Dual Luciferase Activity Assay Research Articles

MiR-125b enhances autophagic flux to improve septic cardiomyopathy via targeting STAT3/HMGB1

We explore the role of miR-125b in septic cardiomyopathy, focusing on miR-125b/STAT3/HMGB1 axis. CLP mouse model and LPS-stimulated primary rat cardiomyocytes (CMs) and H9C2 cell were used as in vivo and in vitro models of septic cardiomyopathy, respectively. qRT-PCR and western blot were performed to measure expression levels of miR-125b, STAT3, HMGB1, and autophagy-related proteins. MTT assay was employed to examine LPS toxicity. Dual luciferase activity assay and CHIP were performed to validate interactions of miR-125b/STAT3 and STAT3/HMGB1 promoter. Immunostaining was used to assess the level of autophagic flux. ROS level was measured by fluorescence assay. Heart functions were examined via intracoronary Doppler ultrasound. miR-125b was diminished while STAT3 and HMGB1 were elevated in the heart tissue following CLP surgery and in LPS-treated H9C2 cells. LPS treatment up-regulated ROS generation and suppressed autophagic flux. Overexpression of miR-125b mimics or knockdown of STAT3 or HMGB1 alleviated LPS-induced hindrance of autophagic flux and ROS production. miR-125b directly targeted STAT3 mRNA and STAT3 bound with HMGB1 promoter. Overexpression of miR-125b mitigated myocardial dysfunction induced by CLP in vivo. Hyperactivation of STAT3/HMGB1 caused by reduced miR-125b contributes to ROS generation and the hindrance of autophagic flux during septic cardiomyopathy, leading to myocardial dysfunction.

CircDTL Functions as an Oncogene and Regulates Both Apoptosis and Ferroptosis in Non-small Cell Lung Cancer Cells.

Background: Circular RNAs (circRNA) play an essential role in the tumorigenesis of non-small cell lung cancer (NSCLC). CircDTL is a novel identified circRNA with little information regarding its biological role. However, the role of circDTL in NSCLC has not been investigated yet.Method: In this study, the levels of circDTL in tissues and cells were measured by RT-PCR. Cell viability was measured by the CCK-8 assay. Cell migration and invasion were evaluated using the wound healing assay and transwell assay, respectively. Cell death was measured by the cell death ELISA kit. The levels of Fe2+, ROS, MDA and GSH were measured using the commercial kits. The interactions between miR-1287-5p and circDTL/3′UTR GPX4 were verified by dual-luciferase activity assay. The effects of circDTL on tumor growth were evaluated in vivo.Results: CircDTL was found to be upregulated and acted as an oncogene in NSCLC cells. Knockdown of circDTL promoted both apoptosis and ferroptosis of NSCLC cells. It was identified that circDTL exerts its oncogenic effects via the circDTL/miR-1287-5p/GPX4 axis and GPX4 inhibits both ferroptosis and apoptosis. Finally, this study showed that silencing of circDTL promoted the sensitivity of NSCLC cells to chemotherapeutic agents and inhibited the growth of tumors in vivo.Conclusion: CircDTL acts as an oncogene and exerts its effects via the miR-1287-5p/GPX4 axis in NSCLC, providing a potential therapeutic target for NSCLC cancer therapy.

MicroRNA-497 induced by Clonorchis sinensis enhances the TGF-\u03b2/Smad signaling pathway to promote hepatic fibrosis by targeting Smad7

BackgroundVarious stimuli, including Clonorchis sinensis infection, can cause liver fibrosis. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) with massive production of extracellular matrix (ECM). Our previous study showed that the TGF-β1-induced Smad signaling pathway played a critical role in the activation of HSCs during liver fibrosis induced by worm infection; however, the mechanisms that modulate the TGF-β/Smad signaling pathway are still poorly understood. Accumulating evidence demonstrates that miRNAs act as an important regulator of activation of HSCs during liver fibrosis.MethodsThe target of miR-497 was determined by bioinformatics analysis combined with a dual-luciferase activity assay. LX-2 cells were transfected with miR-497 inhibitor and then stimulated with TGF-β1 or excretory/secretory products of C. sinensis (CsESPs), and activation of LX-2 was assessed using qPCR or western blot. In vivo, the mice treated with CCl4 were intravenously injected with a single dose of adeno-associated virus serotype 8 (AAV8) that overexpressed anti-miR-497 sequences or their scramble control for 6 weeks. Liver fibrosis and damage were assessed by hematoxylin and eosin (H&E) staining, Masson staining, and qPCR; the activation of the TGF-β/Smad signaling pathway was detected by qPCR or western blot.ResultsIn the present study, the expression of miR-497 was increased in HSCs activated by TGF-β1 or ESPs of C. sinensis. We identified that Smad7 was the target of miR-497 using combined bioinformatics analysis with luciferase activity assays. Transfection of anti-miR-497 into HSCs upregulated the expression of Smad7, leading to a decrease in the level of p-Smad2/3 and subsequent suppression of the activation of HSCs induced by TGF-β1 or CsESPs. Furthermore, miR-497 inhibitor delivered by highly-hepatotropic (rAAV8) inhibited TGF-β/smads signaling pathway by targeting at Smad7 to ameliorate CCL4-induced liver fibrosis.ConclusionsThe present study demonstrates that miR-497 promotes liver fibrogenesis by targeting Smad7 to promote TGF-β/Smad signaling pathway transduction both in vivo and in vitro, which provides a promising therapeutic strategy using anti-miR-497 against liver fibrosis.Graphical

