciferase reporter assay. Interaction between METTL3 and circ_0027791 was determined using methylated RNA immunoprecipitation (MeRIP)-qPCR, RIP-qPCR, and RNA pull-down assays. Circ_0027791, PDL1, and METTL3 expression were upregulated, and miR-496 was decreased in HCC patients and cells. Moreover, circ_0027791 knockdown might repress proliferation, invasion, sphere formation, M2 macrophage polarization, and antitumor immune response. Circ_0027791 knockdown repressed HCC tumor growth in vivo. In mechanism, circ_0027791 functioned as a sponge for miR-496 to increase PDL1 expression. In addition, METTL3 mediated the m6A methylation of circ_0027791 and stabilized its expression. METTL3-induced circ_0027791 facilitated HCC cell progression partly regulating the miR-496/PDL1 axis, which provided a new prognostic and therapeutic marker for HCC.

Circ_0006324 regulates cell proliferation, cell-cycle progression, apoptosis, and glycolysis of non-small cell lung cancer cells through miR-496/TRIM59 axis.

Increasing evidence suggests that circular RNA (circRNA) plays an important role in non-small cell lung cancer (NSCLC) progression. This study aimed to investigate the role and potential molecular mechanism of circ_0006324 in NSCLC. The expression levels of circ_0006324, miR-496, miR-488-5p, and tripartite motif-containing 59 (TRIM59) mRNA were determined by quantitative real-time polymerase chain reaction (PCR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, EdU assay, and flow cytometry were carried out to evaluate cell proliferation and apoptosis. The extracellular acidification rate and lactic acid production were examined to assess cell glycolysis. Western blot assay was used to detect protein levels. The target relationship of circ_0006324/miR-496/TRIM59 axis was validated by RNA pull-down assay, dual luciferase reporter assay, and radio immunoprecipitation assay. Xenograft tumor assay was performed to reveal the function of circ_0006324 in vivo. Circ_0006324 was upregulated in NSCLC and related to tumor node metastasis stage and distant metastasis. Knockdown of circ_00006324 impeded NSCLC cell proliferation, glycolysis, and promoted cell apoptosis. MiR-496 was verified as a target of circ_0006324 and circ_00006324 mediated the altering of cell proliferation, apoptosis, and glycolysis of NSCLC cells through targeting miR-496. TRIM59 was verified as a target of miR-496, and circ_0006324 positively regulated TRIM59 expression by targeting miR-496. Overexpression of TRIM59 could reverse the effects of circ_0006324 silencing on the proliferation, apoptosis, and glycolysis of NSCLC cells. Circ_0006324 knockdown impeded NSCLC tumor growth in vivo. Circ_0006324 functioned as a tumor promoter in NSCLC to promote cell proliferation, cell cycle progression, and glycolysis and inhibit cell apoptosis via miR-496/TRIM59 axis.

LncRNA KCNQ1OT1/miR-496/HMGB1 Signaling Axis Promotes Invasion and Migration of Non-small Cell Lung Cancer Cells.

Enhanced invasion and migration of non-small cell lung cancer (NSCLC) cells is the major cause of metastasis and poor prognosis in NSCLC. This study was conducted to investigate the role and mechanism of lncRNA KCNQ1OT1 in the proliferation, invasion, and migration of NSCLC cells. The expression of KCNQ1OT1 in NSCLC was analyzed in the StarBase database, and the target miRNA of KCNQ1OT1 as well as the target genes of the miRNA was predicted. Then, the mRNA expression levels of KCNQ1OT1, miR-496, and HMGB1 were detected in clinical tissue samples and cells by qRT-PCR assay. Besides, the protein levels of HMGB1 were detected by Western blot. MTT assay, transwell assay, and scratch assay were used to determine the proliferation, invasion, and migration ability of NSCLC cells, respectively. Correlation analysis was performed to assess the correlation between the expression of KCNQ1OT1, miR-496, and HMGB1 in clinical NSCLC samples. Dual-luciferase reporter gene assay was conducted to analyze the interaction between KCNQ1OT1 and miR-496 and between miR-496 and HMGB1. The database results showed that KCNQ1OT1 was highly expressed in NSCLC. Similarly, we found that the expression level of KCNQ1OT1 was significantly higher in NSCLC tissues and cells than that in the corresponding normal tissues and cells. The results of MTT assay, transwell assay, and scratch assay demonstrated that KCNQ1OT1 significantly enhanced the proliferation, invasion, and migration of NSCLC cells. Further mechanism exploration revealed that KCNQ1OT1 could sponge miR-496, and miR-496 directly targeted and regulated the expression of HMGB1. The expression of miR-496 and either KCNQ1OT1 or HMGB1 were negatively correlated in NSCLC, while the expression of KCNQ1OT1 and HMGB1 were positively correlated. Compared with normal paracancer tissues, miR-496 was much lower and HMGB1 was much higher expressed in NSCLC tissues. The results of cotransfection also further demonstrated that miR-496 inhibitor or sh-HMGB1 cotransfected with sh-KCNQ1OT1 could significantly decrease or increase the ability of sh-KCNQ1OT1 to inhibit the proliferation, invasion, and migration of H1299 cells, respectively. In conclusion, lncRNA KCNQ1OT1 promotes the invasion and migration of NSCLC cells through miR-496/HMGB1 signaling axis.

