Direct Target Gene Research Articles

Adipose tissue macrophage-derived exosomal miR-210-5p in modulating insulin sensitivity in rats born small for gestational age with catch-up growth.

Insulin resistance has been implicated in the pathogenesis of children born small for gestational age (SGA) with catch-up growth (CUG). Adipose tissue macrophages (ATMs) regulate insulin resistance by secreting exosomes containing microRNA (miRNA) cargo; however, their pathogenic roles and molecular mechanism are not fully understood. This study aimed to investigate the role of miR-210-5p in rats born SGA with CUG and insulin resistance. The dietary needs of pregnant rats were restricted to ensure the birth of SGA rats. Transmission electron microscopy (TEM) and Western blot analysis were used to identify the exosomes from ATMs of CUG-SGA and adequate-for-gestational-age (AGA) rats. PKH-67 staining was performed to confirm the uptake of exosomes. miR-210-5p expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Glucose uptake and output were detected with glucose uptake and output assays, respectively. Insulin resistance was detected with glucose and insulin tolerance tests in vivo. The interaction between miR-210-5p and SID1 transmembrane family member 2 (SIDT2) was validated with dual-luciferase reporter assay. miR-210-5p was observed to be highly expressed in the exosomes derived from the ATMs of CUG-SGA rats. ATM-derived exosomes can serve as vehicles to deliver miR-210-5p into adipocytes, myocytes, and hepatocytes, where it can enhance cellular insulin resistance. SIDT2 was identified as a direct target gene of miR-210-5p. The miR-210-5p-induced insulin resistance was reversed by the restored SIDT2 expression. However, overexpression of SIDT2 abolished the inhibitory effect of CUG-SGA-ATM-exosomal miR-210-5p on insulin sensitivity in vivo. ATM-derived exosomal miR-210-5p promoted insulin resistance in CUG-SGA rats by targeting SIDT2, which may act as a new potential therapeutic target for children born SGA with CUG.

Hypoxia-induced lncRNA MRVI1-AS1 accelerates hepatocellular carcinoma progression by recruiting RNA-binding protein CELF2 to stabilize SKA1 mRNA

BackgroundLong non-coding RNAs (lncRNAs) perform a vital role during the progression of hepatocellular carcinoma (HCC). Here, we aimed to identify a novel lncRNA involved in HCC development and elucidate the underlying molecular mechanism.MethodsThe RT-qPCR and TCGA dataset analysis were applied to explore the expressions of MRVI1-AS1 in HCC tissues and cell lines. Statistical analysis was applied to analyze the clinical significance of MRVI1-AS1 in HCC. The functions of MRVI1-AS1 in HCC cells metastasis and growth were explored by transwell assays, wound healing assay, MTT assay, EdU assay, the intravenous transplantation tumor model, and the subcutaneous xenograft tumor model. Microarray mRNA expression analysis, dual luciferase assays, and actinomycin D treatment were used to explore the downstream target of MRVI1-AS1 in HCC cells. RIP assay was applied to assess the direct interactions between CELF2 and MRVI1-AS1 or SKA1 mRNA. Rescue experiments were employed to validate the functional effects of MRVI1-AS1, CELF2, and SKA1 on HCC cells.ResultsMRVI1-AS1 was found to be dramatically upregulated in HCC and the expression was strongly linked to tumor size, venous infiltration, TNM stage, as well as HCC patients’ outcome. Cytological experiments and animal experiments showed that MRVI1-AS1 promoted HCC cells metastasis and growth. Furthermore, SKA1 was identified as the downstream targeted mRNA of MRVI1-AS1 in HCC cells, and MRVI1-AS1 increased SKA1 expression by recruiting CELF2 protein to stabilize SKA1 mRNA. In addition, we found that MRVI1-AS1 expression was stimulated by hypoxia through a HIF-1-dependent manner, which meant that MRVI1-AS was a direct downstream target gene of HIF-1 in HCC.ConclusionIn a word, our findings elucidated that hypoxia-induced MRVI1-AS1 promotes metastasis and growth of HCC cells via recruiting CELF2 protein to stabilize SKA1 mRNA, pointing to MRVI1-AS1 as a promising clinical application target for HCC therapy.

Multi-omics analyses reveal the crosstalk between the circadian clock and the response to herbicide application in Oryza sativa.