LncRNA DLGAP1-AS2 regulates miR-503/cyclin D1 to promote cell proliferation in non-small cell lung cancer

BackgroundLncRNA DLGAP1-AS2 plays an oncogenic role in glioma, while its role in other cancers is unknown. This study aimed to study the role of DLGAP1-AS2 in non-small cell lung cancer (NSCLC).MethodsExpression of DLGAP1-AS2 in NSCLC and paired non-tumor tissues from 64 NSCLC patients and the prognostic value of DLGAP1-AS2 for NSCLC were analyzed by performing a 5-year follow-up study. The interaction between DLGAP1-AS2 and miR-503 was confirmed by dual luciferase reporter assay, and their relationship was explored in NSCLC cells transfected with DLGAP1-AS2 expression vector or miR-503 mimic. The roles of DLGAP1-AS2 and miR-503 in regulating cyclin D1 expression were analyzed by RT-qPCR and Western blot. Cell proliferation was analyzed by CCK-8 assay.ResultsDLGAP1-AS2 was upregulated in NSCLC and predicted poor survival. Interaction between DLGAP1-AS2 and miR-503 was confirmed by dual luciferase activity assay. Overexpression experiments showed that DLGAP1-AS2 and miR-503 overexpression failed to significantly affect the expression of each other. Interestingly, DLGAP1-AS2 overexpression upregulated cyclin D1, a target of miR-503, increased cell proliferation and reduced the effects of miR-503 overexpression on cyclin D1 expression and cell proliferation.ConclusionsDLGAP1-AS2 may regulate miR-503/cyclin D1 to promote cell proliferation in NSCLC.

A 192\xa0bp ERV fragment insertion in the first intron of porcine TLR6 may act as an enhancer associated with the increased expressions of TLR6 and TLR1

BackgroundToll-like receptors (TLRs) play important roles in building innate immune and inducing adaptive immune responses. Associations of the TLR genes polymorphisms with disease susceptibility, which are the basis of molecular breeding for disease resistant animals, have been reported extensively. Retrotransposon insertion polymorphisms (RIPs), as a new type of molecular markers developed recently, have great potential in population genetics and quantitative trait locus mapping. In this study, bioinformatic prediction combined with PCR-based amplification was employed to screen for RIPs in porcine TLR genes. Their population distribution was examined, and for one RIP the impact on gene activity and phenotype was further evaluated.ResultsFive RIPs, located at the 3' flank of TLR3, 5' flank of TLR5, intron 1 of TLR6, intron 1 of TLR7, and 3' flank of TLR8 respectively, were identified. These RIPs were detected in different breeds with an uneven distribution among them. By using the dual luciferase activity assay a 192 bp endogenous retrovirus (ERV) in the intron 1 of TLR6 was shown to act as an enhancer increasing the activities of TLR6 putative promoter and two mini-promoters. Furthermore, real-time quantitative polymerase chain reaction (qPCR) analysis revealed significant association (p < 0.05) of the ERV insertion with increased mRNA expression of TLR6, the neighboring gene TLR1, and genes downstream in the TLR signaling pathway such as MyD88 (Myeloid differentiation factor 88), Rac1 (Rac family small GTPase 1), TIRAP (TIR domain containing adaptor protein), Tollip (Toll interacting protein) as well as the inflammatory factors IL6 (Interleukin 6), IL8 (Interleukin 8), and TNFα (Tumor necrosis factor alpha) in tissues of 30 day-old piglet. In addition, serum IL6 and TNFα concentrations were also significantly upregulated by the ERV insertion (p < 0.05).ConclusionsA total of five RIPs were identified in five different TLR loci. The 192 bp ERV insertion in the first intron of TLR6 was associated with higher expression of TLR6, TLR1, and several genes downstream in the signaling cascade. Thus, the ERV insertion may act as an enhancer affecting regulation of the TLR signaling pathways, and can be potentially applied in breeding of disease resistant animals.