Plasma microRNA Profiling in Type 2 Diabetes Mellitus: A Pilot Study.

Type 2 diabetes mellitus (T2D) is a chronic metabolic disease characterized by insulin resistance and ß-cell dysfunction and leading to many micro- and macrovascular complications. In this study we analyzed the circulating miRNA expression profiles in plasma samples from 44 patients with T2D and 22 healthy individuals using next generation sequencing and detected 229 differentially expressed miRNAs. An increased level of miR-5588-5p, miR-125b-2-3p, miR-1284, and a reduced level of miR-496 in T2D patients was verified. We also compared the expression landscapes in the same group of patients depending on body mass index and identified differential expression of miR-144-3p and miR-99a-5p in obese individuals. Identification and functional analysis of putative target genes was performed for miR-5588-5p, miR-125b-2-3p, miR-1284, and miR-496, showing chromatin modifying enzymes and apoptotic genes being among the significantly enriched pathways.

Knockdown of circ_0007290 alleviates oxygen-glucose deprivation-induced neuronal injury by regulating miR-496/PDCD4 axis.

Circular RNAs (circRNAs) are highly enriched in the brain and involved in many types of central nervous system pathologies. Herein, this study aimed to investigate the role and mechanism of circ_0007290 in ischemic stroke. The oxygen-glucose deprivation (OGD) model was established with the HCN-2 cells in vitro. Levels of genes and proteins was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. In vitro experiments were conducted using cell counting kit-8 (CCK-8) assay, EdU (5-ethynyl-2'-deoxyuridine) assay, flow cytometry and ELISA, respectively. The levels of lactate dehydrogenase (LDH) were measured using the commercial kit. RNA pull-down and dual-luciferase reporter assay were used to identify the target relationship between miR-496 and circ_0007290 or PDCD4 (programmed cell death protein 4). Circ_0007290 expression was elevated in acute ischemic stroke (AIS) patients and OGD-induced cell injury model. OGD stimulation induced neuronal apoptosis, promoted LDH release, and enhanced inflammation in HCN-2 cells, which all were reversed by the knockdown of circ_0007290. Mechanistically, circ_0007290 served as a sponge for miR-496 to relieve the repression of miR-496 on the expression of its target PDCD4. Moreover, miR-496 inhibition or PDCD4 overexpression abolished the inhibitory effects of circ_0007290 knockdown OGD-evoked neuronal injury. Knockdown of circ_0007290 alleviated OGD-induced neuronal injury by regulating miR-496/PDCD4 axis, providing a novel insight into the pathology of ischemic stroke.

miR-496 remedies hypoxia reoxygenation-induced H9c2 cardiomyocyte apoptosis via Hook3-targeted PI3k/Akt/mTOR signaling pathway activation.

