Luciferase Activity Reporter Assay Research Articles

Targeting MetaLnc9/miR-143/FSCN1 axis inhibits oxidative stress and myofibroblast transdifferentiation in oral submucous fibrosis

Background/purposePersistent activation of myofibroblasts is attributed to various dysregulated biological events conferring multiple types of fibrosis diseases, including oral submucous fibrosis (OSF). Although the significance of non-coding RNAs (ncRNAs) in the occurrence of fibrosis has been appreciated, the detailed mechanisms still have not been fully elucidated. The aim of this study was to identify key dysregulated ncRNAs and elucidate their pro-fibrotic mechanisms in promoting myofibroblast activation and the pathological development of OSF. Materials and methodsExpression of non-coding RNAs and mRNAs in OSF cohort was determined using RNA sequencing and qRT-PCR. The molecular axis of pro-fibrotic ncRNAs were exploited via luciferase reporter activity assay and RNA expression rescue experiments. Functional assays, including collagen gel contraction, wound healing ability, cell migration, and reactive oxygen species (ROS) production, were conducted to assess the changes in the myofibroblastic phenotypes of primary human buccal mucosal fibroblasts. ResultsHerein, we found that long non-coding RNA MetaLnc9 was upregulated in OSF specimens and positively associated with several fibrosis markers. Silencing of MetaLnc9 diminished the features of activated myofibroblasts and the production of ROS. We not only showed that MetaLnc9 functioned as a competitive endogenous RNA of microRNA (miR)-143, but also demonstrated that the pro-fibrosis effect of MetaLnc9 on myofibroblast activities was mediated by suppression of miR-143. Moreover, our data showed that fascin actin-bundling protein 1 (FSCN1) was a direct target of miR-143 and positively related to MetaLnc9. ConclusionUpregulation of MetaLnc9 may enhance the activation of myofibroblasts by sponging miR-143 and titrating its inhibitory property on FSCN1.

Abstract 376: Targeting the convergence on HIF-1α of CDK4/6 and MAPK pathway: Implications for enhanced anticancer strategies

Abstract Hypoxia-inducible factor 1-alpha (HIF-1α) plays a pivotal role in orchestrating cellular responses to hypoxia, influencing cancer cell survival and progression. Our previous work identified a non-canonical mechanism wherein Smurf2 mediates HIF-1α degradation under CDK4/6 inhibition. Through proteomic analysis, we discovered that serine 451 phosphorylation occurs on HIF-1α but not in palbociclib-treated samples. Point mutations at this site, substituting serine with alanine, resulted in decreased HIF-1α levels and enhanced interaction with Smurf2. Intriguingly, under palbociclib treatment, we observed phosphorylation at the serine 643 site. This site has been previously associated with MAPK-dependent regulation of HIF-1α localization and activity (Ilias Mylonis, et al., 2006), particularly relevant given the reported MAPK reliance in acquired CDK4/6 inhibitor-resistant scenarios (Renée de Leeuw, et al., 2018). To explore therapeutic implications, we investigated the impact of combined CDK4/6 (palbociclib) and MEK1/2 inhibition (trametinib, selumetinib, PD98059, U0126). Dual inhibition robustly reduced HIF-1α expression in colorectal cancer cells (HCT116, SW480) and suppressed HIF-1α activity in luciferase reporter assays. This effect extended to synergistic inhibition of cell viability under both normoxia and hypoxia in HCT116 and SW480 cells. Such effect is also applicable to other cancer types and cell lines (e.g. U251). In summary, our findings unveil a phosphorylation site on HIF-1α associated with CDK4/6 activity, influencing its protein stabilization. This discovery supports the rationale for combining CDK4/6 and MEK1/2 inhibition as a promising strategy in the treatment of solid tumors. Citation Format: Shuai Zhao, Lanlan Zhou, Shengliang Zhang, Wafik S. El-Deiry. Targeting the convergence on HIF-1α of CDK4/6 and MAPK pathway: Implications for enhanced anticancer strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 376.

Clinical significance of long non-coding RNA NORAD in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease that may cause joint deformities and seriously affect the normal life of the patients. In order to enable patients to receive timely attention and treatment, this study developed new diagnostic markers by exploring the expression and molecular mechanism of the long non-coding RNA NORAD (NORAD) in RA.MethodsParticipants including 77 RA patients and 52 healthy persons were enrolled, and the corresponding clinical data and serum samples were obtained. The NORAD and miR-204-5p expression were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The content of inflammatory cytokines (IL-6, TNF-α) were determined through enzyme-linked immunosorbent assay (ELISA). Luciferase activity reporter assay demonstrated the association between NORAD and miR-204-5p. In addition, receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficacy of NORAD, and Pearson’s correlation analysis was applied for the correlation analysis.ResultsNORAD was enriched in RA serum with high diagnostic value. Simultaneously, IL-6 and TNF-α levels were also upregulated (P < 0.001). The C-reactive protein (CRP), rheumatoid factor (RF), erythrocyte sedimentation rate (ESR) and anti-cyclic citrullinated peptide antibody (Anti-CCP) levels in RA patients were generally elevated (P < 0.001). NORAD was positively correlated with the levels of clinical indicators and inflammatory factors (P < 0.0001). Mechanistically, NORAD may affect the progression of RA by targeting and negatively regulating miR-204-5p.ConclusionsThere is a correlation between NORAD and the processes of RA, and NORAD has the potential to predict and diagnose the occurrence of RA.