Plants have evolved circadian clock systems that enable biological processes to occur in tandem with periodic changes in the environment. However, it is largely unknown whether crosstalk occurs between the circadian clock and the response to herbicide in rice. We identified 19 conserved rhythmic metabolites which were response to pesticide application and their metabolic abundance peaked mainly at ZT2 or ZT14-ZT18. We found a series of glyphosate, s-Metolachlor, fenclorim, metcamifen and GA3 response genes were expressed following stable circadian rhythms. In order to determine the patterns of their temporal expression, co-expression network analysis was done on 10,467 genes that were periodically expressed throughout a 24-hour period. Next, we identified 4,031 potential direct target genes of OsCCA1 in using DAP-seq data for OsCCA1. Of these, 339, 22, 53, 53 and 63 genes showed a response to glyphosate, s-Metolachlor, fenclorim, metcamifen and GA3 application, respectively. And they were mainly phased from dusk to midnight. Interestingly, we identified significant OsCCA1 binding peaks in the promoter regions of four herbicide resistance genes, including OsCYP81A12, OsCYP81E22, OsCYP76C2, and OsCYP76C4. Finally, we found that herbicide application could affects the expression of some of the central oscillator genes of the rice circadian clock. Here, we used multi-omics data to reveal the crosstalk between the circadian clock and herbicide response processes at the epigenomics, transcriptome, and metabolome levels in rice. This work will serve as a theoretical guide for identifying rhythmic herbicide targets, leading to the creation of new herbicides or the breeding of crops resistant to herbicides.

The Effect of Hypoxia-inducible Factor Inhibition on the Phenotype of Fibroblasts in Human and Bovine Pulmonary Hypertension.

Hypoxia-inducible factor (HIF) has received much attention as a potential pulmonary hypertension (PH) treatment target because inhibition of HIF reduces the severity of established PH in rodent models. However, the limitations of small-animal models of PH in predicting the therapeutic effects of pharmacologic interventions in humans PH are well known. Therefore, we sought to interrogate the role of HIFs in driving the activated phenotype of PH cells from human and bovine vessels. We first established that pulmonary arteries (PAs) from human and bovine PH lungs exhibit markedly increased expression of direct HIF target genes (CA9, GLUT1, and NDRG1), as well as cytokines/chemokines (CCL2, CSF2, CXCL12, and IL6), growth factors (FGF1, FGF2, PDGFb, and TGFA), and apoptosis-resistance genes (BCL2, BCL2L1, and BIRC5). The expression of the genes found in the intact PAs was determined in endothelial cells, smooth muscle cells, and fibroblasts cultured from the PAs. The data showed that human and bovine pulmonary vascular fibroblasts from patients or animals with PH (termed PH-Fibs) were the cell type that exhibited the highest level and the most significant increases in the expression of cytokines/chemokines and growth factors. In addition, we found that human, but not bovine, PH-Fibs exhibit consistent misregulation of HIFα protein stability, reduced HIF1α protein hydroxylation, and increased expression of HIF target genes even in cells grown under normoxic conditions. However, whereas HIF inhibition reduced the expression of direct HIF target genes, it had no impact on other "persistently activated" genes. Thus, our study indicated that HIF inhibition alone is not sufficient to reverse the persistently activated phenotype of human and bovine PH-Fibs.

Exosomal miR-93-5p regulated the progression of osteoarthritis by targeting ADAMTS9.

Osteoarthritis (OA) is a type of common degenerative joint disorder, in which adipose mesenchymal stem cells (ADSCs) and the secreted exosomes play an important role. The purpose of this study was to investigate the role and mechanism of exosomes derived from ADSCs (ADSC-exos) in OA. The gradient of IL-1β concentration was designed to construct the articular chondrocyte model of arthritic mice. The expression of miR-93-5p and ADAMTS9 in articular chondrocytes was detected by reverse transcription quantitative polymerase chain reaction. Dual luciferase reporter gene assay was performed to verify the interaction between them. Monodansylcadaverine staining was used to visualize the autophagosome formation and cell apoptosis was analyzed by flow cytometry. ADSC-exos were authenticated by transmission electron microscope and western blot assay. miR-93-5p was found to be downregulated in IL-1β-treated articular chondrocytes compared with OA cartilage while ADAMTS9 was upregulated, which was identified as a direct target gene of miR-93-5p. Silencing of ADAMTS9 attenuated the effects of miR-93-5p. Exosomal miR-93-5p can reduce the release of inflammatory factors in mouse arthritis cell models. This study first described the mechanism under that ADSC-exos inhibited inflammation and alleviated OA through the innovative targets miR-93-5p/ADAMTS9 signal axis. This provided a new method for the treatment of OA.

