Dual-luciferase Reporter Assay Research Articles

Circ_0000972 Inhibits Hepatocellular Carcinoma Cell Stemness by Targeting miR-96-5p/PFN1.

Previous study has identified circRNAs as an important factor in cancer stem cells (CSCs) progression, which contributes to tumor initiation and progression. This study aspired to uncover the mechanisms of circ_0000972 on hepatocellular carcinoma (HCC) CSCs. RT-qPCR was utilized to quantify circ_0000972, miR-96-5p, and profilin 1(PFN1) expression in HCC tissues and cells. To evaluate the in vivo functions of circ_0000972, HCC cells with circ_0000972 overexpression were utilized to establish xenograft model through subcutaneous injection. The cell colony and sphere formation assays were adopted to evaluate the impact of circ_0000972 on the stemness characteristics of HCC cells. Additionally, the interaction between circ_0000972, miR-96-5p, and PFN1 was determined through bioinformatics analysis, dual-luciferase reporter assays, and rescue experiments. Circ_0000972 and PFN1 expression was significantly downregulated in HCC tissues and cells, while miR-96-5p exhibited an increased expression level. The overexpression of circ_0000972 was observed to inhibit the cell colony, sphere formation, and EMT of HCC CSCs. In xenograft model, circ_0000972 overexpression restrained the tumor volume and weight. Mechanistically, circ_0000972 stimulated PFN1 expression through the inhibition of miR-96-5p. More importantly, circ_0000972 overexpression could promote PFN1 expression and inhibit the stemness of HCC CSCs. Interestingly, the effect of circ_0000972 overexpression on such progresses was reversed by PFN1 silencing. This study elucidates that circ_0000972, an antitumor factor, sponges miR-96-5p to inhibit oncogenic cellular process in HCC by mediating PFN1 expression.

MiR-148b Caused Liver Injury in Rats with Traumatic Hemorrhagic Shock by Inhibiting SIRT6 Expression.

The purpose of this study was to investigate the role of miR- 148b in liver injury in rats with traumatic hemorrhagic shock (THS) and to elucidate its potential mechanism. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of rats were detected by enzyme-linked immune sorbent assay (ELISA), and the injury of rat liver was analyzed by hematoxylin-eosin (H&E) staining. Apoptosis of rat hepatocytes and normal rat liver cell line (BRL3A) was identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and flow cytometry, respectively. MiR-148b and sirtuin 6 (SIRT6) expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Lactate dehydrogenase (LDH) content and cell viability were measured by commercial kits and cell counting kit-8 (CCK-8) assay, respectively. The binding sites of miR-148b and SIRT6 were predicted by the Starbase database and verified by dual luciferase reporter assay. MiR-148b expression in THS rats or ischemia-reperfusion (I/R)-treated cells was higher than in the control group. Overexpression of miR-148b further promoted the effects of I/R, which enhanced the levels of ALT, AST and LDH, cell apoptosis of liver tissue or BRL3A cells and decreased the expression of SITR6. Besides, miR-148b negatively correlated with SIRT6, and upregulated the expression of SIRT6 could partly reverse the effect of miR-148b. Hepatocyte injury induced by I/R was achieved by regulating miR-148b /SIRT6 axis.

Cannabiorcol as a novel inhibitor of the p38/MSK-1/NF-κB signaling pathway, reducing matrix metalloproteinases in osteoarthritis

BackgroundThe bioactivity and potential medicinal applications of cannabiorcol, a lesser-known derivative of Cannabis sativa, require further investigation. Osteoarthritis (OA) is a chronic joint condition marked by gradual degradation of the cartilage and commonly associated with elevated levels of matrix metalloproteinases (MMPs). However, the influence of cannabiorcol on OA and its underlying mechanisms remains unclear. MethodsIn silico analysis investigated the key transcription factors that regulate MMP expression. A chondrocyte cell model [interleukin (IL)-1β and IL-1⍺-treated C20A4 cell line] was established and treated with cannabiorcol. Associated cytotoxicity was assessed using a WST-8 assay. A monoiodoacetate-induced OA rat model was established and treated with cannabiorcol. Protein translocation and transactivation analyses were conducted using immunofluorescence and dual-luciferase reporter assays, respectively. Western blotting and real-time PCR analyzed relevant markers to examine cannabiorcol's effects on OA and its fundamental mechanisms. ResultsCannabiorcol inhibits the expression of IL-1β-induced MMPs compared to other cannabis-related compounds. In silico analysis revealed that the nuclear factor-kappa β (NF-κβ) and mitogen-activated protein kinase (MAPK) pathways are associated with MMP expression as key regulators. In vitro, cannabiorcol inhibits the NF-κB and p38 MAPK pathways independently cannabinoid receptors and transient receptor potential vanilloids. In vivo, cannabiorcol reduces MMP expression and ameliorates monoiodoacetate-induced OA traits in rats. ConclusionCannabiorcol inhibits IL-1β-induced MMP expression in vitro and alleviates OA in an MIA-induced OA rat model by reducing MMP expression and inhibiting the p65/p38 axis.