Scinderin Promotes Hydrogen Peroxide-induced Lens Epithelial Cell Injury in Age-related Cataract.

Scinderin (SCIN) is a calcium-dependent protein implicated in cell growth and apoptosis by regulating actin cleavage and capping. In this study, we investigated the role of SCIN in hydrogen peroxide-induced lens epithelial cell (LEC) injury related to age-related cataract (ARC). Anterior lens capsules from ARC patients were collected to examine SCIN expression levels. Immortalized human LEC cell line SRA01/04 and lens capsules freshly isolated from mice were induced by H2O2 to mimic the oxidative stress in ARC. The role of SCIN was investigated by gain-of-function (overexpression) and loss-offunction (knockdown) experiments. Flow cytometry (FCM) and Western-blot (WB) assays were performed to investigate the effect of SCIN on apoptosis. The oxidative stress (OS) was examined by detecting malondialdehyde (MDA) level, superoxide dismutase (SOD) and catalase (CAT) activity. The interaction between SCIN mRNA and miR-489-3p was predicted by StarBase and miRDB databases and validated by luciferase reporter activity assay. SCIN was significantly elevated in cataract samples, and the expression levels were positively correlated with the nuclear sclerosis grades. SCIN overexpression promoted OS and apoptosis in H2O2-induced SRA01/04 cells, while SCIN silencing showed the opposite effect. We further showed that miR-489-3p was a negative regulator of SCIN. miR-489-3p overexpression suppressed apoptosis and OS in H2O2-induced SRA01/04 cells by targeting SCIN. Our study identified SCIN as an upregulated gene in ARC, which is negatively regulated by miR-489-3p. Targeting miR-489-3p/SCIN axis could attenuate OS-induced apoptosis in LECs.

MiR-3653 blocks autophagy to inhibit epithelial-mesenchymal transition in breast cancer cells by targeting the autophagy-regulatory genes ATG12 and AMBRA1.

Metastasis is the main cause of tumor-associated death and mainly responsible for treatment failure of breast cancer. Autophagy accelerates tumor metastasis. In our work, we aimed to investigate the possibility of microRNAs (miRNAs) which participate in the regulation of autophagy to inhibit tumor metastasis. MiRNA array and comprehensive analysis were performed to identify miRNAs which participated in the regulation of autophagy to inhibit tumor metastasis. The expression levels of miR-3653 in breast cancer tissues and cells were detected by quantitative real-time polymerase chain reaction. In vivo and in vitro assays were conducted to determine the function of miR-3653. The target genes of miR-3653 were detected by a dual luciferase reporter activity assay and Western blot. The relationship between miR-3653 and epithelial-mesenchymal transition (EMT) was assessed by Western blot. Student's t-test was used to analyze the difference between any two groups, and the difference among multiple groups was analyzed with one-way analysis of variance and a Bonferroni post hoc test. miR-3653 was downregulated in breast cancer cells with high metastatic ability, and high expression of miR-3653 blocked autophagic flux in breast cancer cells. Clinically, low expression of miR-3653 in breast cancer tissues (0.054 ± 0.013 vs. 0.131 ± 0.028, t = 2.475, P = 0.014) was positively correlated with lymph node metastasis (0.015 ± 0.004 vs. 0.078 ± 0.020, t = 2.319, P = 0.023) and poor prognosis (P < 0.001). miR-3653 ameliorated the malignant phenotypes of breast cancer cells, including proliferation, migration (MDA-MB-231: 0.353 ± 0.013 vs. 1.000 ± 0.038, t = 16.290, P < 0.001; MDA-MB-468: 0.200 ± 0.014 vs. 1.000 ± 0.043, t = 17.530, P < 0.001), invasion (MDA-MB-231: 0.723 ± 0.056 vs. 1.000 ± 0.035, t = 4.223, P = 0.013; MDA-MB-468: 0.222 ± 0.016 vs. 1.000 ± 0.019, t = 31.050, P < 0.001), and colony formation (MDA-MB-231: 0.472 ± 0.022 vs. 1.000 ± 0.022, t = 16.620, P < 0.001; MDA-MB-468: 0.650 ± 0.040 vs. 1.000 ± 0.098, t = 3.297, P = 0.030). The autophagy-associated genes autophagy-related gene 12 (ATG12) and activating molecule in beclin 1-regulated autophagy protein 1 (AMBRA1) are target genes of miR-3653. Further studies showed that miR-3653 inhibited EMT by targeting ATG12 and AMBRA1. Our findings suggested that miR-3653 inhibits the autophagy process by targeting ATG12 and AMBRA1, thereby inhibiting EMT, and provided a new idea and target for the metastasis of breast cancer.