Effect of epigenetic activating of Dlk1-Dio3 imprinted cluster on miR-370 expression due to folate deficiency during nerve development

Proper Dlk1-Dio3 imprinting plays a critical role in embryogenesis, and folic acid deficiency may affect the imprinting of this locus through epigenetic regulation. However, whether and how folic acid directly impacts the imprinting status of Dlk1-Dio3 to affect neural development remain unclear. Here, we found decreased IG-DMR (intergenic -differentially methylated regions) methylation in the folate-deficient encephalocele in humans, suggesting that abnormal Dlk1-Dio3 imprinting status is related to neural tube defects (NTDs) caused by folate deficiency. Similar results were obtained with folate-deficient embryonic stem cells. By miRNA chip analysis, folic acid deficiency led to changes in multiple miRNAs, including the upregulation of 15 miRNAs located in the Dlk1-Dio3 locus. Real-time PCR confirmed that seven of these miRNAs were upregulated, especially miR-370. In contrast to normal embryonic development, in which expression of miR-370 is highest at E9.5, the abnormally high and sustained expression of miRNA-370 in folate-deficient E13.5 embryos may contribute to NTDs. In addition, we found that DNMT3A (de novo DNA methyltransferases 3A) is a direct target gene of miR-370 in neural cells, and DNMT3A participates in the role of miR-370 in inhibiting cell migration. Finally, in the folate-deficient mouse model, Dlk1-Dio3 epigenetic activation was found in fetal brain tissue, along with the upregulation of miR-370 and the downregulation of DNMT3A. Collectively, our findings demonstrate a pivotal role of folate in the epigenetic regulation of Dlk1-Dio3 imprinting during neurogenesis, revealing an elegant mechanism for the activation of Dlk1-Dio3 locus miRNAs in folic acid deficiency.

Deletion of the transcriptional regulator TFAP4 accelerates c-MYC-driven lymphomagenesis

Many lymphoid malignancies arise from deregulated c-MYC expression in cooperation with additional genetic lesions. While many of these cooperative genetic lesions have been discovered and their functions characterised, DNA sequence data of primary patient samples suggest that many more do exist. However, the nature of their contributions to c-MYC driven lymphomagenesis have not yet been investigated. We identified TFAP4 as a potent suppressor of c-MYC driven lymphoma development in a previous genome-wide CRISPR knockout screen in primary cells in vivo [1]. CRISPR deletion of TFAP4 in Eµ-MYC transgenic haematopoietic stem and progenitor cells (HSPCs) and transplantation of these manipulated HSPCs into lethally irradiated animals significantly accelerated c-MYC-driven lymphoma development. Interestingly, TFAP4 deficient Eµ-MYC lymphomas all arose at the pre-B cell stage of B cell development. This observation prompted us to characterise the transcriptional profile of pre-B cells from pre-leukaemic mice transplanted with Eµ-MYC/Cas9 HSPCs that had been transduced with sgRNAs targeting TFAP4. This analysis revealed that TFAP4 deletion reduced expression of several master regulators of B cell differentiation, such as Spi1, SpiB and Pax5, which are direct target genes of both TFAP4 and MYC. We therefore conclude that loss of TFAP4 leads to a block in differentiation during early B cell development, thereby accelerating c-MYC-driven lymphoma development.

Transcriptional Response to Hypoxia: The Role of HIF-1-Associated Co-Regulators.

The Hypoxia Inducible Factor 1 (HIF-1) plays a major role in the cellular response to hypoxia by regulating the expression of many genes involved in adaptive processes that allow cell survival under low oxygen conditions. Adaptation to the hypoxic tumor micro-environment is also critical for cancer cell proliferation and therefore HIF-1 is also considered a valid therapeutical target. Despite the huge progress in understanding regulation of HIF-1 expression and activity by oxygen levels or oncogenic pathways, the way HIF-1 interacts with chromatin and the transcriptional machinery in order to activate its target genes is still a matter of intense investigation. Recent studies have identified several different HIF-1- and chromatin-associated co-regulators that play important roles in the general transcriptional activity of HIF-1, independent of its expression levels, as well as in the selection of binding sites, promoters and target genes, which, however, often depends on cellular context. We review here these co-regulators and examine their effect on the expression of a compilation of well-characterized HIF-1 direct target genes in order to assess the range of their involvement in the transcriptional response to hypoxia. Delineating the mode and the significance of the interaction between HIF-1 and its associated co-regulators may offer new attractive and specific targets for anticancer therapy.