The hypoxia-reoxygenation (H/R) model helps analyze myocardial infarction triggered by acute myocardial ischemia, which induces cardiomyocyte proliferation and apoptosis. The Gene Expression Omnibus database was used to obtain the GSE74205 and GSE3866 microarray data, including microRNA (miRNA) and messenger RNA profiles, to catalog potential key miRNAs and genes. The role of rno-mir-496 expression in cardiomyocyte proliferation within 10 days of birth was established. The microRNA Target Prediction Database (miRDB) database-via Gene Ontology annotation-predicted hook microtubule tethering protein 3 (Hook3), a key target gene of rno-mir-496, was closely related to cell proliferation. Upregulation of miR-496 related to a significant reduction in apoptosis of H9c2 and human cardiomyocytes treatment with H/R. Moreover, transfection of H9c2 cells with miR-496 mimics, which were pretreated with H/R for 12 hours, increased Ki67 levels, proliferating cell nuclear antigen and Bcl-2 proteins; and decreased cleaved caspase-3 and Bax protein levels, as determined by reverse transcription-polymerase chain reaction and Western blot assays. A dual-luciferase reporter system confirmed that miR-496 targets the Hook3 suppressor. Hook3 overexpression stimulated apoptosis in H/R-treated cells, thus reducing cell proliferation. Upregulated miR-496 activated phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling, while Hook3 exhibited the inverse trend in H/R-treated H9c2 cells. In summary, with Hook3 functionality's aid, miR-496 upregulation defends cells from H/R-induced apoptosis and stimulates cell proliferation. miR-496 targets Hook3 to trigger the PI3K/Akt/mTOR signaling pathway for antiapoptotic and proliferative effects.

NNT-AS1 modulates prostate cancer cell proliferation, apoptosis and migration through miR-496/DDIT4 axis.

BACKGROUND: Emerging studies have disclosed long non-coding RNAs (lncRNAs) as pivotal modulators in the progression of prostate cancer (PCa). Current research planned to figure out the involvement of lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in PCa. METHODS: RNA expression was examined using RT-qPCR in PCa cells. Functional assays assessed the viability, proliferation, apoptosis and migration of PCa cells. RNA pull down and luciferase reporter experiments detected the interplay between miRNA and lncRNA or mRNA. RESULTS: NNT-AS1 was apparently upregulated in PCa cells. NNT-AS1 deficiency abrogated PCa cell viability, proliferation and migration but promoted apoptosis. Besides, miR-496 could be sequestered by NNT-AS1 to elevate the expression of DNA damage inducible transcript 4 (DDIT4) in PCa. Rescue assays indicated that overexpressed DDIT4 or restrained miR-496 could reverse the influence of NNT-AS1 depletion on malignant processes in PCa cells. CONCLUSION: NNT-AS1 contributes to the malignant phenotypes of PCa cells through targeting miR-496 to boost DDIT4 expression.

LINC00689 promotes prostate cancer progression via regulating miR-496/CTNNB1 to activate Wnt pathway.

BACKGROUND: Accumulating evidence has proved the significant influence of long non-coding RNAs (lncRNAs) in cancer formation and development, including PCa. METHODS: The role of LINC00689 in PCa was confirmed by RT-qPCR, MTT, colony formation, flow cytometry, western blot and transwell assays. Besides, the binding ability between LINC00689 and miR-496 was validated by using luciferase reporter assay. Then RT-qPCR, RIP and luciferase reporter and western blot assays were employed to verify the interactions among LINC00689, miR-496 and CTNNB1. Furthermore, the rescuing role of CTNNB1 in Wnt pathway was proved by RT-qPCR, TOP/FOP Flash and western blot assays. RESULTS: LINC00689 was upregulated in PCa tissues and cells as well as at the terminal stage. Further, knock down of LINC00689 repressed PCa cell proliferation, migration and invasion, and initiated PCa cell apoptosis. Additionally, miR-496 inhibitor and pcDNA3.1/CTNNB1 could neutralize the prohibitive effects of LINC00689 silencing on cell proliferation, migration and invasion, meanwhile, could offset the encouraging role of knocking down LINC00689 in cell apoptosis. Moreover, CTNNB1 upregulation exerted redemptive function in Wnt pathway inhibited by LINC00689 depletion. CONCLUSIONS: To sum up, LINC00689 promotes PCa progression via regulating miR-496/CTNNB1 to activate Wnt pathway, which may contribute to research about new targets for PCa treatment.