FOXO 1 deletion in chondrocytes rescues diabetes-impaired fracture healing by restoring angiogenesis and reducing apoptosis.

Diabetes mellitus is associated with higher risks of long bone and jaw fractures. It is also associated with a higher incidence of delayed union or non-union. Our previous investigations concluded that a dominant mechanism was the premature loss of cartilage during endochondral bone formation associated with increased osteoclastic activities. We tested the hypothesis that FOXO1 plays a key role in diabetes-impaired angiogenesis and chondrocyte apoptosis. Closed fractures of the femur were induced in mice with lineage-specific FOXO1 deletion in chondrocytes. The control group consisted of mice with the FOXO1 gene present. Mice in the diabetic group were rendered diabetic by multiple streptozotocin injections, while mice in the normoglycemic group received vehicle. Specimens were collected 16 days post fracture. The samples were fixed, decalcified, and embedded in paraffin blocks for immunostaining utilizing anti cleaved caspase-3 or CD31 specific antibodies compared with matched control IgG antibody, and apoptosis by the TUNEL assay. Additionally, ATDC5 chondrocytes were examined in vitro by RT-PCR, luciferase reporter and chromatin immunoprecipitation assays. Diabetic mice had ~ 50% fewer blood vessels compared to normoglycemic mice FOXO1 deletion in diabetic mice partially rescued the low number of blood vessels (p < 0.05). Additionally, diabetes increased caspase-3 positive and apoptotic chondrocytes by 50%. FOXO1 deletion in diabetic animals blocked the increase in both to levels comparable to normoglycemic animals (p < 0.05). High glucose (HG) and high advanced glycation end products (AGE) levels stimulated FOXO1 association with the caspase-3 promoter in vitro, and overexpression of FOXO1 increased caspase-3 promoter activity in luciferase reporter assays. Furthermore, we review previous mechanistic studies demonstrating that tumor necrosis factor (TNF) inhibition reverses impaired angiogenesis and reverses high levels of chondrocyte apoptosis that occur in fracture healing. New results presented here, in combination with recent studies, provide a comprehensive overview of how diabetes, through high glucose levels, AGEs, and increased inflammation, impair the healing process by interfering with angiogenesis and stimulating chondrocyte apoptosis. FOXO1 in diabetic fractures plays a negative role by reducing new blood vessel formation and increasing chondrocyte cell death which is distinct from its role in normal fracture healing.

LncRNA DLEU1 promotes angiogenesis in diabetic foot ulcer wound healing by regulating miR-96-5p.

Diabetic foot ulcer (DFU) carries high rates of major amputation and mortality. The goals of this study were to identify expression of circulating lncRNA DLEU1 and miR-96-5p in patients with diabetic foot ulcer (DFU) and to explore the function of lncRNA DLEU1/miR-96-5p axis in DFU. Matched patients with DFU and healthy individuals were randomly selected. Serum samples from all subjects were used for circulating lncRNA DLEU1 and miR-96-5p assessment by RT-qPCR. Receiver operating characteristic (ROC) curve was plotted to assess the discriminative capacity of lncRNA DLEU1 and miR-96-5p in identifying DFU. Cell proliferation was detected by CCK-8 assay. Cell apoptosis was assayed by Annexin V-FITC/PI staining method. Bioinformatics, luciferase reporter activity assay, and in vitro cell experiments were used to explore the relationship between lncRNA DLEU1 and miR-96-5p. LncRNA DLEU1 and miR-96-5p were significantly up- and downregulated in patients with DFU, respectively, compared with controls. After ROC assessment, lncRNA DLEU1 and miR-96-5p were found to discriminate DFU from miR-96-5p. Furthermore, lncRNA DLEU1 inhibited human umbilical vein endothelial cells (HUVECs) cell proliferation and increased HUVECs apoptosis and oxidative stress through sponging miR-96-5p. Our findings suggest lncRNA DLEU1 and miR-96-5p as circulating biomarkers for DFU. Also, we provide the clue for the pathogenic significance of lncRNA DLEU1/miR-96-5p in DFU, as well as insights for new potential targets.

BLZ8 activates a plastidial peroxiredoxin and a ferredoxin to protect Chlamydomonas reinhardtii against oxidative stress.