CircOMA1 modulates cabergoline resistance by downregulating ferroptosis in prolactinoma.

Prolactinomas are one of the most common pituitary neuroendocrine tumors (PitNETs), accounting for approximately 50% of all pituitary tumors. Dopamine agonists are the main treatment for prolactinoma, but a small number of patients are still resistant to pharmacotherapy. Recent discoveries have revealed that ferroptosis is involved in regulating tumor drug resistance. However, the role of ferroptosis in prolactinoma has not been reported. In this study, we aimed to explore the mechanism of a circRNA in ferroptosis in prolactinoma. The expression of circOMA1 in prolactinoma tissues was examined by quantitative reverse transcription PCR (qRT-PCR). The biological function of circOMA1 was evaluated in vitro and in vivo. To explore the role of ferroptosis in prolactinoma, we used qRT-PCR and western blotting. Glutamate-cysteine ligase, modifier subunit (GCLM) was predicted to be a direct target gene of miR-145-5p by bioinformatics analysis, which was confirmed by luciferase reporter assays. circOMA1 was overexpressed in drug-resistant prolactinoma tissues compared with sensitive prolactinoma samples. We further found that circOMA1 promoted MMQ cells growth in vivo and in vitro. In addition, GCLM was directly targeted by miR-145-5p and indirectly regulated by circOMA1. Importantly, circOMA1 induced ferroptosis resistance through the increased expression of Nrf2, GPX4, and xCT, and circOMA1 attenuated CAB-induced ferroptosis in MMQ cells in vivo and in vitro. The present study demonstrates that circOMA1 attenuates CAB efficacy through ferroptosis resistance and may be a new therapeutic target for the individualized treatment of DA-resistant prolactinoma patients.

Plexin-B3 expression stimulates MET signaling, breast cancer stem cell specification, and lung metastasis

Intratumoral hypoxia is a microenvironmental feature that promotes breast cancer progression and is associated with cancer mortality. Plexin B3 (PLXNB3) is highly expressed in estrogen receptor-negative breast cancer, but the underlying mechanisms and consequences have not been thoroughly investigated. Here, we report that PLXNB3 expression is increased in response to hypoxia and that PLXNB3 is a direct target gene of hypoxia-inducible factor 1 (HIF-1) in human breast cancer cells. PLXNB3 expression is correlated with HIF-1α immunohistochemistry, breast cancer grade and stage, and patient mortality. Mechanistically, PLXNB3 is required for hypoxia-induced MET/SRC/focal adhesion kinase (FAK) and MET/SRC/STAT3/NANOG signaling as well as hypoxia-induced breast cancer cell migration, invasion, and cancer stem cell specification. PLXNB3 knockdown impairs tumor formation and lung metastasis in orthotopic breast cancer mouse models.

MYC promotes fibroblast osteogenesis by regulating ALP and BMP2 to participate in ectopic ossification of ankylosing spondylitis

BackgroundEctopic ossification is an important cause of disability in patients with ankylosing spondylitis (AS). Whether fibroblasts can transdifferentiate into osteoblasts and contribute to ossification remains unknown. This study aims to investigate the role of stem cell transcription factors (POU5F1, SOX2, KLF4, MYC, etc.) of fibroblasts in ectopic ossification in patients with AS.MethodsPrimary fibroblasts were isolated from the ligaments of patients with AS or osteoarthritis (OA). In an in vitro study, primary fibroblasts were cultured in osteogenic differentiation medium (ODM) to induce ossification. The level of mineralization was assessed by mineralization assay. The mRNA and protein levels of stem cell transcription factors were measured by real-time quantitative PCR (q-PCR) and western blotting. MYC was knocked down by infecting primary fibroblasts with lentivirus. The interactions between stem cell transcription factors and osteogenic genes were analysed by chromatin immunoprecipitation (ChIP). Recombinant human cytokines were added to the osteogenic model in vitro to evaluate their role in ossification.ResultsWe found that MYC was elevated significantly in the process of inducing primary fibroblasts to differentiate into osteoblasts. In addition, the level of MYC was remarkably higher in AS ligaments than in OA ligaments. When MYC was knocked down, the expression of the osteogenic genes alkaline phosphatase (ALP) and bone morphogenic protein 2 (BMP2) was decreased, and the level of mineralization was reduced significantly. In addition, the ALP and BMP2 were confirmed to be the direct target genes of MYC. Furthermore, interferon-γ (IFN-γ), which showed high expression in AS ligaments, was found to promote the expression of MYC in fibroblasts in the process of ossification in vitro.ConclusionsThis study demonstrates the role of MYC in ectopic ossification. MYC may act as the critical bridge that links inflammation with ossification in AS, thus providing new insights into the molecular mechanisms of ectopic ossification in AS.