Genome-wide identification of the bHLH transcription factor family and the regulatory roles of PbbHLH74 in response to drought stress in Phoebe bournei.

Phoebe species constitute a large portion of subtropical forestry, which are key players in biomass resources. However, abiotic stresses such as drought stress severely limit the growth and development of P. bournei, and even lead to its death. It has been shown that basic helix-loop-helix (bHLH) as the second largest transcription factor family plays essential roles in response to multiple stresses in plants. However, little information of bHLH family is available in P. bournei. In this study, 130 PbbHLHs were identified and classified into 24 subfamilies. Then, the bHLH domain, conserved motifs and gene structures, evolutionary patterns and protein structural features were probed. The expression levels of 17 PbbHLHs were differentially induced by PEG and ABA by RT-qPCR analysis, indicating that they may be involved in drought stress response. Characterization of the drought candidate gene PbbHLH74 showed that it was transcriptionally active and localized in the nucleus. Heterologous transformation of PbbHLH74 into yeast improved cellular tolerance to drought stress. Meanwhile, overexpression of PbbHLH74 in Arabidopsis showed higher seed germination, plant biomass and expression levels of stress-related genes under drought conditions. Through the hairy root technique, overexpression of PbbHLH74 in P. bournei improved drought tolerance by enhancing root development and expression levels of genes involved in ABA-dependent and ROS scavenging pathways. Moreover, PbbHLH74 might positively regulate the expression of PbPOD by Y1H and dual-luciferase reporter assays. Overall, these results elucidated the structure and evolution of the PbbHLH family, in which PbbHLH74 could be applied to molecular assisted breeding for drought tolerance in P. bournei.

CircPWWP2A promotes renal interstitial fibrosis through modulating miR-182/ROCK1 axis.

Renal fibrosis is a long-term and progressively worsening condition that impacts kidney function during aging and in the context of chronic kidney disease (CKD). CKD and renal fibrosis affect approximately 10% of the global population and are prevalent in about half of individuals over the age of 70. Despite ongoing research, the mechanisms underlying renal fibrosis are still not well understood, and there is currently a lack of effective treatments available. In the present study, we demonstrated a significant increase of circPWWP2A in renal tubular cells both in vivo and in vitro models of renal fibrosis. Suppressing circPWWP2A has the potential to reduce mitochondrial dysfunction and the production of mitochondrial reactive oxygen species (mtROS), ultimately leading to the inhibition of renal fibrosis. Whereas, supplementation of circPWWP2A led to more serve mitochondrial dysfunction, mtROS production and renal fibrosis. Mechanistically, we found the expression of circPWWP2A was negatively correlated with the expression of miR-182. And we further confirmed miR-182 was the direct target of circPWWP2A by dual-luciferase reporter assay and RIP assay. Then, we found miR-182 suppressed the expression of ROCK1 in both in vitro and in vivo models of renal fibrosis. Luciferase microRNA target reporter assay further indicated ROCK1 as a direct target of miR-182. Knockdown of ROCK1 inhibits renal fibrosis and mitochondrial dysfunction, suggesting ROCK1 not only served as an injurious role in mitochondrial homeostasis but also a pro-fibrotic factor in CKD. Taking together, our findings suggest that circPWWP2A may promote renal interstitial fibrosis by modulating miR-182/ROCK1-mediated mitochondrial dysfunction.