Salicylic Acid-Responsive Factor TcWRKY33 Positively Regulates Taxol Biosynthesis in Taxus chinensis in Direct and Indirect Ways.

Taxol is a rare secondary metabolite that accumulates considerably in Taxus species under salicylic acid (SA) and methyl jasmonate treatment. However, the molecular mechanism of its accumulation remains unclear. We investigated TcWRKY33, a nuclear-localized group I WRKY transcription factor, as an SA-responsive regulator of taxol biosynthesis. Overexpression and RNA interference of TcWRKY33 confirmed that TcWRKY33 regulates the expression of most taxol biosynthesis genes, especially 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) and taxadiene synthase (TASY), which were considered as key enzymes in taxol biosynthesis. Transient overexpression of TcWRKY33 in Taxus chinensis leaves resulted in increased taxol and 10-deacetylbaccatin accumulation by 1.20 and 2.16 times compared with the control, respectively. Furthermore, TcWRKY33, DBAT, and TASY were confirmed to respond positively to SA signals. These results suggested that TcWRKY33 was the missing component of taxol biosynthesis that responds to SA. The sequence analysis identified two W-box motifs in the promoter of DBAT but not in the TASY. Yeast one-hybrid and dual-luciferase activity assays confirmed that TcWRKY33 can bind to the two W-boxes in the promoter of DBAT, upregulating its expression level. Hence, DBAT is a direct target of TcWRKY33. Furthermore, TcERF15, encoding a TASY activator, also contains two W-boxes in its promoter. Yeast one-hybrid and dual-luciferase activity assays further confirmed that TcWRKY33 can upregulate TASY expression through the activation of TcERF15. In summary, TcWRKY33 transmits SA signals and positively regulates taxol biosynthesis genes in two ways: directly and through the activation of other activators. Therefore, TcWRKY33 is an excellent candidate for genetically engineering regulation of taxol biosynthesis in Taxus plants.

Circular RNA hsa_circ_0081343 promotes trophoblast cell migration and invasion and inhibits trophoblast apoptosis by regulating miR-210-5p/DLX3 axis

BackgroundVarious circular RNAs (circRNAs) are dysregulated in the placenta of fetal growth restriction (FGR) fetuses, but their roles and regulatory mechanisms have not been fully elucidated. Herein, we aimed to elucidate the role of hsa_circ_0081343 in regulating the migration, invasion, and apoptosis of human extravillous trophoblast HTR-8 cells.MethodsCircRNA and miRNA levels were examined by quantitative reverse transcription PCR (qRT-PCR). Overexpression plasmid constructs and siRNAs were used to overexpress and knockdown hsa_circ_0081343, respectively. Transwell assays and flow cytometry analyses were performed to evaluate the effects of hsa_circ_0081343 or miR-210-5p on migration, invasion, and apoptosis. Protein levels were analyzed by western blotting. Dual luciferase activity and anti-AGO2 RNA immunoprecipitation (RIP) assays were performed to identify the relationship between miR-210-5p and hsa_circ_0081343.ResultsHsa_circ_0081343 expression was significantly downregulated in 37 FGR placental tissues compared to healthy placental control tissues. Hsa_circ_0081343 overexpression may inhibit apoptosis by downregulating the expression of cleaved caspase 3 and caspase 9 and alleviating the migration and invasion of HTR-8 cells by inducing the expression of MMP2 and MMP9. The dual luciferase activity and anti-AGO2 RIP assay results showed that hsa_circ_0081343 binds to miR-210-5p. miR-210-5p overexpression eliminated the effect of hsa_circ_0081343 overexpression in HTR-8 cells. Finally, DLX3 was identified as a direct target of miR-210-5p.Conclusionshsa_circ_0081343 expression levels are significantly downregulated in FGR placental tissues. Hsa_circ_0081343 regulates the migration, invasion, and apoptosis of HTR-8 cells via the hsa-miR-210-5p/DLX3 axis.

Downregulation of long noncoding RNA SNHG7 protects against inflammation and apoptosis in Parkinson's disease model by targeting the miR-425-5p/TRAF5/NF-κB axis.

