MiR-23a-5p alleviates chronic obstructive pulmonary disease through targeted regulation of RAGE-ROS pathway.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common respiratory disease and represents the third leading cause of death worldwide. This study aimed to investigate miRNA regulation of Receptor for Advanced Glycation End-products (RAGE), a causal receptor in the pathogenesis of cigarette smoke (CS)-related COPD, to guide development of therapeutic strategies. METHODS: RAGE expression was quantified in lung tissue of COPD patients and healthy controls, and in mice with CS-induced COPD. RNA-sequencing of peripheral blood from COPD patients with binding site prediction was used to screen differentially expressed miRNAs that may interact with RAGE. Investigation of miR-23a-5p as a potential regulator of COPD progression was conducted with miR-23a-5p agomir in COPD mice in vivo using histology and SCIREQ functional assays, while miR-23a-5p mimics or RAGE inhibitor were applied in 16-HBE human bronchial epithelial cells in vitro. RNA-sequencing, ELISA, and standard molecular techniques were used to characterize downstream signaling pathways in COPD mice and 16-HBE cells treated with cigarette smoke extract (CSE). RESULTS: RAGE expression is significantly increased in lung tissue of COPD patients, COPD model mice, and CSE-treated 16-HBE cells, while inhibiting RAGE expression significantly reduces COPD severity in mice. RNA-seq analysis of peripheral blood from COPD patients identified miR-23a-5p as the most significant candidate miRNA interaction partner of RAGE, and miR-23a-5p is significantly downregulated in mice and cells treated with CS or CSE, respectively. Injection of miR-23a-5p agomir leads to significantly reduced airway inflammation and alleviation of symptoms in COPD mice, while overexpressing miR-23a-5p leads to improved lung function. RNA-seq with validation confirmed that reactive oxygen species (ROS) signaling is increased under CSE-induced aberrant upregulation of RAGE, and suppressed in CSE-stimulated cells treated with miR-23a-5p mimics or overexpression. ERK phosphorylation and subsequent cytokine production was also increased under RAGE activation, but inhibited by increasing miR-23a-5p levels, implying that the miR-23a-5p/RAGE/ROS axis mediates COPD pathogenesis via ERK activation. CONCLUSIONS: This study identifies a miR-23a-5p/RAGE/ROS signaling axis required for pathogenesis of COPD. MiR-23a-5p functions as a negative regulator of RAGE and downstream activation of ROS signaling, and can inhibit COPD progression in vitro and in vivo, suggesting therapeutic targets to improve COPD treatment.

TFAP2C inhibits cell autophagy to alleviate myocardial ischemia/reperfusion injury by regulating miR-23a-5p/SFRP5/Wnt5a axis.

Myocardial ischemia/reperfusion (MI/R) injury contributes to severe injury for cardiomyocytes. In this study, we aimed to explore the underlying mechanism of TFAP2C on cell autophagy in MI/R injury. MTT assay measured cell viability. The cells injury was evaluated by commercial kits. IF detected the level of LC3B. Dual luciferase reporter gene assay, ChIP or RIP assay were performed to verify the interactions between crucial molecules. We found that TFAP2C and SFRP5 expression were decreased while miR-23a-5p and Wnt5a increased in AC16 cells in response to H/R condition. H/R induction led to cell injury and induced autophagy, which were reversed by TFAP2C overexpression or 3-MA treatment (an autophagy inhibitor). Mechanistically, TFAP2C suppressed miR-23a expression through binding to miR-23a promoter, and SFRP5 was a target gene of miR-23a-5p. Moreover, miR-23a-5p overexpression or rapamycin reversed the protective impacts of TFAP2C overexpression on cells injury and autophagy upon H/R condition. In conclusion, TFAP2C inhibited autophagy to improve H/R-induced cells injury by mediating miR-23a-5p/SFRP5/Wnt5a axis.

The linear ANRIL transcript P14AS regulates the NF-κB signaling to promote colon cancer progression.