Application of hydrogen-rich water maintains red pitaya fruit quality through regulation of ROS and energy metabolism

Hydrogen-rich water (HRW) treatment has been shown to enhance the shelf life of fruit and vegetables. This study aimed to investigate the effects of HRW on improving the quality of red pitaya and its underlying regulatory mechanisms. The results revealed that applying HRW at a 70% concentration reduced decay and weight loss rates, slowed the respiratory rate, and delayed the decline in titratable acidity and total soluble solids. Additionally, HRW treatment mitigated the increase in superoxide anion production and hydrogen peroxide levels, while enhancing antioxidant enzyme activities and gene expression, including those of peroxidase, superoxide dismutase, glutathione peroxidase, catalase, ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase. Non-enzymatic antioxidant compounds such as ascorbate and glutathione also increased. Moreover, RT-qPCR analysis showed that HRW treatment upregulated HcMYB6 and downregulated HcR2R3MYB. Dual-luciferase reporter assays indicated that HcMYB6 activated the expression of HcSOD and HcCAT, whereas HcR2R3MYB repressed their expression. These findings suggest that HcMYB6 and HcR2R3MYB may be involved in reactive oxygen species metabolism, with HcMYB6 being induced and HcR2R3MYB inhibited by HRW treatment. Furthermore, HRW treatment led to increased adenosine triphosphate and adenosine diphosphate levels, a decrease in adenosine monophosphate content, and elevated gene expression and activity of Ca2⁺-ATPase, succinate hydrogenase, H⁺-ATPase, and cytochrome C. Collectively, these results demonstrate that HRW treatment effectively reduces oxidative damage and improves energy status, thereby preserving the quality of red pitaya fruit.

Circular RNA circ_0004630 promotes malignancy and glycolysis of nonsmall cell lung cancer by sponging microRNA-1208 and regulating leucine-rich repeat kinase 2 expression.

Emerging evidence has discovered that circular RNAs play important regulators of nonsmall cell lung cancer (NSCLC), but the role and potential molecular mechanism of hsa_circ_100549 (circ_0004630) involved in NSCLC is poorly defined. In this study, circ_0004630, microRNA-1208 (miR-1208), and leucine-rich repeat kinase 2 (LRRK2) expression were detected using real-time quantitative polymerase chain reaction. Cell proliferation, colony formation, apoptosis, and invasion were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine, colony formation, flow cytometry, and transwell assays. Protein levels of glucose transporter 1, Hexokinase 2, and LRRK2 were detected using western blot assay. Glucose consumption, lactate production, and adenosine triphosphatecontent were assessed using the corresponding kits. After predicting via bioinformatics software Circinteractome and Targetscan, the binding between miR-1208 and circ_0004630 or LRRK2 was verified by a dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assay. The xenograft tumor model analyzed the biological role circ_000460 on tumor growth in vivo. It was found that circ_0004630 and LRRK2 were increased, and miR-1208 was low expression in NSCLC tissues and cells. Functionally, the downregulation of circ_0004630 inhibited NSCLC cell proliferation, invasion, glycolysis, and accelerated apoptosis in vitro. In mechanism, circ_0004630 might work as a sponge of miR-1208 to modulate LRRK2 expression. In addition, DUXAP8 deficiency cured neuroblastoma tumor growth in vivo. In conclusion, circ_0004630 knockdown might suppress NSCLC cell proliferation, metastasis, and glycolysis partly by the miR-1208/LRRK2 axis. Our findings hinted at an important theoretical basis for further elucidating the pathogenesis of NSCLC and targeted therapy.

FAM83H regulated by glis3 promotes triple-negative breast cancer tumorigenesis and activates the NF-κB signaling pathway.

Triple-negative breast cancer (TNBC) is a highly aggressive and invasive form of breast cancer (BC) with a high mortality rate and a lack of effective targeted drugs. Family with sequence similarity 83 member H (FAM83H) is critically implicated in tumorigenesis. However, the potential role of FAM83H in TNBC remains elusive. Here, we discovered that FAM83H exhibited high expression in tumor tissues of patients with TNBC and was associated with TNM stage. Gain- or loss-of-function experiments were conducted to explore the biological role of FAM83H in TNBC. Subsequently, functional enrichment analysis confirmed that FAM83H overexpression promoted TNBC cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT), accompanied by upregulation of cyclin E, cyclin D, Vimentin, N-cadherin and Slug. As observed, FAM83H knockdown showed anti-cancer effects, such as fostering apoptosis and inhibiting tumorigenicity and metastasis of TNBC cells. Mechanistically, FAM83H activated the NF-κB signaling pathway. Moreover, a dual-luciferase reporter assay demonstrated that GLIS family zinc finger 3 (GLIS3) bound to the promoter of FAM83H and enhanced its transcription. Notably, overexpression of GLIS3 significantly stimulated TNBC cell proliferation and invasion, and all of this was reversed by rescue experiments involving the knockdown of FAM83H. Overall, FAM83H exacerbates tumor progression, and in-depth understanding of FAM83H as a therapeutic target for TNBC will provide clinical translational potential for intervention therapy.