Reactive oxygen species (ROS) cause damage to various cellular processes in almost all organisms, in particular photosynthetic organisms that depend on the electron transfer chain for CO2 fixation. However, the detoxifying process to mitigate ROS damage has not been studied intensively in microalgae. Here, we characterized the ROS detoxifying role of a bZIP transcription factor, BLZ8, in Chlamydomonas reinhardtii. To identify downstream targets of BLZ8, we carried out comparative genome-wide transcriptomic profiling of BLZ8 OX and its parental CC-4533 under oxidative stress conditions. Luciferase reporter activity assays and RT-qPCR were performed to test whether BLZ8 regulates downstream genes. We performed an in silico functional gene network analysis and an in vivo immunoprecipitation assay to identify the interaction between downstream targets of BLZ8. Comparative transcriptomic analysis and RT-qPCR revealed that overexpression of BLZ8 increased the expression levels of plastid peroxiredoxin1 (PRX1) and ferredoxin-5 (FDX5) under oxidative stress conditions. BLZ8 alone could activate the transcriptional activity of FDX5 and required bZIP2 to activate transcriptional activity of PRX1. Functional gene network analysis using FDX5 and PRX1 orthologs in A. thaliana suggested that these two genes were functionally associated. Indeed, our immunoprecipitation assay revealed the physical interaction between PRX1 and FDX5. Furthermore, the complemented strain, fdx5 (FDX5), recovered growth retardation of the fdx5 mutant under oxidative stress conditions, indicating that FDX5 contributes to oxidative stress tolerance. These results suggest that BLZ8 activates PRX1 and FDX5 expression, resulting in the detoxification of ROS to confer oxidative stress tolerance in microalgae.

The transcription factor Stat-1 is essential for Schwann cell differentiation, myelination and myelin sheath regeneration

BackgroundMyelin sheath is a crucial accessory to the functional nerve-fiber unit, its disruption or loss can lead to axonal degeneration and subsequent neurodegenerative diseases (NDs). Notwithstanding of substantial progress in possible molecular mechanisms underlying myelination, there is no therapeutics that prevent demyelination in NDs. Therefore, it is crucial to seek for potential intervention targets. Here, we focused on the transcriptional factor, signal transducer and activator of transcription 1 (Stat1), to explore its effects on myelination and its potential as a drug target.MethodsBy analyzing the transcriptome data obtained from Schwann cells (SCs) at different stages of myelination, it was found that Stat1 might be involved in myelination. To test this, we used the following experiments: (1) In vivo, the effect of Stat1 on remyelination was observed in an in vivo myelination mode with Stat1 knockdown in sciatic nerves or specific knockdown in SCs. (2) In vitro, the RNA interference combined with cell proliferation assay, scratch assay, SC aggregate sphere migration assay, and a SC differentiation model, were used to assess the effects of Stat1 on SC proliferation, migration and differentiation. Chromatin immunoprecipitation sequencing (ChIP-Seq), RNA-Seq, ChIP-qPCR and luciferase activity reporter assay were performed to investigate the possible mechanisms of Stat1 regulating myelination.ResultsStat1 is important for myelination. Stat1 knockdown in nerve or in SCs reduces the axonal remyelination in the injured sciatic nerve of rats. Deletion of Stat1 in SCs blocks SC differentiation thereby inhibiting the myelination program. Stat1 interacts with the promoter of Rab11-family interacting protein 1 (Rab11fip1) to initiate SC differentiation.ConclusionOur findings demonstrate that Stat1 regulates SC differentiation to control myelinogenic programs and repair, uncover a novel function of Stat1, providing a candidate molecule for clinical intervention in demyelinating diseases.

Histone deacetylase inhibitor belinostat regulates metabolic reprogramming in killing KRAS-mutant human lung cancer cells.

Kirsten rat sarcoma virus (KRAS) oncogene, found in 20%-25% of lung cancer patients, potentially regulates metabolic reprogramming and redox status during tumorigenesis. Histone deacetylase (HDAC) inhibitors have been investigated for treating KRAS-mutant lung cancer. In the current study, we investigate the effect of HDAC inhibitor (HDACi) belinostat at clinically relevant concentration on nuclear factor erythroid 2-related factor 2 (NRF2) and mitochondrial metabolism for the treatment of KRAS-mutant human lung cancer. LC-MS metabolomic study of belinostat on mitochondrial metabolism was performed in G12C KRAS-mutant H358 non-small cell lung cancer cells. Furthermore, l-methionine (methyl-13 C) isotope tracer was used to explore the effect of belinostat on one-carbon metabolism. Bioinformatic analyses of metabolomic data were performed to identify the pattern of significantly regulated metabolites. To study the effect of belinostat on redox signaling ARE-NRF2 pathway, luciferase reporter activity assay was done in stably transfected HepG2-C8 cells (containing pARE-TI-luciferase construct), followed by qPCR analysis of NRF2 and its target gene in H358 cells, which was further confirmed in G12S KRAS-mutant A549 cells. Metabolomic study reveals significantly altered metabolites related to redox homeostasis, including tricarboxylic acid (TCA) cycle metabolites (citrate, aconitate, fumarate, malate, and α-ketoglutarate); urea cycle metabolites (Arginine, ornithine, argino-succinate, aspartate, and fumarate); and antioxidative glutathione metabolism pathway (GSH/GSSG and NAD/NADH ratio) after belinostat treatment. 13 C stable isotope labeling data indicates potential role of belinostat in creatine biosynthesis via methylation of guanidinoacetate. Moreover, belinostat downregulated the expression of NRF2 and its target gene NAD(P)H:quinone oxidoreductase 1 (NQO1), indicating anticancer effect of belinostat is mediated, potentially via Nrf2-regulated glutathione pathway. Another HDACi panobinostat also showed potential anticancer effect in both H358 and A549 cells via Nrf2 pathway. In summary, belinostat is effective in killing KRAS-mutant human lung cancer cells by regulating mitochondrial metabolism which could be used as biomarkers for preclinical and clinical studies.