BACKGROUND: The linear long non-coding RNA P14AS has previously been reported to be dysregulated in colon cancer, but the mechanistic role that P14AS plays in colon cancer progression has yet to be clarified. Accordingly, this study was developed to explore the regulatory functions of ANRIL linear transcript-P14AS in cancer. METHODS: The expression of P14AS, ANRIL, miR-23a-5p and their target genes were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell supernatants of IL6 and IL8 were measured by Enzyme linked immunosorbent (ELISA) assay. Dual-luciferase reporter assays, RNA immunoprecipitation, or pull-down assays were used to confirm the target association between miR-23a-5p and P14AS or UBE2D3. Cell proliferation and chemosensitivity of NF-κB inhibitor BAY 11-7085 were evaluated by cell counting kit 8 (CCK8). RESULTS: When P14AS was overexpressed in colon cancer cell lines, enhanced TNF-NF-κB signaling pathway activity was observed together with increases in IL6 and IL8 expression. The Pita, miRanda, and RNA hybrid databases revealed the ability of miR-23a-5p to interact with P14AS, while UBE2D3 was further identified as a miR-23a-5p target gene. The results of dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation experiments confirmed these direct interactions among P14AS/miR-23a-5p/UBE2D3. The degradation of IκBa mediated by UBE2D3 may contribute to enhanced NF-κB signaling in these cells. Moreover, the beneficial impact of P14AS on colon cancer cell growth was eliminated when cells were treated with miR-23a-5p inhibitors or UBE2D3 was silenced. As such, these findings strongly supported a role for the UBE2D3/IκBa/NF-κB signaling axis as a mediator of the ability of P14AS to promote colon cancer progression. CONCLUSIONS: These data suggested a mechanism through which the linear ANRIL transcript P14AS can promote inflammation and colon cancer progression through the sequestration of miR-23a-5p and the modulation of NF-κB signaling activity, thus highlighting P14AS as a promising target for therapeutic intervention efforts.

Identification lncRNA LOC102551149/miR-23a-5p pathway in hepatic fibrosis.

BACKGROUND: Hepatic fibrosis is a worldwide incurable disease; due to the complex and unclear mechanism, there lack the effective therapeutic targets. However, the mechanism of miR-23a-5p underling this pathological process is largely not clear. The purpose of this study was to investigate the role of miR-23a-5p in hepatic fibrosis and HSC activation. METHODS: The content of miR-23a-5p in hepatic fibrosis induced by N-nitrosodimethylamine (NDMA) and HSC activation induced by platelet-derived growth factor (PDGF) was detected by qRT-PCR. H&E staining, Masson staining and Shear wave electrography (SWE) were used to detect the degree of hepatic fibrosis. Immunohistochemistry staining, qRT-PCR and Western blot detect the related markers of liver fibrosis or HSC activation, as well as the related pathway genes and proteins. Dual-luciferase reporter system verifies the interaction between miR-23a-5p with PTEN or miR-23a-5p with lncRNA LOC102551149 in HSC-T6. siRNA and miRNA mimic transfer to HSC-T6 to detect the function of lncRNA LOC102551149 and miR-23a-5p on HSC activation. RESULTS: After hepatic fibrosis and HSC activation happened, the expression of miR-23a-5p was up-regulated, whereas anti-miR-23a-5p can alleviate hepatic fibrosis and HSC activation. Further research shows miR-23a-5p can target PTEN and degrade it, causing activation of PI3K/Akt/mTOR/Snail pathway. lncRNA LOC102551149 can be used as a competition endogenous RNA (ceRNA) targeting miR-23a-5p through base pairing, and siRNA LOC102551149 or exogenous miR-23a-5p can induce HSC activation through PI3K/Akt/mTOR/Snail pathway. CONCLUSION: We demonstrate mechanism pathway of miR-23a-5p on hepatic fibrosis and HSC activation, which may develop a therapeutic target for hepatic fibrosis.

miR-23a-3p and miR-23a-5p target CiGadd45ab to modulate inflammatory response and apoptosis in grass carp.