MicroRNA-142-5p promotes the proliferation and metastasis of nasopharyngeal carcinoma

Growing pieces of evidence reported abnormal expression of microRNA in various cancer. Our research aimed to ascertain the miR-142-5p expression and its potential function in the growth and metastasis of human nasopharyngeal carcinoma (NPC). In human NPC tissues and cell lines, miR-142-5p expression was quantified via the real-time qPCR assay. Functionally, the potential effect of miR-142-5p in human CNE-1 and SUNE-1 cells through MTT assay, colony formation assay, Transwell assay, and cell cycle assay. In addition, the potential target gene of miR-142-5p was determined by the dual-luciferase reporter assay. MiR-142-5p expression was remarkably elevated in human NPC tissues, CNE-1 and SUNE-1 cells. MiR-142-5p overexpression obviously enhanced the ability of cell proliferative and colony formation, and prevented G1 phase arrest in CNE-1 and SUNE-1 cells. Further, the migration number of NPC cells was increased compared to NP69 cells. BTG3 was identified as the direct target gene of miR-142-5p. Inhibition of BTG3 expression could reverse the cell proliferation by miR-142-5p-induced. Overall, miR-142-5p could strengthen the NPC cell’s proliferation and migration by directly targeting BTG3. Hence, miR-142-5p may provide a new strategy and program for future clinical treatment of NPC.

Anti-miR-96 and Hh pathway inhibitor MDB5 synergistically ameliorate alcohol-associated liver injury in mice

Alcohol-associated liver disease (ALD) and its complications are significant health problems worldwide. Several pathways in ALD are influenced by alcohol that drives inflammation, fatty acid metabolism, and fibrosis. Although miR-96 has become a key regulator in several liver diseases, its function in ALD remains unclear. In contrast, sonic hedgehog (SHH) signaling has a well-defined role in liver disease through influencing the activation of hepatic stellate cells (HSCs) and the inducement of liver fibrosis. In this study, we investigated the expression patterns of miR-96 and Hh molecules in mouse and human liver samples. We showed that miR-96 and Shh were upregulated in ethanol-fed mice. Furthermore, alcoholic hepatitis (AH) patient specimens also showed upregulated FOXO3a, TGF-β1, SHH, and GLI2 proteins. We then examined the effects of Hh inhibitor MDB5 and anti-miR-96 on inflammatory and extracellular matrix (ECM)-related genes. We identified FOXO3 and SMAD7 as direct target genes of miR-96. Inhibition of miR-96 decreased the expression of these genes in vitro in AML12 cells, HSC-T6 cells, and in vivo in ALD mice. Furthermore, MDB5 decreased HSCs activation and the expression of ECM-related genes, such as Gli1, Tgf-β1, and collagen. Lipid nanoparticles (LNPs) loaded with the combination of MDB5, and anti-miR-96 ameliorated ALD in mice. Our study demonstrated that this combination therapy could serve as a new therapeutic target for ALD.

The Long Noncoding RNA Cytoskeleton Regulator RNA (CYTOR)/miRNA-24-3p Axis Facilitates Nasopharyngeal Carcinoma Progression by Modulating GAD1 Expression.