Accumulated evidence has manifested that long noncoding RNA (lncRNA) is involved in the progress of Parkinson's disease (PD). SNHG7, a novel lncRNA, has been found to be involved in tumorigenesis. However, SNHG7 expression and its functional effects on PD remain uncharted. Rotenone (Rot) was adopted to construct PD models in Sprague-Dawley (SD) rats and SH-SY5Y cells, respectively. The expression levels of caspase 3, tyrosine hydroxylase (TH), ionized calcium-binding adapter molecule 1 (Iba1) in SD rat striatum were measured via immunohistochemistry and western blot. Additionally, the expressions of inflammatory cytokines (interleukin 1β [IL-1β], IL-6, tumor necrosis factor α) and oxidative stress factors (malondialdehyde, superoxide dismutase, and glutathione peroxidase) in the brain tissues were examined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Moreover, the protein levels of tumor necrosis factor receptor-associated factor (TRAF5), I-κB, nuclear factor-κB (NF-κB), HO-1, Nrf2 were detected via western blot. Bioinformatics was applied to predict the targeting relationship between SNHG7, miR-425-5p, and TRAF5. Dual-luciferase activity assay and RNA immunoprecipitation assays were conductedto verify their interactions. In comparison to healthy donors, SNHG7 was found upregulated while miR-425-5p expression was downregulated in PD patients. Functional experiments confirmed that SNHG7 downregulation or miR-425-5p overexpression attenuated neuronal apoptosis in the Rot-mediated PD model, TH-positive cell loss, and microglial activation by mitigating inflammation and oxidative stress. Mechanistically, SNHG7 served as a competitive endogenous RNA by sponging miR-425-5p and promoted TRAF5 mediated inflammation and oxidative stress. Inhibition of SNHG7 ameliorated neuronal apoptosis in PD through relieving miR-425-5p/TRAF5/NF-κB signaling pathway modulated inflammation and oxidative stress, and similar results were observed in the Rot-mediated rat model of PD.

Circ-RBMS1 Knockdown Alleviates CSE-Induced Apoptosis, Inflammation and Oxidative Stress via Up-Regulating FBXO11 Through miR-197-3p in 16HBE Cells.

BackgroundEmerging evidence has reported that circular RNAs (circRNAs) are aberrantly expressed and act as significant regulators in pathological processes of chronic obstructive pulmonary disease (COPD). Here, the purpose of this article was to evaluate and clarify the biological functions and mechanism of circRNA single stranded interacting protein 1 (circ-RBMS1) in cigarette smoke (CS)-induced COPD.MethodsHuman bronchial epithelial cells 16HBE treated with or without cigarette smoke extract (CSE) were used in the experimental group in vitro. Levels of circ-RBMS1, microRNA (miR)-197-3p, and F-box only protein 11 (FBXO11) were detected using quantitative real-time polymerase chain reaction and Western blot. The present study used cell counting kit-8 (CCK-8), 5-ethynyl-2ʹ-deoxyuridine (EDU), flow cytometry and Western blot assays to determine the survival of 16HBE cells. The activity of interleukin (IL)-1β, tumor necrosis factor (TNF-α), malondialdehyde (MDA) and superoxide dismutase (SOD) was evaluated using the relative commercial kits. Dual-luciferase activity and RIP assays were used to identify the target relationship between miR-197-3p and circ-RBMS1 or FBXO11.ResultsCirc-RBMS1 was highly expressed in COPD patients, and CSE induced an increased expression of circ-RBMS1 in a dose-dependent manner. Functionally, knockdown of circ-RBMS1 attenuated CSE-induced apoptosis, inflammation and oxidative stress in 16HBE cells. Circ-RBMS1 directly targeted miR-197-3p, and miR-197-3p inhibition reversed the effects of circ-RBMS1 knockdown on CSE-induced 16HBE cells. FBXO11 was a target of miR-197-3p. MiR-197-3p overexpression or FBXO11 silencing reduced the apoptosis, inflammation and oxidative stress in CSE-induced 16HBE cells. Moreover, miR-197-3p exerted its effects by targeting FBXO11. Additionally, circ-RBMS1 acted as a sponge for miR-197-3p to positively regulate FBXO11 expression in 16HBE cells.ConclusionCirc-RBMS1 knockdown alleviated CSE-induced apoptosis, inflammation and oxidative stress in 16HBE cells via miR-197-3p/FBXO11 axis, suggesting a new insight into the pathogenesis of CS-induced COPD.

Exosomes miR-22-3p Derived from Mesenchymal Stem Cells Suppress Colorectal Cancer Cell Proliferation and Invasion by Regulating RAP2B and PI3K/AKT Pathway.