Molecular mechanism of METTL14-mediated m6A modification regulating microglial function post ischemic stroke

This study explores the molecular mechanism of METTL14 regulating microglial function post ischemic stroke. A murine model was established by tMCAO. The neurological function was evaluated by mNSS. The cerebral infarct size and pathological changes were observed by TTC and H&E staining. M1 and M2 microglia in brain tissues were detected by flow cytometry. BV2 cells were subjected to OGD/R to establish an in vitro model. qRT-PCR and Western blot were used for detecting METTL14, PAX6, YTHDF2, TREM2, iNOS, and Arg1 expressions. The m6A level was quantitatively analyzed, and the binding of YTHDF2 or m6A to PAX6 was analyzed by RIP. PAX6 mRNA stability was assessed after actinomycin D treatment. ChIP was utilized for determining the enrichment of PAX6 on TREM2 promoter. The binding relationship between TREM2 and PAX6 was verified by dual-luciferase reporter assay. METTL14 was highly expressed after tMCAO, and silence of METTL14 alleviated symptoms of tmcao mice and promoted microglial M2 polarization. METTL14 enhanced PAX6 mRNA m6A modification to promote YTHDF2 binding to PAX6 mRNA and its degradation. PAX6 bound to TREM2 promoter and facilitated its transcription and expression. In conclusion, METTL14-mediated m6A modification aggravates ischemic stroke by promoting microglial M1 polarization via YTHDF2/PAX6/TREM2 axis.

HNF-1β alleviates podocyte injury in lupus nephritis by maintaining endoplasmic reticulum homeostasis

ObjectiveThe current study aims to elucidate the critical function of hepatocyte nuclear factor 1-beta (HNF1-β) in lupus nephritis (LN) by investigating its modulation of the Derlin-1/valosin-containing protein (VCP)/VCP-interacting membrane selenoprotein...

Effects of miR-214-5p and miR-21-5p in hypoxic endometrial epithelial-cell-derived exosomes on human umbilical cord mesenchymal stem cells

BACKGROUND Thin endometrium seriously affects endometrial receptivity, resulting in a significant reduction in embryo implantation, and clinical pregnancy and live birth rates, and there is no gold standard for treatment. The main pathophysiological characteristics of thin endometrium are increased uterine arterial blood flow resistance, angiodysplasia, slow growth of the glandular epithelium, and low expression of vascular endothelial growth factor, resulting in endometrial epithelial cell (EEC) hypoxia and endometrial tissue aplasia. Human umbilical cord mesenchymal stem cells (HucMSCs) promote repair and regeneration of damaged endometrium by secreting microRNA (miRNA)-carrying exosomes. However, the initiation mechanism of HucMSCs to repair thin endometrium has not yet been clarified. AIM To determine the role of hypoxic-EEC-derived exosomes in function of HucMSCs and explore the potential mechanism. METHODS Exosomes were isolated from normal EECs (EEC-exs) and hypoxia-damaged EECs (EECD-exs), before characterization using Western blotting, nanoparticle-tracking analysis, and transmission electron microscopy. HucMSCs were cocultured with EEC-exs or EECD-exs and differentially expressed miRNAs were determined using sequencing. MiR-21-5p or miR-214-5p inhibitors or miR-21-3p or miR-214-5p mimics were transfected into HucMSCs and treated with a signal transducer and activator of transcription 3 (STAT3) activator or STAT3 inhibitor. HucMSC migration was assessed by Transwell and wound healing assays. Differentiation of HucMSCs into EECs was assessed by detecting markers of stromal lineage (Vimentin and CD13) and epithelial cell lineage (CK19 and CD9) using Western blotting and immunofluorescence. The binding of the miRNAs to potential targets was validated by dual-luciferase reporter assay. RESULTS MiR-21-5p and miR-214-5p were lowly expressed in EECD-ex-pretreated HucMSCs. MiR-214-5p and miR-21-5p inhibitors facilitated the migratory and differentiative potentials of HucMSCs. MiR-21-5p and miR-214-5p targeted STAT3 and protein inhibitor of activated STAT3, respectively, and negatively regulated phospho-STAT3. MiR-21-5p- and miR-214-5p-inhibitor-induced promotive effects on HucMSC function were reversed by STAT3 inhibition. MiR-21-5p and miR-214-5p overexpression repressed HucMSC migration and differentiation, while STAT3 activation reversed these effects. CONCLUSION Low expression of miR-21-5p/miR-214-5p in hypoxic-EEC-derived exosomes promotes migration and differentiation of HucMSCs into EECs via STAT3 signaling. Exosomal miR-214-5p/miR-21-5p may function as valuable targets for thin endometrium.