Nasopharyngeal carcinoma (NPC) is a head and neck epithelial carcinoma that is unusually prevalent in Southeast Asia. Noncoding RNAs, including lncRNA and miRNA, and their target genes are considered vital regulators of tumorigenesis and the progression of NPC. However, the detailed underlying mechanisms of GAD1 involved in the regulation of NPC need to be further elucidated. In the present study, we identified that GAD1 was significantly upregulated in NPC tissues. GAD1 overexpression is promoted, while genetic knockdown of GAD1 suppresses proliferation, colony formation, migration, and invasion of NPC cells. Bioinformatics analysis and a luciferase reporter assay demonstrated that GAD1 is a direct target gene of miR-24-3p. In NPC tissues, miR-24-3p was downregulated and the lncRNA CYTOR was upregulated. CYTOR was sponged to suppress the function of miR-24-3p. CYTOR regulates GAD1 expression via modulating miR-24-3p. The CYTOR/miR-24-3p/GAD1 axis is converged to modulate the growth, migration, and invasion of NPC cells. In conclusion, the study identified a novel axis for the regulation of NPC cell growth, providing new insights into the understanding of NPC.

MiR-26a-5p alleviates CFA-induced chronic inflammatory hyperalgesia through Wnt5a/CaMKII/NFAT signaling in mice.

Inflammation often leads to the occurrence of chronic pain, and many miRNAs have been shown to play a key role in the development of inflammatory pain. However, whether miR-26a-5p relieves pain induced by inflammation and its possible mechanism are still unclear. The complete Freund's adjuvant (CFA)-induced inflammatory pain mouse model was employed. Intrathecal or subcutaneous injection of miR-26a-5p agomir was performed after modeling to study its antinociceptive effect and the comparison of different administration methods. Bioinformatics analysis of miRNAs was performed to study the downstream mechanisms of miR-26a-5p. HE staining, RT-qPCR, Western blotting, and immunofluorescence were used for further validation. A single intrathecal and subcutaneous injection of miR-26a-5p both reversed mechanical hypersensitivity and thermal latency in the left hind paw of mice with CFA-induced inflammatory pain. HE staining and immunofluorescence studies found that both administrations of miR-26a-5p alleviated inflammation in the periphery and spinal cord. Bioinformatics analysis and dual-luciferase reporter gene analysis identified Wnt5a as a direct downstream target gene of miR-26a-5p. Wnt5a was mainly expressed in neurons and microglia in the spinal cord of mice with inflammatory pain. Intrathecal injection of miR-26a-5p could significantly reduce the expression level of Wnt5a and inhibit the downstream molecules of noncanonical Wnt signaling Camk2/NFAT, inhibiting the release of spinal cord inflammatory factors and alleviating the activation of microglia. In addition, miR-26a-5p could also inhibit lipopolysaccharide (LPS)-stimulated BV2 cell inflammation in vitro through a noncanonical Wnt signaling pathway. miR-26a-5p is a promising therapy for CFA-induced inflammatory pain. Both intrathecal and subcutaneous injections provide relief for inflammatory pain. miR-26a-5p regulated noncanonical Wnt signaling to be involved in analgesia partly through antineuroinflammation, suggesting a pain-alleviating effect via noncanonical Wnt signaling pathway in the CFA-induced inflammatory pain model in vivo.

MiR-29b level-mediated regulation of Klotho methylation via DNMT3A targeting in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by chronic bronchitis and emphysema. Cigarette smoke extract (CSE) is the predominant cause of COPD. This study aimed to investigate the effects of miR-29b and their underlying mechanisms in a COPD cell model. MiR-29b and DNMT3A expression in lung tissue samples (taken at least 5 cm away from the tumor lesion) of NSCLC cases with smoking (n = 30), without smoking (n = 30), and with COPD (with smoking) (n = 30) was researched by qRT-PCR. A medium containing 10 % CSE was employed to induce murine alveolar macrophage MH-S cells to establish COPD cells. 5-Aza-cdr (5-AZA-2′-deoxycytidine) was used to block DNMT3A. The relationship and interaction between miR-29b and DNMT3A were validated through the dual luciferase reporter assay. The expression levels of macrophage M1 polarization marker proteins iNOS and TNF-α, DNMT3A, and Klotho protein were monitored using western blotting. The methylation levels of the miR-29b precursor gene and Klotho promoter were detected by quantitative methylation-specific PCR (MS-qPCR). The levels of IL-1β, IL-6, and TNF-α in cell culture medium were detected via ELISA. It was found that the expression of miR-29b was downregulated, as a result of increased DNA methylation, and that of DNMT3A was upregulated in the lung tissues of NSCLC cases with COPD (with smoking). DNMT3A expression was negatively correlated with miR-29b expression in the lung tissues of NSCLC cases with COPD (with smoking). In addition, miR-29b expression was distinctly downregulated in CSE-induced MH-S cells and inhibited CSE-induced M1 polarization and inflammation. Importantly, DNMT3A was identified as a direct target gene of miR-29b. MiR-29b is negatively regulated by DNMT3A-mediated DNA methylation. Moreover, Klotho expression was downregulated and the Klotho promoter methylation level was increased in lung tissues of NSCLC cases with COPD (with smoking). The negative feedback between miR-29b and DNMT3A modulates CSE-induced M1 polarization and inflammation in macrophages as well as Klotho promoter methylation in CSE-mediated MH-S. Collectively, these findings indicate that the miR-29b level in COPD controls Klotho methylation via DNMT3, which maybe a promising target for the treatment of COPD.