Ctenopharyngodon idella growth arrest and DNA damage-inducible 45 ab (CiGadd45ab) is a subtype of the Gadd45a gene of the Gadd45 family in grass carp. There is increasing evidence that microRNAs (miRNAs) are involved in the regulation of inflammatory and apoptotic responses. However, little is known about the regulatory effects of miRNAs on CiGadd45ab expression. In the present study, CiGadd45ab was identified as a target gene of miR-23a-3p and miR-23a-5p, based on miRNA expression profiling and a dual-luciferase reporter assay. In addition, miR-23a-3p and miR-23a-5p were both confirmed to be involved in the inflammatory response following infection with Aeromonas hydrophila by targeting CiGadd45ab. Transfection with miR-23a-3p and miR-23a-5p mimics and inhibitor altered proinflammatory gene expression and apoptosis rate, thereby suggesting that miRNAs regulate immune response and anti-apoptosis by targeting CiGadd45ab in grass carp. Our results provide a theoretical basis for exploring the molecular mechanisms by which miR-23a-3p and miR-23a-5p target CiGadd45ab to regulate inflammation and apoptosis against bacterial infection in grass carp.

Circular RNA expression profile in human fibroblast premature senescence after repeated ultraviolet B irradiations revealed by microarray.

Circular RNA (circRNA) is a class of noncoding RNA that regulates the activity of microRNAs and gene expression. Altered circRNA expression is associated with human diseases. The present study profiled differentially expressed circRNAs in the ultraviolet B stress-induced human fibroblast premature senescence (UVB-SIPS) model, and assessed the role of circRNA_100797 in UVB-SIPS. The UVB-SIPS model was confirmed by ß-galactosidase staining, cell viability CCK-8 assay, and flow cytometric cell cycle distribution assay, and subjected to circRNA gene chip profiling. These differentially expressed circRNAs were analyzed using the clusterProfiler R package for Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathways. The selected circRNAs were confirmed using quantitative reverse transcription polymerase chain reaction (qRT-PCR), the relationship of circRNA_100797 with miR-23a-5p was assessed using luciferase reporter assay, and their functions were determined by qRT-PCR and western blot analysis. A total of 472 differentially expressed circRNAs occurred in the UVB-SIPS. qRT-PCR confirmed five of eight differentially expressed circRNAs. The GO and KEGG analyses revealed that these differently expressed circRNAs function in biology process, cell component, and molecular function. Furthermore, it was found that circRNA_100797 had a low expression in UVB-SIPS. However, when circRNA_100797 was overexpressed, the acceleration of cell proliferation and alleviation of cell cycle arrest were observed. Moreover, circRNA_100797 could target miR-23a-5p, their expression levels were inversely associated in fibroblasts, and the miR-23a-5p overexpression blocked the effect of the overexpression of circRNA_100797 in UVB-SIPS. The present study demonstrated that circRNA_100797 acts as a sponge of miR-23a-5p, and has a photoprotection role in UVB-irradiated fibroblasts.

MicroRNA-23a-5p promotes atherosclerotic plaque progression and vulnerability by repressing ATP-binding cassette transporter A1/G1 in macrophages.

Disruption of carotid vulnerable atherosclerotic plaque is responsible for acute ischemic stroke (AIS) and the early detection and intervention approach are greatly limited. Undertaking a microarray of microRNAs (miRNAs) in the plasma of AIS patients with carotid vulnerable plaques, miR-23a-5p was markedly elevated and was positively correlated with the plaque progression and vulnerability. Correspondingly, we found that miR-23a-5p expression was significantly increased in both plasma and macrophages from atherosclerosis mice. Bioinformatics analysis and in vitro knockdown experiments identified that ATP-binding cassette transporter A1/G1 as a novel target of miR-23a-5p. Luciferase reporter assays showed that miR-23a-5p repressed the 3' untranslated regions (UTR) activity of ABCA1/G1. Moreover, functional analyses demonstrated that transfection of miR-23a-5p inhibitor enhanced cholesterol efflux and decreased foam cell formation through upregulating ABCA1/G1 expression levels. Furthermore, long term in vivo systemically delivered miR-23a-5p antagomir significantly increased ABCA1/G1 expression in the aorta of ApoE-/- mice. Importantly, the miR-23a-5p antagomir therapy significantly reduced atherosclerosis progression and promoted plaque stability. Our observations indicate that miR-23a-5p promotes macrophage-derived foam cell formation and might be a key regulator contributing to atherosclerotic plaque progression and vulnerability.