MiR-219-5p attenuates cisplatin resistance of ovarian cancer by inactivating Wnt/β-catenin signaling and autophagy via targeting HMGA2.

Our previous study confirmed that miR-219-5p inhibits the progression of ovarian cancer (OC) by targeting high mobility group AT-hook 2 (HMGA2), while the role of miR-219-5p on the chemoresistance of OC is unclear. HMGA2 and miR-219-5p expression in OC tumors and various types of OC cells were determined by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. The miRNA profiles in A2780 and cisplatin-resistant A2780 cells were investigated via bulk miRNA sequencing, and the interactions of miR-219-5p and HMGA2 were determined by luciferase reporter activity assay. Cell function was verified through Cell Counting Kit-8, invasion assay, wound-healing, and TUNEL assays. HMGA2 level is highly expressed in cisplatin-resistant OC cell lines compared to normal OC cells, while the expression trend of miR-219-5p is the opposite. In addition, we found that miR-219-5p is one of the miRNAs that have the most significant reduction in levels in the cisplatin-resistant A2780/DDP cell line compared to A2780 cells. Then, we reveal that miR-219-5p directly targets HMGA2 in cisplatin-resistant OC cells, and upregulation of miR-219-5p significantly reduces the resistance of OC cells to cisplatin both in vitro and in vivo. Finally, our results suggest that Wnt/β-catenin signaling and autophagy pathway is involved in the role of miR-219-5p/HMGA2 on resistance of OC cells to cisplatin via gain-of-function experiments. Collectively, the present study shows that miR-219-5p decreases the resistance of OC cells to cisplatin via Wnt/β-catenin signaling and autophagy by regulating HMGA2, which provides a feasible solution for the resistance of OC to chemotherapy.

Inhibition of miR-146a-5p and miR-8114 in Insulin-Secreting Cells Contributes to the Protection of Melatonin against Stearic Acid-Induced Cellular Senescence by Targeting Mafa.

Chronic exposure to elevated levels of saturated fatty acids results in pancreatic β-cell senescence. However, targets and effective agents for preventing stearic acid-induced β-cell senescence are still lacking. Although melatonin administration can protect β-cells against lipotoxicity through anti-senescence processes, the precise underlying mechanisms still need to be explored. Therefore, we investigated the anti-senescence effect of melatonin on stearic acid-treated mouse β-cells and elucidated the possible role of microRNAs in this process. β-Cell senescence was identified by measuring the expression of senescence-related genes and senescence-associated β-galactosidase staining. Gain- and loss-of-function approaches were used to investigate the involvement of microRNAs in stearic acid-evoked β-cell senescence and dysfunction. Bioinformatics analyses and luciferase reporter activity assays were applied to predict the direct targets of microRNAs. Long-term exposure to a high concentration of stearic acid-induced senescence and upregulated miR-146a-5p and miR- 8114 expression in both mouse islets and β-TC6 cell lines. Melatonin effectively suppressed this process and reduced the levels of these two miRNAs. A remarkable reversibility of stearic acid-induced β-cell senescence and dysfunction was observed after silencing miR-146a-5p and miR-8114. Moreover, V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) was verified as a direct target of miR-146a-5p and miR-8114. Melatonin also significantly ameliorated senescence and dysfunction in miR-146a-5pand miR-8114-transfected β-cells. These data demonstrate that melatonin protects against stearic acid-induced β-cell senescence by inhibiting miR-146a- 5p and miR-8114 and upregulating Mafa expression. This not only provides novel targets for preventing stearic acid-induced β-cell dysfunction, but also points to melatonin as a promising drug to combat type 2 diabetes progression.

MiRNA-124 regulates palmitic acid-induced epithelial-mesenchymal transition and cell migration in human retinal pigment epithelial cells by targeting LIN7C.