Oxidized low-density lipoprotein promotes vascular endothelial cell dysfunction by stimulating miR-496 expression and inhibiting the Hippo pathway effector YAP.

Oxidized low-density lipoprotein (ox-LDL) can damage vascular endothelial cells and cause atherosclerosis, but its epigenetic regulatory mechanism has not been fully elucidated. We show that ox-LDL induced significant apoptosis and loss of function in human umbilical vascular endothelial cells (HUVECs). At the same time, ox-LDL significantly decreased the expression of Hippo-YAP/ZAP (Yes-associated protein/YLP motif-containing 1) pathway proteins as compared to that of the control. The luciferase reporter system confirmed that microRNA (miR)-496 silenced YAP gene expression by binding to its 3' untranslated region (3' UTR). Ox-LDL-treated miR-496 overexpression HUVECs had a higher apoptosis rate and more severe dysfunction compared to the control cells. This in-depth study shows that ox-LDL inhibits YAP protein expression by inducing miR-496 expression, leading to its inability to enter the nucleus, thereby losing its function as a transcriptional cofactor for activating the downstream genes. Our findings reveal that, through epigenetic modification, ox-LDL can inhibit the normal expression of Hippo-YAP/ZAP pathway proteins via miR-496 expression and induce vascular endothelial cell dysfunction.

LncRNA-DANCR contributes to lung adenocarcinoma progression by sponging miR-496 to modulate mTOR expression.

Long non-coding RNAs (lncRNAs) have emerged as new and important regulators of pathological processes including tumour development. In this study, we demonstrated that differentiation antagonizing non-protein coding RNA (DANCR) was up-regulated in lung adenocarcinoma (ADC) and that the knockdown of DANCR inhibited tumour cell proliferation, migration and invasion and restored cell apoptosis rescued; cotransfection with a miR-496 inhibitor reversed these effects. Luciferase reporter assays showed that miR-496 directly modulated DANCR; additionally, we used RNA-binding protein immunoprecipitation (RIP) and RNA pull-down assays to further confirm that the suppression of DANCR by miR-496 was RISC-dependent. Our study also indicated that mTOR was a target of miR-496 and that DANCR could modulate the expression levels of mTOR by working as a competing endogenous RNA (ceRNA). Furthermore, the knockdown of DANCR reduced tumour volumes in vivo compared with those of the control group. In conclusion, this study showed that DANCR might be an oncogenic lncRNA that regulates mTOR expression through directly binding to miR-496. DANCR may be regarded as a biomarker or therapeutic target for ADC.

Amentoflavone attenuates cell proliferation and induces ferroptosis in human gastric cancer by miR-496/ATF2 axis.

Amentoflavone (AF) is a natural multifunctional biflavonoid that has been revealed to possess multiple biological activities, including anticancer activity. Here, this work focused on exploring the functions and mechanism of AF in gastric cancer (GC). Levels of genes and proteins were examined by quantitative real-time PCR and western blotting. Cell proliferation and cell death were analyzed using cell counting kit-8, colony formation, and lactate dehydrogenase (LDH) release assay, respectively. Cell ferroptosis was evaluated by detecting the levels of malondialdehyde (MDA), reduced glutathione (GSH), Fe2+ , and intracellular reactive oxygen species (ROS). The binding between miR-496 and activating transcription factor 2 (ATF2) was confirmed by using dual-luciferase reporter assay. Murine xenograft assay was conducted for in vivo experiments. The results showed that AF suppressed the proliferation and induced ferroptotic cell death in GC cells. MiR-496 expression was decreased in GC tissues and cells, and AF treatment increased miR-496 expression level in GC cells. Functionally, miR-496 inhibition reversed the inhibitory effects of AF on GC cell proliferation and promoting effects on ferroptotic cell death. Mechanistically, ATF2 was targeted by miR-496. ATF2 expression was increased in GC tissues and cells, which was decreased by AF treatment and subsequently rescued by miR-496 downregulation in GC cells. Moreover, miR-496 overexpression suppressed the proliferation and induced ferroptotic cell death in GC cells via targeting ATF2. In all, AF suppressed the proliferation and induced ferroptotic cell death in GC cells via miR-496/ATF2 axis, indicating a novel therapeutic approach for GC patients.