ATF3 regulates CDC42 transcription and influences cytoskeleton remodeling, thus inhibiting the proliferation, migration and invasion of malignant skin melanoma cells.

Cutaneous malignant melanoma (CMM) is one of the most aggressive and lethal types of skin cancer. Cytoskeletal remodeling is a key factor in the progression of CMM. Previous research has shown that activating transcription factor 3 (ATF3) inhibits metastasis in bladder cancer by regulating actin cytoskeleton remodeling through gelsolin. However, whether ATF3 plays a similar role in cytoskeletal remodeling in CMM cells remains unknown. Various gene and protein expression analyses were performed using techniques such as reverse transcription quantitative PCR, western blot, immunofluorescent staining, and immunohistochemical staining. CMM viability, migration, and invasion were examined through cell counting kit-8 and transwell assays. The interactions between cell division cycle 42 (CDC42) and ATF3 were investigated using chromatin immunoprecipitation and dual-luciferase reporter assays. CDC42 was upregulated in CMM tissues and cells. Cytoskeletal remodeling of CMM cells, as well as CMM cell proliferation, migration, and invasion, were inhibited by CDC42 or ATF3. ATF3 targeted the CDC42 promoter region to regulate its transcriptional activity. ATF3 suppresses cytoskeletal remodeling in CMM cells, thereby inhibiting CMM progression and metastasis through CDC42. This research may provide a foundation for using ATF3 as a therapeutic target for CMM.

Endothelial FOSL1 drives angiotensin II-induced myocardial injury via AT1R-upregulated MYH9.

Vascular remodeling represents a pathological basis for myocardial pathologies, including myocardial hypertrophy and myocardial infarction, which can ultimately lead to heart failure. The molecular mechanism of angiotensin II (Ang II)-induced vascular remodeling following myocardial infarction reperfusion is complex and not yet fully understood. In this study, we examined the effect of Ang II infusion on cardiac vascular remodeling in mice. Single-cell sequencing showed Ang IIinduced cytoskeletal pathway enrichment and that FOS like-1 (FOSL1) affected mouse cardiac endothelial dysfunction by pseudotime analysis. Myosin heavy chain 9 (MYH9) was predominantly expressed in primary cardiac endothelial cells. The Ang II type I receptor blocker telmisartan and the protein kinase C inhibitor staurosporine suppressed Ang II-induced upregulation of MYH9 and FOSL1 phosphorylation in human umbilical vein endothelial cells. Silencing MYH9 abolished Ang II-mediated inhibition of angiogenesis in human umbilical vein endothelial cells, and attenuated AngII-induced vascular hyperpermeability. We found that FOSL1 directly bound to the MYH9 promoter and thus activated transcription of MYH9 by the dual luciferase reporter and chromatin immunoprecipitation assays, leading to vascular dysfunction. In vivo, 6 weeks after injecting adeno-associated virus-ENT carrying the TEK tyrosine kinase (tie) promoter-driven short hairpin RNA for silencing FOSL1 (AAV-tie-shFOSL1), cardiac function represented by the ejection fraction and fractional shortening was improved, myocardial fibrosis was decreased, protein levels of phosphorylated FOSL1, MYH9, and collagen type I alpha were reduced, and cardiac vascular density was recovered in mice with endothelial Fosl1-specific knockdown in Ang II-infused mice. In ischemia-reperfusion mice, AAV-shFosl1 mice had a reduced infarct size and preserved cardiac function compared with control AAV mice. Our findings suggest a critical role of the FOSL1/MYH9 axis in hindering Ang II-induced vascular remodeling, and we identified FOSL1 as a potential therapeutic target in endothelial cell injuries induced by myocardial ischemia-reperfusion.