The present study revealed that palmitic acid (PA) treatment induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, which are involved in the progression of proliferative vitreoretinopathy (PVR). ARPE-19 cells were treated with PA followed by miRNA screening and EMT marker detection using qRT-PCR. Then, miR-124 mimic or inhibitor was transfected into ARPE-19 cells to explore the role of miR-124 on the EMT of ARPE-19 cells using transwell assay. The underlying mechanism of miRNA were predicted by bioinformatics method and confirmed by luciferase activity reporter assay. Furthermore, gain-of-function strategy was also used to explore the role of LIN7C in the EMT of ARPE-19 cells. The expression of miRNA or mRNA expression was determined by qRT-PCR and the protein expression was determined using western blot assay. The result presented that PA reduced the expression of E-cadherin/ZO-1 whilst increasing the expression of fibronectin/α-SMA. In addition, PA treatment enhanced the expression of microRNA (miR)-124 in ARPE-19 cells. Overexpression of miR-124 enhanced PA-induced upregulation of E-cadherin and ZO-1 expression and downregulation of fibronectin and α-SMA. Moreover, miR-124 mimic also enhanced the migration of ARPE-19 cells induced by PA treatment. Inversely, miR-124 inhibitor presented opposite effect on PA-induced EMT and cell migration in ARPE-19 cells. Luciferase activity reporter assay confirmed that Lin-7 homolog C (LIN7C) was a direct target of miR-124 in ARPE-19 cells. Overexpression of LIN7C was found to suppress the migration ability and expression of fibronectin and α-SMA, while increasing expression of E-cadherin and ZO-1; miR-124 mimic abrogated the inhibitive effect of LIN7C on the EMT of ARPE-19 cells and PA further enhanced this abolishment. Collectively, these findings suggest that miR-124/LIN7C can modulate EMT and cell migration in RPE cells, which may have therapeutic implications in the management of PVR diseases.

Tanshinone IIA protects against chronic obstructive pulmonary disease via exosome‑shuttled miR‑486‑5p

Chronic obstructive pulmonary disease (COPD) is one of the major causes of death worldwide today, and its related morbidity has been predicted to show an increase in subsequent years. Recent studies have shown that Danshen, a Chinese herbal medicine, is a potential drug in the treatment of inflammation-related lung diseases. COPD was induced in this study using cigarette smoke (CS) exposure plus intranasal inhalation of lipopolysaccharide to ascertain whether the main pharmacological component from Danshen, tanshinone IIA (TIIA), and its water soluble form, sodium tanshinone IIA sulfonate (STS), protect against the development of COPD. The weight, lung function, hematoxylin and eosin staining, and Masson Trichrome determinations revealed that TIIA inhalation attenuated lung dysfunction in COPD mice induced by cigarette smoke and lipopolysaccharide exposure. In addition, exosomes derived from TIIA-treated COPD mice exerted similar protective effects against COPD, suggesting that TIIA may protect against COPD through exosome-shuttled signals. miR-486-5p was found to be a key molecule in mediating the protective effects of exosomes derived from TIIA-treated COPD mice using miRNA sequencing and cellular screening. Treatment of COPD mice with an agomiR of miR-486-5p protected lung function in COPD mice, and treatment of COPD mice with an antagomir of miR-486-5p abolished the protective effects of TIIA. Moreover, luciferase activity reporter assay, RT-qPCR, and western blot analyses showed that miR-486-5p exerted protective effects against COPD via targeting phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1). These results suggest that STS protects against COPD through upregulation of miR-486-5p, and that TIIA or miR-486-5p is a potential drug for the treatment of COPD.

Transcriptional Regulation of the Synaptic Vesicle Protein Synaptogyrin-3 (SYNGR3) Gene: The Effects of NURR1 on Its Expression.

Synaptogyrin-3 (SYNGR3) is a synaptic vesicular membrane protein. Amongst four homologues (SYNGR1 to 4), SYNGR1 and 3 are especially abundant in the brain. SYNGR3 interacts with the dopamine transporter (DAT) to facilitate dopamine (DA) uptake and synaptic DA turnover in dopaminergic transmission. Perturbed SYNGR3 expression is observed in Parkinson’s disease (PD). The regulatory elements which affect SYNGR3 expression are unknown. Nuclear-receptor-related-1 protein (NURR1) can regulate dopaminergic neuronal differentiation and maintenance via binding to NGFI-B response elements (NBRE). We explored whether NURR1 can regulate SYNGR3 expression using an in silico analysis of the 5′-flanking region of the human SYNGR3 gene, reporter gene activity and an electrophoretic mobility shift assay (EMSA) of potential cis-acting sites. In silico analysis of two genomic DNA segments (1870 bp 5′-flanking region and 1870 + 159 bp of first exon) revealed one X Core Promoter Element 1 (XCPE1), two SP1, and three potential non-canonical NBRE response elements (ncNBRE) but no CAAT or TATA box. The longer segment exhibited gene promoter activity in luciferase reporter assays. Site-directed mutagenesis of XCPE1 decreased promoter activity in human neuroblastoma SH-SY5Y (↓43.2%) and human embryonic kidney HEK293 cells (↓39.7%). EMSA demonstrated NURR1 binding to these three ncNBRE. Site-directed mutagenesis of these ncNBRE reduced promoter activity by 11–17% in SH-SY5Y (neuronal) but not in HEK293 (non-neuronal) cells. C-DIM12 (Nurr1 activator) increased SYNGR3 protein expression in SH-SY5Y cells and its promoter activity using a real-time luciferase assay. As perturbed vesicular function is a feature of major neurodegenerative diseases, inducing SYNGR3 expression by NURR1 activators may be a potential therapeutic target to attenuate synaptic dysfunction in PD.