Circular RNA Ubiquitin-associated Protein 2 Silencing Suppresses Bladder Cancer Progression by Downregulating DNA Topoisomerase 2-alpha Through Sponging miR-496.

Background: Circular RNAs (circRNAs) have been uncovered to be implicated in the malignant development of bladder cancer (BC). Objective: Herein, this work aimed to investigate the role and mechanism of circRNA ubiquitin-associated protein 2 (circUBAP2) in BC progression. Design setting and participants: Quantitative real-time polymerase chain reaction and Western blotting were used for the detection of genes and proteins. Outcome measurements and statistical analysis: In vitro functional experiments were conducted using colony formation, 5-ethynyl-2'-deoxyuridine (EdU), Transwell, wound healing, and flow cytometry assays, respectively. A glycolysis analysis was conducted by assessing glucose uptake and lactate production. A murine xenograft model was established to perform in vivo experiments. The binding interaction between miR-496 and circUBAP2 or DNA topoisomerase 2-alpha (TOP2A) was verified using a dual-luciferase reporter assay. Results and limitations: CircUBAP2 was highly expressed in BC patients, and high circUBAP2 expression showed a shorter survival rate. Functionally, knockdown of circUBAP2 could suppress BC cell growth, migration, invasion, and aerobic glycolysis in vitro, as well as impede BC growth in nude mice. Mechanistically, circUBAP2 acted as a sponge for miR-496, which targeted TOP2A. Moreover, circUBAP2 could indirectly regulate TOP2A expression through sequestering miR-496. Furthermore, a series of rescue experiments showed that miR-496 inhibition reversed the anticancer action of circUBAP2 knockdown on BC cells. Moreover, miR-496 could attenuate BC cell malignant phenotypes and aerobic glycolysis, which were abolished by TOP2A overexpression. Conclusions: Silencing of circUBAP2 could suppress BC growth, invasion, migration, and aerobic glycolysis by the miR-496/TOP2A axis, suggesting a promising target for the molecular targeted therapies of BC. Patient summary: Circular RNA ubiquitin-associated protein 2 (circUBAP2) was found to be associated with poor prognosis in bladder cancer (BC). Knockdown of circUBAP2 might suppress BC growth, invasion, migration, and aerobic glycolysis, indicating that it may be a new target for the development of molecular targeted therapy for BC.

The long noncoding RNA noncoding RNA activated by DNA damage (NORAD)-microRNA-496-Interleukin-33 axis affects carcinoma-associated fibroblasts-mediated gastric cancer development.

Carcinoma-associated fibroblasts (CAFs) are one of the crucial parts of in the tumor microenvironment and contribute to tumor progression. Interleukin-33 (IL-33), a tissue-derived nuclear cytokine from the IL-1 family, has been found abnormally expressed in tumor cells and Fibroblast. However, the role and mechanism of IL-33 in the interaction between gastric cancer (GC) cells and CAFs need investigation. Presently, we inquire into the function of lncRNA NORAD-miR-496 axis-mediated IL-33 in modulating the GC-CAFs interaction. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was adopted to gauge the expression of NORAD, miR-496, and IL-33 in GC tissues and cells, and gain- or loss-of-function assays were conducted to investigate the role of them in GC. A GC cell-CAFs co-culture model was established to explore the interaction between CAFs and GCs. As exhibited, NORAD was up-regulated in GC tissues and cells, while miR-496 was remarkably down-regulated. Overexpressing NORAD substantially promoted the proliferation, migration, invasion, and EMT of GC cells and repressed cell death, while overexpressing miR-496 had the opposite effects. Additionally, NORAD enhanced the IL-33 expression and the release of IL-33 from GC cells. The dual-luciferase reporter assay confirmed that miR-496 was a target of NORAD and targeted IL-33. CAFs aggravated the malignant behaviors of GC cells as indicated by both experiments. However, NORAD knockdown in CAFs reversed CAFs-mediated promotive effects on GC cells. In conclusion, NORAD enhanced the promotive effect of CAFs in GC cells by up-regulating IL-33 and targeting miR-496, which provided new insights into the microenvironment of GC cells and CAFs.Abbreviation ANOVA: Analysis of Variance; BCA:Bicinchoninic acid; CAFs: carcinoma-associated fibroblasts; CCK-8: cell counting kit-8; ceRNA: competing endogenous RNA; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's minimal essential medium/Ham's; ECL: enhanced chemiluminiscent; ELISA: Enzyme-Linked Immunosorbent Assay; EMT: epithelial-mesenchymal transition; FBS: fetal bovine serum; FISH:Fluorescence in situ hybridization; FITC:fluorescein isothiocyanate; FSP:fibroblast-specific protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GC: gastric cancer; IHC: immunohistochemistry; IL: Interleukin; lncRNA: long Noncoding RNA; miR-496: microRNA-496; MMP-14:matrix metalloproteinase-14; MUT:mutant; MYH9: myosin heavy chain 9; NFs: normal fibroblasts; NORAD: Noncoding RNA activated by DNA damage; ORF: open reading frame; PBS: phosphate-buffered saline; PMSF: Phenylmethylsulfonyl fluoride; PVDF: polyvinylidene difluoride; RIPA: Radio-Immunoprecipitation Assay; RT-PCR: Real-time reverse transcription polymerase chain reaction; S100A4:S100 calcium binding protein A4; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; sh-NC: short-hairpin RNA negative control; sh-NORAD: short-hairpin RNA of NORAD; α-SMA: α-smooth muscle actin; TBST: Tris-buffered saline with Tween-20; TGF-ß1: Transforming growth factor ß1; TUNEL: TdT-mediated dUTP Nick-End Labeling; TWIST1: the twist-related protein 1; VEGF-C: vascular endothelial growth factor C; WT: Wildtype.