MiR-23a-5p modulates mycobacterial survival and autophagy during mycobacterium tuberculosis infection through TLR2/MyD88/NF-κB pathway by targeting TLR2.

Autophagy plays a pivotal role in activating the antimicrobial host defense against Mycobacterium tuberculosis (M.tb.). The emerging roles of microRNAs (miRNAs) in regulating immune responses have attracted increasing attention in recent years. Appreciating the potential of host-directed therapies designed to control autophagy during mycobacterial infection, we focused on the influence of miR-23a-5p on the activation of macrophage autophagy during M.tb. infection in bone marrow-derived macrophages (BMDMs) and murine RAW264.7 cells. Here, we demonstrated that M.tb.-infection of macrophages lead to markedly enhanced expression of miR-23a-5p in a time- and dose-dependent manner. Furthermore, forced expression of miR-23a-5p accelerated the survival rate of intracellular mycobacteria, while transfection with miR-23a-5p inhibitors attenuated mycobacterial survival. More importantly, overexpression of miR-23a-5p dramatically prevented M.tb.-induced activation of autophagy in macrophages, whereas inhibitors of miR-23a-5p remarkably accelerated M.tb.-induced autophagy. Mechanistically, miR-23a-5p is able to modulate TLR2/MyD88/NF-κB signaling activity by targeting TLR2 in RAW264.7 cells in response to M.tb.-infection. Collectively, these findings demonstrated that miR-23a-5p modulated the innate host defense by promoting mycobacteria survival and inhibiting the activation of autophagy against M.tb. through TLR2/MyD88/NF-κB pathway by targeting TLR2, which may provide a promising therapeutic target for tuberculosis.

Exosome-derived miR-23a-5p inhibits HCC proliferation and angiogenesis by regulating PRDX2 expression: MiR-23a-5p/PRDX2 axis in HCC progression.

microRNAs (miRNAs) are closely related to the progression of hepatocellular carcinoma (HCC). Cancer-derived exosomes play an essential role in the establishment of the HCC microenvironment. However, the possible effects and underlying mechanisms of exosome (exo) microRNA-23a-5p (miR-23a-5p) in the progression of HCC remain unknown. In this study, we aimed to determine the role and specific molecular mechanism of exo miR-23a-5p in regulating HCC progression and to investigate whether exo miR-23a-5p levels can serve as an indicator of the prognosis of transarterial chemoembolization in patients with HCC. Our findings illustrated that miR-23a-5p was downregulated in exosomes separated from the serum of HCC patients and that miR-23a-5p carried by exosomes inhibited HCC cell proliferation and angiogenesis. Mechanistically, miR-23a-5p negatively targeted peroxiredoxin-2 (PRDX2). Functionally, PRDX2 overexpression relieved exosome-induced inhibition of HCC cell proliferation and angiogenesis by promoting vascular endothelial growth factor (VEGF) expression. In conclusion, Exo miR-23a-5p inhibited HCC proliferation and angiogenesis by regulating PRDX2 expression. Our results revealed the role and specific molecular mechanism of exo miR-23a-5p in regulating HCC progression.

[Retracted] Oncogenic miR-23a-5p is associated with cellular function in RCC.

Following the publication of this article, a concerned reader drew to the Editor's attention that, for the invasion and migration assay data shown in Fig. 4 on p. 2314, three pairs of data panels were overlapping, such that data which were intended to show the results from differently performed experiments were obtained from a smaller number of original sources. Moreover, after having conducted an internal investigation, the Editorial Office also observed that some of the flow cytometric data shown in Fig. 6 were duplicated in Fig. 7. Considering the number of overlapping data panels that have been identified in this published paper, the Editor of Molecular Medicine Reports has concluded that the article should be retracted from the publication on account of a lack of confidence in the integrity of the data. Upon contacting the authors about this matter, they accepted the decision to retract this paper. The Editor apologizes to the readership for any inconvenience caused, and thanks the interested reader for drawing this matter to our attention. [Molecular Medicine Reports 16: 2309-2317, 2017; DOI: 10.3892/mmr.2017.6829].