Objective Exosomes (exo) which contain proteins, microRNAs (miRNAs), and other bioactive substances can participate in intercellular signal transduction and material transport. Bone marrow mesenchymal stem cells (BMSCs) have a strong ability to produce exosomes. The purpose of this study was to observe the effect of hBMSCs-derived-exo miR-22-3p on proliferation and invasion of colorectal cancer (CRC) cells and to explore its mechanism. Methods miR-22-3p and RAS oncogene family (RAP2B) expression was detected using qRT-PCR or Western blotting. Their interaction was confirmed by dual luciferase activity assay. Effects of miR-22-3p on cell proliferation and invasion were evaluated by CCK-8 and Transwell assay, respectively. Exosomes were extracted by the ultracentrifugation and identified through electron microscopy and Western blotting. Results In CRC tissues and cells, downregulation of miR-22-3p and upregulation of RAP2B were observed. According to the analysis of dual luciferase activity, RAP2B was a target gene of miR-22-3p. In addition, miR-22-3p obviously repressed the cells proliferation and invasion via mediating RAP2B/PI3K/AKT pathway. Coculture experiments indicated that miR-22-3p derived from hBMSCs-exo had inhibition effects on SW480 cell proliferation and invasion. Conclusions Collectively, miR-22-3p from hBMSCs-exo might impede CRC progression, which emphasized the potential of hBMSCs-exo-miR-22-3p as CRC treatment in the future.

CircELK4 Contributes to Lupus Nephritis by Acting as a miR-27b-3p Sponge to Regulate STING/IRF3/IFN-I Signaling.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease and a common complication of SLE is lupus nephritis (LN) during which lupus autoantibodies and proinflammatory cytokines attack the kidney and cause renal dysfunction. The current treatments to LN are limited due to a poor understanding of the pathogenesis. Here, we studied the molecular mechanisms of LN by investigating the function of circELK4/miR-27b-3p axis. MRL/lpr mice and LPS-treated HK-2 cells were used as the mouse model and cell model of LN, respectively. Blood samples were collected from LN patients. qRT-PCR and western blot were used to measure expression levels of circELK4, miR-27b-3p, apoptosis-related proteins, cytokines, and STING/IRF-3/IFN-I signaling. ELISA was performed to examine levels of cytokines including IL-6 and TNF-α. H&E staining was used to examine kidney morphology. TUNEL staining and flow cytometry were used to determine cell apoptosis. Dual luciferase activity assay and RNA pull down were employed to validate the interactions of circELK4/miR-27b-3p and miR-27b-3p/STING. CircELK4 was elevated in LN mice, patients, and LPS-treated HK-2 cells. Knockdown of circELK4 attenuated renal injury in LN mice and LPS-induced HK-2 cell injury. CircELK4 directly bound to miR-27b-3p while miR-27b-3p targeted STING. Moreover, overexpression of circELK4 could partially reverse the effects of miR-27b-3p mimics on cell apoptosis and inflammation. Furthermore, circELK4/miR-27b-3p regulated renal cell damage via modulating STING/IRF3/IFN-I signaling. CircELK4 contributes to renal injury by promoting inflammation and cell apoptosis via acting as a miR-27b-3p sponge to modulate STING/IRF3/IFN-I signaling in LN.

LncRNA TUG1 ameliorates diabetic nephropathy via inhibition of PU.1/RTN1 signaling pathway.

Diabetic nephropathy (DN) is a leading cause of end-stage renal failure. The study aimed to investigate whether long noncoding RNA taurine-upregulated gene 1 (TUG1) can ameliorate the endoplasmic reticulum stress (ERS) and apoptosis of renal tubular epithelial cells in DN, and the underlying mechanism. The DN mouse model was established by streptozocin injection, and the human renal tubular epithelial cell line HK-2 was treated with high glucose (HG) to mimic DN in vitro. The molecular mechanism was explored through dual-luciferase activity assay, RNA pull-down assay, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (CHIP) assay. The expression of TUG1 was significantly decreased in the renal tubules of DN model mice. Overexpression of TUG1 reduced the levels of ERS markers and apoptosis markers by inhibiting reticulon-1 (RTN1) expression in HG-induced HK-2 cells. Furthermore, TUG1 down-regulated RTN1 expression by inhibiting the binding of transcription factor PU.1 to the RTN1 promoter, thereby reducing the levels of ERS markers and apoptosis markers. Meanwhile, TUG1-overexpression adenovirus plasmids injection significantly alleviated tubular lesions, and reduced RTN1 expression, ERS markers and apoptosis markers, whereas these results were reversed by injection of PU.1-overexpression adenovirus plasmids. TUG1 restrains the ERS and apoptosis of renal tubular epithelial cells and ameliorates DN through inhibition of transcription factor PU.1.