miR-496 inhibits proliferation via LYN and AKT pathway in gastric cancer.

MicroRNAs (miRNAs) operate as tumor suppressor or carcinogen to regulate cell proliferation, metastasis, invasion, differentiation, apoptosis, and metabolic process. In the present research, we investigated the effect and mechanism of miR-496 in human gastric cancer cells. miR-496 was downregulated in two gastric cancer cell lines, AGS and MKN45, compared with normal gastric epithelial cell line GES-1. miR-496 mimics inhibited the proliferation of AGS cells after the transfection for 48 and 72 h. The migration and invasion of AGS cells were also inhibited by the transfection of miR-496 mimics. miR-496 mimics induced the apoptosis through upregulating the levels of Bax and Active Caspase 3 and downregulating the levels of Bcl-2 and Total Caspase 3. Bioinformatics analysis showed that there was a binding site between miR-496 and Lyn kinase (LYN). miR-496 mimics could inhibit the expression of LYN in AGS cells. LYN overexpression blocked the inhibition of tumor cell growth, as well as the inhibition of AKT/mTOR signaling pathway induced by miR-496. In conclusion, miR-496 inhibited the proliferation through the AKT/mTOR signaling pathway via targeting LYN in gastric cancer cells. Our research provides a new potential target for clinical diagnosis and targeted treatment for gastric cancer.

miR-496/MMP10 Is Involved in the Proliferation of IL-1ß-Induced Fibroblast-Like Synoviocytes Via Mediating the NF-κB Signaling Pathway.

Rheumatoid arthritis (RA) is a common chronic autoimmune disease featured by synovial inflammation. miR-496 is closely involved in various pathologic conditions. However, its role in RA has not yet been elucidated. Expression of miR-496 and MMP10 was determined based on the clinical samples with RA retrieved from the Gene Expression Omnibus (GEO) datasets. In vitro model of RA was constructed in MH7A cells stimulated by IL-1ß (10 ng/mL). Cell counting kit 8 (CCK-8) and flow cytometry experiments were implemented to investigate the cell viability and apoptosis rate of MH7A cells. TargetScan was applied to identify the targets of miR-496, and the regulation of miR-496 on MMP10 expression was validated by a dual-luciferase reporter gene assay. qRT-PCR and western blot analyses were conducted to examine the expression. miR-496 expression was decreased in RA tissues and MH7A cells after IL-1ß treatment. Overexpression of miR-496 significantly inhibited IL-1ß-treated MH7A cell viability. MMP10 was identified as a target of miR-496 and its expression was negatively regulated by miR-496. The effects of miR-496 on MH7A cell proliferation and apoptosis were reversed by MMP10. The activity of NF-κB pathway was associated with the miR-496/MMP10 axis in IL-1ß-stimulated MH7A cells. To summarize, this study demonstrated that miR-496 can impair the proliferative ability and facilitate the apoptosis of IL-1ß-treated MH7A through regulating MMP10 expression and NF-κB signaling pathway.