LncRNA B4GALT1-AS1 promotes non-small cell lung cancer cell growth via increasing ZEB1 level by sponging miR-144-3p.

BackgroundLong noncoding RNAs (lncRNAs) are emerging as key players in the development and progression of cancer. Several malignancies involve dysregulated long noncoding ribonucleic acids (lncRNAs) in non-small cell lung cancer cell growth and their aggressive phenotypes. LncRNA B4GALT1-AS1 is important in the advancement of various malignancies, although its contribution to non-small cell lung cancer (NSCLC) remains unexplored.MethodsLncRNA B4GALT1-AS1 in NSCLC tissues was detected and further validated in a cohort of non-small cell lung cancer tissues. The effects of lncRNA B4GALT1-AS1 on proliferation were determined by in vitro experiments. The B4GALT1-AS1-miR-144-3p-ZEB1 axis was assessed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Furthermore, the mechanism of B4GALT1-AS1 was investigated using loss-of-function assays in vitro.ResultsWe showed significant upregulation of B4GALT1-AS1 in cell lines and tissues of NSCLC. B4GALT1-AS1 knockdown impeded the in vitro proliferation-related characteristics of the NSCLC cells. The demonstration of the binding capacity of B4GALT1-AS1 and miR-144-3p was predicted by bioinformatics and luciferase reporter activity assay. The B4GALT1-AS1 and miR-144-3p interaction was shown by using rescue experiments. NSCLC has a positive association with its target, zinc finger e-box binding homeobox 1 (ZEB1).ConclusionsIn summary, the progression of NSCLC was facilitated by lncRNA B4GALT1-AS1 via interaction with miR-144-3p and positive regulation of ZEB1 expression.

ZNF191 alters DNA methylation and activates the PI3K-AKT pathway in hepatoma cells via transcriptional regulation of DNMT1.

BackgroundAlteration of DNA methylation is an important event in pathogenesis and progression of hepatocellular carcinoma (HCC). DNA methyltransferase (DNMT) 1, the foremost contributor in DNA methylation machinery, was revealed elevated in HCC and significantly correlates with poor prognosis. However, the transcriptional regulation of DNMT1 in HCC remains unknown.MethodsReal‐time PCR and immunohistochemistry were performed to detect DNMT1 and zinc finger transcription factor 191 (ZNF191) expressions in HCCs. Transcription activity of DNMT1promoter was analyzed with Luciferase reporter activity assay. The binding capacity of ZNF191 protein to DNMT1 promoter was examined with chromatin immunoprecipitation‐qPCR (ChIP‐qPCR) and electrophoretic mobility shift assay (EMSA). DNA methylation level of hepatoma cells was detected with Methylation array.ResultsZNF191 can regulate DNMT1 mRNA and protein expression positively, and increase the transcription activity of the DNMT1 promoter. ChIP‐qPCR and EMSA revealed that ZNF191 protein directly binds to the DNMT1 promoter at nt‐240 AT(TCAT)3TC. Moreover, DNMT1 and ZNF191 expression correlate positively in human HCCs. With methylation array, DNA methylation alteration was observed in hepatoma cells with ZNF191 knockdown, and the differential methylation sites are enriched in the PI3K‐AKT pathway. Furthermore, we proved DNMT1 contributes the effect of ZNF191 on hepatoma cell growth via the PI3K‐AKT pathway.ConclusionZNF191 is a novel transcription regulator for DNMT1, and the pro‐proliferation effect of ZNF191/DNMT1/p‐AKT axis in hepatoma cells implies that ZNF191 status in HCCs may affect the therapeutic effect of DNMTs inhibitors and PI3K inhibitors for precise treatment of the disease.

MicroRNA-17-5p, a novel endothelial cell modulator, controls vascular re-endothelialization and neointimal lesion formation.