LncRNA APOC1P1-3 promoting anoikis-resistance of breast cancer cells

BackgroundAnoikis resistance plays a critical role in the tumor metastasis by allowing survival of cancer cells in the systemic circulation. We previously showed that long non-coding RNAs APOC1P1-3 (lncRNA APOC1P1-3) inhibit apoptosis of breast cancer cells. In this study, we explored its role in anoikis resistance.MethodsWe induced anoikis resistance in two breast cancer cell lines (MCF-7 and MDA-MB-231) under anchorage-independent culture conditions and studied lncRNA APOC1P1-3 effects on apoptosis. Using Dual-Luciferase activity assay, we determined whether it specifically binds to miRNA-188-3P. We further explored its role in lung metastasis by injecting MDA-MB-231 and MDA-MB-231-APOC1P1-3-knock-down cells in female BALB/c nude mice.ResultsWe found that lncRNA APOC1P1-3 suppressed early apoptosis of these cells (demonstrated by gain or loss of their function, respectively) and promoted anoikis resistance via reducing activated- Caspase 3, 8, 9 and PARP. Moreover, it specifically binds to the target miRNA-188-3p acting as a “sponge” to block the inhibition of Bcl-2 (an anti-apoptosis protein).ConclusionsOur study supports a theory that lncRNA APOC1P1-3 can promote development of breast cancer metastasis via anoikis resistance by specifically binding to miRNA-188-3p to block the inhibition of Bcl-2.

Long Non-Coding RNA LINC01929 Accelerates Progression of Oral Squamous Cell Carcinoma by Targeting the miR-137-3p/FOXC1 Axis.

Recently, additional long noncoding RNAs (lncRNAs) have been identified and their possible roles were investigated in a variety of human tumors. One of these lncRNAs, LINC01929, promoted the progression of some cancers, whereas its expression and biological function in human oral squamous cell carcinoma (OSCC) remains still mostly uncertain. The LINC01929 expression in OSCC tissues or cell lines was identified via quantitative real-time polymerase chain reaction. The cell counting kit-8, transwell migration, wound-healing, and flow cytometry assays were utilized to characterize the functions of LINC01929 in OSCC cells. The interactive relationships between LINC01929 and miR-137-3p, miR-137-3p and Forkhead box C1 (FOXC1) were investigated by the dual-luciferase activity assay. Our findings demonstrated that LINC01929 was highly expressed in OSCC tissue samples and cell lines, whereas miR-137-3p expression was downregulated. LINC01929 acted as a carcinogenic lncRNA with accelerated OSCC cell proliferation, migration and invasion, and suppression of apoptosis. We further indicated that LINC01929 facilitated tumor growth in xenograft mouse models. Mechanistically, LINC01929 acted as a sponge for miR-137-3p to elevate FOXC1 expression, which is the target of miR-137-3p. In addition, downregulated miR-137-3p expression rescued the suppressive behaviors of LINC01929 knockdown on the biological behaviors of OSCC cells. Taken together, LINC01929 functioned as a tumor-promoting lncRNA via the miR-137-3p/FOXC1 axis in OSCC, suggesting novel targets for OSCC therapy.

LncRNA MYCNOS promotes glioblastoma cell proliferation by regulating miR-216b/FOXM1 axis.

MYCNOS is an oncogenic lncRNA in liver cancer, but its role in glioblastoma (GBM) is unknown. We predicted that MYCNOS might interact with miR-216b, which targets FOXM1 to perform tumor suppressive roles. This study was performed to analyze the role of MYCNOS in GBM and explore its potential interactions with miR-216b and FOXM1. MYCNOS expression in paired GBM and non-tumor tissues from 62 GBM patients was analyzed by RT-qPCR. The interaction between MYCNOS and miR-216b was predicted by IntaRNA 2.0 and confirmed by dual luciferase activity assay. Overexpression of MYCNOS, miR-216b, and FOXM1 was achieved in GBM cells, followed by performing RT-qPCR and Western blot to explore the relationship among them. CCK-8 assay was performed to explore the role of MYCNOS, miR-216b, and FOXM1 in regulating GBM cell proliferation. MYCNOS was upregulated in GBM tissues compared to the paired non-tumor tissues. MYCNOS is predicted to interact with miR-216b, but overexpression of MYCNOS and miR-216b failed to affect each other's expression significantly. Dual luciferase activity assay showed that MYCNOS and miR-216b could directly interact with each other. MYCNOS overexpression increased the expression of FOXM1, which is a direct target of miR-216b. Cell proliferation assay showed that MYCNOS and FOXM1 overexpression resulted in an increased proliferation rate of GBM cells, while miR-216b overexpression suppressed cell proliferation. Moreover, MYCNOS overexpression suppressed the role of miR-216b. MYCNOS may regulate FOXM1 expression of by serving as an endogenous sponge of miR-216b axis to promote the proliferation of GBM cells.