SNHG9 promotes Hepatoblastoma Tumorigenesis via miR-23a-5p/Wnt3a Axis.

Background: Hepatoblastoma is a common hepatic tumor occurring in children between 0-5 years. Accumulating studies have shown lncRNA's potential role in distinct cancer progression and development, including hepatoblastoma. SnoRNA host gene 9 (SNHG9) is associated with the progression of distinct human cancers, but, its specific molecular mechanisms in hepatoblastoma is not unknown. Methods: In this study, we estimated SNHG9 expression in hepatoblastoma tissue and cell lines by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Next, we downregulated and upregulated SNHG9 expression in hepatoblastoma cell lines and then determined cell proliferation (CCK-8), colony formation, and cellular apoptosis activity. The dual luciferase reporter activity, RNA immunoprecipitation (RIP), biotin RNA pull down and Spemann's Pearson correlation coefficient assay were performed to establish the interaction between SNHG9, WNt3a and miR- 23a-5p. A xenograft in-vivo tumorgenicity test was performed to elucidate the role of SNHG9 hepatoblastoma in tumorigenesis. SNHG9 role in Cisplatin drug resistance in hepatoblastoma was also determined. Results: SNHG9 was significantly upregulated in hepatoblastoma tissue and cell lines. SNHG9 overexpression on HUH6 & HepG2 resulted in a significant increase in cell proliferation and clonogenic activity while SNHG9 knock down resulted in a sustained inhibition of cell proliferation and clonogenic activity. Dual luciferase activity, RNA immunoprecipitation and biotin pull down confirmed the direct interaction of miR-23a-5p with SNHG9. The xenograft tumorgenicity test showed SNHG9 downregulation significantly inhibited the tumor growth in BALB/c mice. ROC and Kaplan-Meier analysis showed potential prognostic and diagnostic importance of SNHG9 in hepatoblastoma. Conclusion: We concluded that SNHG9/miR-23a-5p/Wnt3a axis promotes the progression hepatoblastoma tumor.

LncRNA TPT1-AS1 Sponges miR-23a-5p in Glioblastoma to Promote Cancer Cell Proliferation.

Background: Long noncoding RNA (LncRNA) TPT1-AS1 is an oncogene in ovarian cancer and cervical cancer, while its role in glioblastoma (GBM) is unknown. The bioinformatics analysis in this study showed that miR-23a-5p may bind to TPT1-AS1. This study was performed to investigate the interactions between miR-23a-5p and TPT1-AS1 in GBM. Materials and Methods: A total of 60 GBM patients (40 males and 20 females, 24 to 60 years old, mean age 41.7 ± 7.8 years old) were enrolled at the First Hospital of Jilin University between April 2016 and April 2018. Gene expression levels were determined by qPCR and Western blot. Cell transfections were performed to analyze the interactions between TPT1-AS1, miR-23a-5p, and extracellular matrix protein 1 (ECM1). Cell proliferation was detected by cell proliferation assay. Results: The authors found miR-23a-5p was downregulated in GBM and TPT1-AS1 was upregulated in GBM, whereas the expression of these two was not significantly correlated. In GBM cells, overexpression of TPT1-AS1 did not affect the expression of miR-23a-5p, but upregulated ECM1. In cell proliferation assay, overexpression of TPT1-AS1 and ECM1 resulted in increased proliferation rate of GBM cells. Overexpression of miR-23a-5p attenuated the effects of overexpressing TPT1-AS1. Conclusions: TPT1-AS1 may sponge miR-23a-5p in GBM to promote cancer cell proliferation by upregulating ECM1.

Osteoclast-derived miR-23a-5p-containing exosomes inhibit osteogenic differentiation by regulating Runx2.