The functions of miR-17-5p in tumorigenesis have been explored. However, their functionalities in arterial endothelial cells (ECs) have not been investigated. Besides, the issue of vascular remodelling is barely addressed. The study aimed to determine the effect of overexpression or inhibition of miR-17-5p on arterial endothelial cells' (ECs) function and vascular remodelling in vitro and the rat carotid arteries model. Quantitative RT-PCR analysis was performed to examine the expression of miR-17-5p. Then, gain-of-function and loss-of-function approaches were employed to investigate the functional roles of miR-17-5p in cultured human coronary artery endothelial cells (HCAECs); further, TargetScan software analysis and luciferase reporter activity assay were performed to investigate the potential mechanism. Lastly, the results of the cell segment were verified in a rat carotid artery balloon injury model by Western blot analysis, measurement of the vascular cGMP level and plasma 8-iso-prostaglandin F2 (8-iso-PGF2) testing. Moreover, morphometric analysis was implemented to detect the re-endothelialization and neointimal formation in rat carotid artery after balloon injury. This study firstly found that miR-17-5p expression was upregulated in the injured vascular walls and highly expressive in ECs; overexpression of miR-17-5p inhibited HCAECs' proliferation and migration, whereas miR-17-5p knockdown strengthened its proliferative and migratory roles, influenced inflammatory response, through regulating VEGRA and VEGFR2. It was found that miR-17-5p bind to VEGFA and VEGFR2 at the 3'UTR. Next, downregulation of miR-17-5p promotes re-endothelialization, and attenuates neointimal formation as measured by the I/M ratio (0.63±0.05 vs 1.45±0.06, antagomiR-17-5p vs. Lenti-NC, p < 0.05). In addition, the functional recovery of the endothelium was also accelerated by miR-17-5p knockdown. Our study suggests that miR-17-5p is a feasible strategy for the selective modulation of endothelialization and vascular remodelling through regulating VEGFA and VEGFR2.

MiR-223-3p targets FBXW7 to promote epithelial-mesenchymal transition and metastasis in breast cancer.

BackgroundBreast cancer is the most common malignant tumor diagnosed in women. It is the second leading cause of cancer‐related death among women in the world. Aberrant expression of microRNAs (miRNAs) have been identified to be involved in the development and progression of breast cancer. The aim of this study was to investigate the function of miR‐223‐3p in breast cancer progression and metastasis.MethodsqRT‐PCR was used to analyze the expression levels of miR‐223‐3p in breast cancer tissues and cell lines. Wound healing and Matrigel assays were used to examine cell motility and invasiveness. FBXW7 3′‐UTR construct and luciferase reporter assays were performed for the target gene.ResultsmiR‐223‐3p was overexpressed in breast cancer tissue and cell lines. A high level of miR‐223‐3p was associated with poor prognosis in breast cancer patients. In addition, overexpressed miR‐223‐3p promoted the migration and invasion of breast cancer cells in vitro and in vivo. Mechanistically, we found that tumor suppressor gene FBXW7 is a target of miR‐223‐3p. Luciferase activity reporter assay indicated miR‐223‐3p could directly bind with the 3′‐UTR of FBXW7. miR‐223‐3p exhibited its oncogenic role partly by decreasing FBXW7 expression, and consequently promoted the invasion and metastasis of breast cancer cells.ConclusionsOur study revealed a physical and functional relationship among miR‐223‐3p and FBXW7. By negatively regulating FBXW7 expression, miR‐223‐3p exerts a tumor promotion role promoting cell invasion and metastasis in breast cancer.

Long non-coding RNA NORAD aggravates acute myocardial infarction by promoting fibrosis and apoptosis via miR-577/COBLL1 axis.

Acute myocardial infarction (AMI) is one of the most common and serious cardiovascular diseases. With high morbidity and mortality, AMI has attracted the most attention. Emerging studies indicated that long noncoding RNAs (lncRNAs) play an important role in the progression of AMI. However, the role of NORAD in AMI remained unclear. The current study aimed to investigate the function and mechanism of NORAD in AMI. Bioinformatics tools and a wide range of assays including RT-qPCR, flow cytometry, TTC staining, western blot, luciferase reporter and caspase-3 activity assays were conducted to investigate the function and mechanism of NORAD in AMI. We found out that NORAD was significantly upregulated in AMI rats. Knockdown of NORAD alleviated H9c2 cell injury by reducing apoptosis and decreasing expression levels of fibrogenic factors. In addition, NORAD inhibition ameliorated AMI in a rat model by decreasing infarct size and fibrosis. We confirmed that NORAD bound to miR-577, which was downregulated in ischemia-reperfusion (I/R) rats and hypoxia-exposed H9c2 cells. Additionally, miR-577 combined with the 3'UTR of COBLL1, which was upregulated in I/R rats and hypoxia-exposed H9c2 cells. At last, rescue assay validated that the suppressive effects of NORAD knockdown on apoptosis and expression levels of fibrogenic factors were counteracted by COBLL1 overexpression. Overall, NORAD aggravates acute myocardial infarction by promoting fibrosis and apoptosis via the miR-577/COBLL1 axis. This novel discovery suggested that NORAD may serve as a potential therapeutic target for AMI patients.