Identification and functional study of GATA4 gene regulatory variants in type 2 diabetes mellitus

BackgroundType 2 diabetes mellitus (T2D) is a common and complex disease. Dysfunction of pancreatic β cells, which cannot release sufficient insulin, plays a central role in T2D. Genetics plays a critical role in T2D etiology. Transcription factor GATA4 is required for the pancreatic development, and GATA4 gene mutations are implicated in neonatal or childhood-onset diabetes. In this study, we aimed to investigate whether regulatory variants in GATA4 gene may change GATA4 levels, conferring susceptibility to T2D development.MethodsThe promoter region of GATA4 gene was analyzed by targeted sequencing in T2D patients (n = 255) and ethnic-matched controls (n = 371). Dual luciferase activity assay was used for functional study, and EMSA (electrophoretic mobility shift assay) was performed for detecting transcription factor binding.ResultsThirteen regulatory variants including 5 SNPs were identified. A novel heterozygous variant (32124C > T) and one SNP [31487C > G (rs1053351749)] were only identified in T2D. Both regulatory variants significantly affected GATA4 gene promoter activity in cultured HEK-293 and INS-1 cells. Furthermore, the variant (32124C > T) evidently enhanced the binding of unknown transcriptional activator.ConclusionsOur data suggested that GATA4 gene regulatory variants may contribute to T2D development as a rare risk factor.

Overexpression of miR-365a-3p relieves sepsis-induced acute myocardial injury by targeting MyD88/NF-κB pathway.

Sepsis often leads to systemic multiple organ dysfunction, with the majority of deaths attributable to acute myocardial injury (AMI). In this study, we aimed to explore the functional role of miR-365a-3p in sepsis-induced AMI. The sepsis myocardial injury model was constructed using lipopolysaccharide (LPS) both in vitro and in vivo with selective regulation of miR-365a-3p expression. Real-time PCR or Western blot was employed to detect the expressions of miR-365a-3p, inflammatory cytokines (tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6), and inflammation-related proteins (nuclear factor-κB (NF-κB), I-κB, myeloid differentiation factor 88 (MyD88)) in myocardial tissues and cells. Also, cell counting kit-8 (CCK8) and flow cytometry assays were used to measure cardiomyocyte proliferation and apoptosis, respectively. Furthermore, the targeting relationship between miR-365a-3p and MyD88 was verified with the dual luciferase activity assay. miR-365a-3p was downregulated in LPS-induced myocardial injury model. miR-365a-3p overexpression attenuated cardiomyocyte apoptosis and suppressed the expressions of inflammatory cytokines and proteins. Inhibiting miR-365a-3p, however, produced the opposite effects. Mechanistically, miR-365a-3p targeted the 3'-untranslated region of MyD88, thereby inactivating MyD88-mediated NF-κB pathway. miR-365a-3p overexpression mitigated sepsis-mediated myocardial injury by inhibiting MyD88-mediated NF-κB activation.

Novel LncRNA OXCT1-AS1 indicates poor prognosis and contributes to tumorigenesis by regulating miR-195/CDC25A axis in glioblastoma

BackgroundLong noncoding RNAs (lncRNAs) contribute to multiple biological processes in human glioblastoma (GBM). However, identifying a specific lncRNA target remains a challenge. In this study, bioinformatics methods and competing endogenous RNA (ceRNA) network regulatory rules were used to identify GBM-related lncRNAs and revealed that OXCT1 antisense RNA 1 (OXCT1-AS1) is a potential therapeutic target for the treatment of glioma.MethodsBased on the Gene Expression Omnibus (GEO) dataset, we identified differential lncRNAs, microRNAs and mRNAs and constructed an lncRNA-associated ceRNA network.The novel lncRNA OXCT1-AS1 was proposed to function as a ceRNA, and its potential target miRNAs were predicted through the database LncBase Predicted v.2. The expression patterns of OXCT1-AS1 in glioma and normal tissue samples were measured. The effect of OXCT1-AS1 on glioma cells was checked using the Cell Counting Kit 8 assay, cell colony formation assay, Transwell assay and flow cytometry in vitro. The dual-luciferase activity assay was performed to investigate the potential mechanism of the ceRNA network. Finally, orthotopic mouse models of glioma were created to evaluate the influence of OXCT1-AS1 on tumour growth in vivo.ResultsIn this study, it was found that the expression of lncRNA OXCT1-AS1 was upregulated in both The Cancer Genome Atlas (TCGA) GBM patients and GBM tissue samples, and high expression of OXCT1-AS1 predicted a poor prognosis. Suppressing OXCT1-AS1 expression significantly decreased GBM cell proliferation and inhibited cell migration and invasion. We further investigated the potential mechanism and found that OXCT1-AS1 may act as a ceRNA of miR-195 to enhance CDC25A expression and promote glioma cell progression. Finally, knocking down OXCT1-AS1 notably attenuated the severity of glioma in vivo.ConclusionOXCT1-AS1 inhibits glioma progression by regulating the miR-195-5p/CDC25A axis and is a specific tumour marker and a novel potential therapeutic target for glioma treatment.