MicroRNA-496 suppresses tumor cell proliferation by targeting BDNF in osteosarcoma.

MicroRNAs (miRNAs) are integrally involved in biological and pathobiological development. Many studies have demonstrated the abnormal expression of microRNA-496 (miR-496) in various human malignant tumors. The present study was designed to investigate the functions and the underlying mechanisms of miR-496 in osteosarcoma (OS) progression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression of miR-496 in OS tissues and cell lines. Luciferase activity was used to confirm the interaction between miR-496 and brain derived neurotrophic factor (BDNF), a downstream gene of miR-496. RT-qPCR was also used to quantify BDNF mRNA expression, and the BDNF protein expression level was detected by western blot analysis. In addition, the Cell Counting Kit-8 (CCK-8) was used to detect cell viability. The results revealed that the level of miR-496 expression was significantly reduced in osteosarcoma tissues and cell lines. BDNF was verified to be a direct target gene of miR-496 and was found to be negatively regulated by miR-496. Overall, it was demonstrated that miR-496 inhibits osteosarcoma cell proliferation via inhibition of BDNF. Thus, the miR-496/BDNF axis may be a novel strategy for the clinical treatment of OS.

Upregulation of miR-496 Rescues Propofol-induced Neurotoxicity by Targeting Rho Associated Coiled-coil Containing Protein Kinase 2 (ROCK2) in Prefrontal Cortical Neurons.

OBJECTIVE: Early exposure to general anesthesia in children might be a potentially highrisk factor for learning and behavioral disorders. The mechanism of neurotoxicity induced by general anesthesia was not defined. miR-496 could regulate cerebral injury, while the roles of miR- 496 in neurotoxicity were not elucidated. Therefore, we aimed to investigate the effects of miR- 496 in neurotoxicity induced by propofol. METHODS: Primary Prefrontal Cortical (PFC) neurons were isolated from neonatal rats and treated with propofol to induce neurotoxicity. Cell viability was detected by (3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The target relationship of miR-496 and Rho Associated Coiled-Coil Containing Protein Kinase 2 (ROCK2) was explored using luciferase assays. RESULTS: Propofol decreased cell viability, promoted cell apoptosis, and decreased the expression of miR-496 in PFC neurons in a dose-dependent manner. Overexpression of miR-496 attenuated neurotoxicity induced by propofol in PFC neurons. ROCK2 was a target of miR-496, and miR-496 oppositely modulated the expression of ROCK2. Besides, propofol increased the expression of ROCK2 through inhibiting miR-496 in PFC neurons. Overexpression of miR-496 attenuated propofol- induced neurotoxicity by targeting ROCK2 in PFC neurons. CONCLUSION: miR-496 was decreased in PFC neurons treated with propofol, and overexpression of miR-496 attenuated propofol-induced neurotoxicity by targeting ROCK2. miR-496 and ROCK2 may be promising targets for protecting propofol-induced neurotoxicity.

Long non-coding RNA HCG11 modulates glioma progression through cooperating with miR-496/CPEB3 axis.