BACKGROUND: Some microRNAs (miRNAs) are involved in osteogenic differentiation. In recent years, increasing evidences have revealed that exosomes contain specific miRNAs. However, the effect and mechanism of miR-23a-5p-containing exosomes in osteoblast remain largely unclear. METHODS: We extracted exosomes from RANKL-induced RAW 264.7 cells, and identified exosomes via transmission electron microscopy, western blot and flow cytometry analysis. In addition, exosome secretion was inhibited by GW4869 and Rab27a siRNAs. miR-23a-5p expression was analyzed by qRT-PCR, and the related protein levels were examined by western blot assay. Furthermore, the number and distribution of osteoclasts were detected by TRAP staining, and early osteogenesis was evaluated by ALP staining. Combination of YAP1 and Runx2 was verified by Co-IP assay, and the regulation of miR-23a-5p and Runx2 was measured by dual luciferase reporter assay. RESULTS: We successfully extracted exosomes from RANKL-induced RAW 264.7 cells, and successfully verified exosomes morphology. We also indicated that miR-23a-5p was highly expressed in exosomes from RANKL-induced RAW 264.7 cells, and osteoclast-derived miR-23a-5p-containing exosomes inhibited osteoblast activity, while its inhibition weakened osteoclasts. In mechanism, we demonstrated that Runx2 was a target gene of miR-23a-5p, YAP interacted with Runx2, and YAP or Runx2 inhibited MT1DP expression. In addition, we proved that knockdown of MT1DP facilitated osteogenic differentiation by regulating FoxA1 and Runx2. CONCLUSIONS: We demonstrated that osteoclast-derived miR-23a-5p-containing exosomes could efficiently suppress osteogenic differentiation by inhibiting Runx2 and promoting YAP1-mediated MT1DP. Therefore, we suggested miR-23a-5p in exosomes might provide a novel mechanism for osteoblast function.

MiR-23a-5p exacerbates intestinal ischemia-reperfusion injury by promoting oxidative stress via targeting PPAR alpha.

MiR-23a-5p is involved in the occurrence and development of some serious diseases, but its effects on intestinal ischemia-reperfusion (II/R) injury is unclear. In this research, the hypoxia/reoxygenation (H/R) model on IEC-6 cells and II/R model in mice were used. The data showed that the ROS level in model group was significantly increased compared with control group. The level of intestinal MPO was increased and serum SOD was decreased in mice compared with sham group. Moreover, the expression levels of miR-23a-5p in model groups were obviously increased in vitro and in vivo, while the expression levels of PPARα, FOXO3α, PGC-1α, Nrf2, CAT, NQO1, HO-1 and SOD2 were significantly decreased. The double luciferase reporter gene assay showed that there was binding site between miR-23a-5p and PPARα. When miR-23a-5p was inhibited or PPARα gene was overexpressed, H/R-caused cell damage was alleviated and ROS level was decreased compared with NC group. PPARα expression level was increased, accompanied by the increased levels of FOXO3α, PGC-1α, Nrf2, CAT, NQO1, HO-1 and SOD2. After enhancing miR-23a-5p expression or silencing PPARα gene, H/R-caused cell damage was further aggravated compared with NC group, and ROS level was increased associated with the decreased levels of FOXO3α, PGC-1α, Nrf2, CAT, NQO1, HO-1 and SOD2. Our study demonstrated that miR-23a-5p exacerbated II/R injury by promoting oxidative stress via targeting PPARα, which should be considered as one new drug target to treat II/R injury.

miR-23a-5p inhibits cell proliferation and invasion in pancreatic ductal adenocarcinoma by suppressing ECM1 expression.

Pancreatic ductal adenocarcinoma (PDAC) is a genetic disease and a leading cause of cancer-related mortality. However, the molecular mechanism underlying PDAC progression remains unclear. In this study, we first confirmed that ECM1 is significantly upregulated in PDAC tissues and that its high levels of expression are closely associated with an advanced histologic grade and a poor prognosis using The Cancer Genome Atlas (TCGA) dataset and the Gene Expression Omnibus (GEO) database. We then found that miR-23a-5p binds directly to the ECM1 3'-untranslated region (3'-UTR), thereby inhibiting ECM1 expression. Functional studies revealed that the induced expression of ECM1 promoted oncogenic abilities and reversed the suppressive effects induced by miR-23a-5p. Collectively, our findings indicate that ECM1 is a proto-oncogene and show that targeting the miR-23a-5p/ECM1 axis may represent a promising therapeutic strategy for PDAC.