Effect of MiR-424-5p on the Drug Resistance of Diffuse Large B-Cell Lymphoma Cells by Regulating PD-1/PD-L1 Signaling Pathway

To explore the effect of microRNA-424-5p (miR-424-5p) on the drug resistance of diffuse large B-cell lymphoma (DLBCL) cells by regulating the programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling pathway. Human DLBCL cell line CRL2631 cells were induced to construct CRL2631-CHOP resistant cell line. RT-qPCR and Western blot were used to detect the expression levels of MiR-424-5p, PD-L1 mRNA and protein, and multidrug resistance gene-1 (MDR-1) protein in CRL2631 cells and CRL2631-CHOP cells, respectively. The target genes of MiR-424-5p was verified by dual luciferase reporter assay. The miRNA simulation/interference technology and thiazole blue (MTT) method were used to detect the resistance of CRL2631 cells and CRL2631-CHOP cells to CHOP. Compared with CRL2631 cells, the drug resistance of CRL2631-CHOP cells to CHOP and the levels of MDR-1 protein (P<0.05), PD-L1 mRNA and protein in the cells were significantly increased (both P<0.001), while the relative level of MiR-424-5p was significantly reduced (P<0.001). The result of the dual luciferase reporter assay showed that PD-L1 was the direct downstream target gene of MiR-424-5p (P<0.001). After transfection of MiR-424-5p inhibitor, the resistance of CRL2631 cells to CHOP drugs increased, and the expression level of MDR-1 protein (P<0.01), PD-L1 mRNA and protein also increased significantly (both P<0.01). After transfection of MiR-424-5p mimics, the resistance of CRL2631-CHOP cells to CHOP drugs decreased, and the expression level of MDR-1 protein (P<0.001), PD-L1 mRNA and protein also decreased significantly (both P<0.001). Overexpression of PD-L1 could reverse the inhibitory effect of upregulating MiR-424-5p on PD-L1 (P<0.001). Down-regulation of MiR-424-5p enhances the drug resistance of DLBCL cells by regulating the PD-1/PD-L1 signaling pathway.

Transcriptional regulatory mechanism of a variant transcription factor CsWRKY23 in citrus fruit resistance to Penicillium digitatum

As the most productive fruit in the world, there is a lack of research on disease resistance mechanisms in citrus. WRKY transcription factors are mediators of disease resistance in plants. In this research, CsWRKY23, which could respond to exogenous salicylic acid (SA) was identified. As a variant WRKY, the transient overexpression of CsWRKY23 in citrus peel enhanced the resistance to Penicillium digitatum. The potential targets of CsWRKY23 were identified by using DNA affinity purification sequencing (DAP-seq). In combination with RNA sequencing (RNA-seq) data, 48 direct target genes of CsWRKY23 were identified. In addition, five genes for EMSA and dual-luciferase report system analysis were selected and the results showed that CsWRKY23 activate CsAAE12, CsRbohD, CsSARD1, CsWRKY22 and CsIQM6 expressions. Further analysis revealed that CsWRKY23 contributed to SA synthesis pathway, reactive oxygen species (ROS) accumulation, and cell wall strengthening. This study presented a possible regulatory network for disease resistance involving the CsWRKY23.