Ozone oil promotes wound healing via increasing miR-21-5p-mediated inhibition of RASA1.

Skin wound is a very common type of injury and the healing process greatly affects the life quality of individuals. Ozone has been shown beneficial to wound healing with unclear mechanisms. Here, we tested the effect of ozone oil (OZ) on wound healing and investigated the underlying mechanisms. Mouse skin wound model and Masson staining were used to evaluate the effect of OZ on wound healing. Primary fibroblast culture was employed to assess the functions of OZ, miR-21-5p, and RASA1. QRT-PCR and western blot were used to determine expression levels of miR-21-5p, RASA1, α-SMA, and collagen I. CCK-8 assay and scratch wound healing assay were used to measure viability and migration of fibroblasts. Dual luciferase activity assay was performed to validate miR-21-5p/RASA1 interaction. OZ accelerated wound healing in mice and promoted proliferation and migration abilities of fibroblasts. miR-21-5p was increased while RASA1 was reduced during the wound healing and OZ treatment augmented those changes, as well as increased levels of α-SMA and collagen I. Knockdown of miR-21-5p suppressed those effects of OZ on fibroblasts. Furthermore, miR-21-5p directly targeted RASA1 mRNA and negatively regulated its expression. Overexpression of RASA1 inhibited fibroblast proliferation and migration as well as diminished α-SMA and collagen I protein expression. Additionally, RASA1 overexpression blocked the promotion of miR-21-5p overexpression on fibroblast viability and migration. In vivo, miR-21-5p facilitated wound healing while overexpression of RASA1 reversed the effect. OZ promoted wound healing by enhancing miR-21-5p-mediated RASA1 inhibition to increase fibroblast proliferation and migration.

Downregulating lncRNA PVT1 Relieves Astrocyte Overactivation Induced Neuropathic Pain Through Targeting miR-186-5p/CXCL13/CXCR5 Axis.

Spinal cord injury (SCI) is one of the main causes leading to neuropathic pain. Here, we aim to explore the molecular mechanism and function of lncRNA PVT1 in neuropathic pain induced by SCI. The expression of lncRNA PVT1, microRNA (miR) - 186-5p was measured via quantitative reverse transcription PCR (qRT-PCR), and the activation of astrocytes (labeled by GFAP) was detected by immunohistochemistry. Western blot was conducted to detect the expression of chemokine ligand 13 (CXCL13), chemokine receptor 5 (CXCR5), cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP) in spinal cord injury lesions. The levels of inflammatory cytokines (including IL-1β and IL-6) and MDA in tissues were examined via Enzyme-linked immunosorbent assay (ELISA). In vitro experiments were also conducted in primary cultured astrocyte to explore the response of astrocyte to lipopolysaccharide (LPS). What's more, the PVT1-miR-186-5p interaction was verified via the dual luciferase activity assay and RNA immunoprecipitation (RIP) assay. The results demonstrated that the levels of PVT1, CXCL13 and CXCR5 were upregulated, while miR-186-5p were decreased in SCI rats' spinal cord and LPS-mediated astrocytes. In the SCI model, PVT1 depletion significantly alleviated neuropathic pain, astrocytic activation and reduced the expression of neuroinflammatory factors and proteins. The relevant mechanism studies confirmed that PVT1 is a competitive endogenous RNA (ceRNA) of miR-186-5p, targets and inhibits its expression and promotes the expression of CXCL13/CXCR5, while miR-186-5p targets CXCL13. In conclusion, inhibition of lncRNA PVT1 alleviates neuropathic pain in SCI rats by upregulating miR-186-5p and down-regulating CXCL13/CXCR5. The PVT1/miR-186-5p/CXCL13/CXCR5 axis can be used as a new therapeutic target for neuropathic pain.