OBJECTIVES: It has been widely reported that long non-coding RNAs (lncRNAs) can participate in multiple biological processes of human cancers. lncRNA HLA complex group 11 (HCG11) has been reported in human cancers as a tumour suppressor. This study focused on investigating the function and mechanism of HCG11 in glioma. MATERIALS AND METHODS: Based on The Cancer Genome Atlas (TCGA) data set and qRT-PCR analysis, the expression pattern of HCG11 was identified in glioma samples. The mechanism associated with HCG11 downregulation was determined by mechanism experiments. Gain-of-function assays were conducted for the identification of HCG11 function in glioma progression. Mechanism investigation based on the luciferase reporter assay, RIP assay and pull-down assay was used to explore the downstream molecular mechanism of HCG11. The role of molecular pathway in the progression of glioma was analysed in accordance with the rescue assays. RESULTS: HCG11 was expressed at low level in glioma samples compared with normal samples. FOXP1 could bind with HCG11 and transcriptionally inactivated HCG11. Overexpression of HCG11 efficiently suppressed cell proliferation, induced cell cycle arrest and promoted cell apoptosis. HCG11 was predominantly enriched in the cytoplasm of glioma cells and acted as a competing endogenous RNAs (ceRNAs) by sponging micro-496 to upregulate cytoplasmic polyadenylation element binding protein 3 (CPEB3). CEPB3 and miR-496 involved in HCG11-mediated glioma progression. CONCLUSIONS: HCG11 inhibited glioma progression by regulating miR-496/CPEB3 axis.

Upregulation of miR-496 decreases cerebral ischemia/reperfusion injury by negatively regulating BCL2L14.

Neurological functions were seriously impaired by cerebral ischemia-reperfusion (I/R) injury following ischemic stroke and its molecular mechanism is still not fully understood. MicroRNA-496 (miR-496) has been reported to be deregulated in several diseases but it still remains unknown about the function of miR-496 in cerebral I/R injury. Here, Middle cerebral artery occlusion/reperfusion (MCAO/R) was performed to induce cerebral I/R injury in rats. Then, neurological deficits were assessed by Bederson and Longa score system. The 2,3,5-triphenyltetrazolium chloride (TTC) and terminal deoxynucleotidyltransferase UTP nick end labeling (TUNEL) staining were used to evaluate the rat brain pathology. The oxygen-glucose deprivation and reperfusion (OGD/R) induced in vitro I/R injury was constructed in SH-SY5Y cells. The expression of miR-496 was determined using Real-time PCR. Bioinformatics analysis and dual luciferase reporter assay were utilized to confirm the target gene of miR-496. CCK-8, EdU staining, flow cytometry and Hoechst 33258 staining were respectively utilized to measure cell viability, proliferation and apoptosis. The results showed the expression of miR-496 was found to be down-regulated by cerebral I/R in rats and OGD/R-inducedSH-SY5Y cell model. MiR-496 overexpression alleviated the OGD/R-induced injury in SH-SY5Y cells. In addition, pro-apoptosis factor Bcl-2-like protein 14 (BCL2L14) was predicted and verified as the direct target of miR-496 and suppressed by miR-496. Furthermore, BCL2L14 knockdown exhibited similar effects similar to those of miR-496 overexpression, and the restored BCL2L14expression reversed the protective effects of miR-496 on SH-SY5Y cells. In conclusions, our results suggest that miR-496 alleviates cerebral I/R injury possibly via inhibiting BCL2L14 expression.

Role of Circular RNA DLEU2 in Human Acute Myeloid Leukemia.

In the current study, we were interested in exploring the molecular mechanism of circular RNA DLEU2 (circRNA-DLEU2) (hsa_circ_0000488) and microRNA 496 (miR-496), as well as PRKACB, in human acute myeloid leukemia (AML) cell activities. The RNA expression levels of circRNA-DLEU2, hsa-miR-496, and PRKACB were assessed by quantitative real-time PCR (qRT-PCR). The proliferation and apoptosis abilities of the cells were determined by CCK8 assay and flow cytometry analysis. Target relationships between circRNA-DLEU2 and miR-496, as well as PRKACB, were analyzed by luciferase reporter assay and probe assay. Immunoblotting assays were used to detect the protein expression level of PRKACB. We also did in vivo experiments to observe tumor formation after overexpression of circRNA-DLEU2. Our data showed that circRNA-DLEU2 was upregulated in AML tissues and cells, which promoted AML cell proliferation and inhibited cell apoptosis. circRNA-DLEU2 promoted AML tumor formation in vivo miR-496 was inhibited by circRNA-DLEU2 and was downregulated in AML tissues. circRNA-DLEU2 inhibited miR-496 expression and promoted PRKACB expression. miR-496 antagonized the effects of PRKACB on MOLM-13 cell proliferation and apoptosis. Collectively, circRNA-DLEU2 accelerated human AML by suppressing miR-496 and promoting PRKACB expression.