Lncfos/miR-212-5p/CASP7 Axis-Regulated miR-212-5p Protects the Brain Against Ischemic Damage.

miR-212-5p has been reported to be involved in many biological processes. However, the role of miR-212-5p in ischemic stroke remains unclear. This study explored the biological role and potential mechanism of miR-212-5p in ischemic stroke by investigating the lncfos/miR-212-5p/CASP7 axis. A total of 32 patients with ischemic stroke and 32 age- and sex-matched healthy controls (HCs) were enrolled in this study. In addition, 336 rats were used in this study. The rats were subjected to middle cerebral artery occlusion (MCAO) and intracerebroventricular injection of a microRNA (miRNA) agomir, a miRNA antagomir, a short hairpin RNA (shRNA) lentiviral vector, or a negative control. The neurological deficit score was calculated; the infarct volume was measured; histopathological assays were performed; the neuronal apoptosis rate was determined; and the lncfos, miR-212-5p, and CASP7 expression levels in the peri-infarct area were assessed. In this study, we found that the expression level of miR-212-5p was significantly downregulated in the peri-infarct area and blood of the MCAO model rats and the blood of patients with ischemic stroke. A double-luciferase experiment showed that CASP7 was a direct target gene of miR-212-5p and that miR-212-5p was a target miRNA of lncfos. Lateral ventricular injection of the miR-212-5p agomir effectively inhibited the apoptosis induced by ischemic brain damage, reduced the infarct volume, attenuated the neurological deficit symptoms, and downregulated the expression of CASP7 in the peri-infarct area of the MCAO model rats. Suppressing lncfos with sh-fos led to the upregulated expression of miR-212-5p and played a neuroprotective role in the rat MCAO models. We concluded that miR-212-5p plays a neuroprotective role in ischemic stroke and that its function is regulated by the lncfos/miR-212-5p/CASP7 axis. Moreover, miR-212-5p may be a potential biomarker and therapeutic target for ischemic stroke.

OP31 High fat diet exacerbates colitis through microbial metabolite deoxycholic acids induced-ferroptosis

Abstract Background High-fat diet (HFD) is closely related to the occurrence and development of IBD, which increases the level of deoxycholic acid (DCA). DCA-treated mice showed pro-inflammatory cell infiltration and colonic inflammation. Ferroptosis, a newly recognized form of cell death characterized by iron overload and the generation of reactive oxygen species (ROS) has recently been observed in colitis. Whether DCA affects ferroptosis and whether it contributes to colitis has not been explored. Methods Twenty-four Female C57BL/6 mice were randomly divided into 4 groups: control group, DCA group, DSS group, and DSS plus DCA group. Real-time PCR and Western blotting were used to detect the transcription and protein expression of ferroptosis-related genes. RNA sequencing was used to analyze the changes of differential genes in the intestinal tissue of mice treated with DCA. Transmission electron microscope was employed to observed the changes of intestinal epithelial cell morphology and intracellular mitochondria. Finally, the binding force between DCA and HIF-2α was calculated by molecular dynamics to explore the structural biological basis of the interaction between DCA and HIF-2α, and screen innovative drugs. Results We found that DCA exacerbated the disease activity index, histological damage and cell death in mice with colitis. In vivo, ferroptosis KEGG pathway was enriched by RNA sequencing in colonic tissues. DCA treatment significantly induced ferroptosis in DSS-induced mice, as evidenced by iron overload, ROS and MDA production, accompanied by decreased expression of GPX4 and increased expression of ACSL4. Transmission electron microscopy also confirmed the occurrence of ferroptosis in colonic tissues. In vitro, DCA induced ferroptosis were also observed in IEC-6 cells and Caco-2 cells under the combined treatment of Lipopolysaccharide (LPS). Interestingly, RNA sequencing revealed that the expression levels of the apical iron divalent metal transporter-1 (DMT1) were significantly upregulated in DCA treated-mice with colitis. DMT1 is a direct target gene of hypoxia inducible factor (HIF)-2α. We also found that DCA could increase the level of DMT1 and HIF-2α in the intestinal epithelial cell lines. Pharmacological inhibition of HIF-2α significantly inhibited the expression of DMT1 and blocked the promotion of ferroptosis by DCA. These data provide a mechanistic basis for the widely reported link between ferroptosis and colitis risk. Conclusion Taken together, these findings suggest that DCA aggravates colitis through the induction of ferroptosis in the HIF2α-DMT1 signaling pathway and provides a new perspective for UC prevention in the future.