Transcription factor AP-4 reactivates telomerase reverse transcriptase independently of genome alteration in non-HBV associated hepatocellular carcinoma

Recurrent genome alterations have been identified in the telomerase reverse transcriptase (TERT) promoter region, a common occurrence in various types of cancer. In addition to epigenetic alterations at the TERT promoter region, the recruitment of transcription factors to this region, which potentially drives the reactivation of TERT gene transcription in human hepatocellular carcinoma (HCC) remains unknown. In this study, using in-silico DNA binding sequence analysis at the TERT promoter and with confirmation with dual luciferase reporter and chromatin immunoprecipitation assays, we found that Transcription Factor AP-4 (TFAP4) physically interacted with the TERT promoter, driving telomerase reactivation. Furthermore, TFAP4 mRNA is upregulated in human HCCs and positively correlates with TERT mRNA expression, according to TCGA-LIHC database, our in-house clinical samples, and HCC cell lines. Interestingly, the knockdown of TFAP4 only suppressed TERT expression levels and telomere length in HCC cells which are not associated with HBV infection. Additionally, we identified that the CCCTC-binding factor (CTCF) physically interacted with the TFAP4 promoter, leading to induced TFAP4 gene transcription in HCC. Clinically, TFAP4 mRNA expression significantly correlated with TERT mRNA expression in alcohol-related HCCs, but not in HBV-associated HCCs, according to TCGA-LIHC cohort. Consistently, ethanol enhanced both TFAP4 and TERT mRNA expression in non-HBV-associated HCC cells but not HBV-associated HCC cells. In conclusion, our findings demonstrate that TFAP4 directly regulates TERT gene transcription via CTCF in non-HBV-associated HCCs. However, its role in regulating telomerase expression or activity through HBV DNA integration in HBV-associated cells might be limited.

Forkhead box P1 transcriptionally activates IGF-1 to lighten ox-LDL-induced endothelial cellular senescence by inactivating NLRP3 inflammasome.

Endothelial cell (EC) senescence is a major contributor in atherosclerosis (AS) development. Herein, the role of forkhead box P transcription factor 1 (FOXP1) and insulin-like growth factor (IGF)-1 in regulating EC senescence during AS progression was investigated. The mRNA and protein expressions were assessed using qRT-PCR and western blot. IL-1β and IL-18 secretion levels were analyzed by ELISA. Cell viability and pyroptosis were determined by MTT assay and flow cytometry, respectively. SA β-Gal staining was used to measure cell senescence. Tube formation assay was adopted to detect the angiogenesis ability. Dual-luciferase reporter and ChIP assays were used to investigate the relationship between FOXP1 and IGF‑1. ox-LDL stimulation significantly reduced FOXP1 and IGF-1 expression levels in human aortic endothelial cells (HAECs). FOXP1 or IGF-1 overexpression both mitigated ox-LDL-induced cellular senescence and NLRP3 activation in HAECs. It was subsequently revealed that FOXB1 transcriptionally activated IGF-1 expression in HAECs by binding to IGF-1 promoter. Rescue experiments demonstrated that IGF-1 silencing abolished the inhibitory impact of FOXP1 overexpression on ox-LDL-induced cellular senescence and NLRP3 activation in HAECs. FOXP1 transcriptionally activated IGF-1 to lighten ox-LDL-induced endothelial cellular senescence by inactivating NLRP3 inflammasome.

The Molecular Mechanism of Farnesoid X Receptor Alleviating Glucose Intolerance in Turbot (Scophthalmus maximus)

To explore the molecular targets for regulating glucose metabolism in carnivorous fish, the turbot (Scophthalmus maximus) was selected as the research object to study. Farnesoid X receptor (FXR; NR1H4), as a ligand-activated transcription factor, plays an important role in glucose metabolism in mammals. However, the mechanisms controlling glucose metabolism mediated by FXR in fish are not understood. It was first found that the protein levels of FXR and its target gene, small heterodimer partner (SHP), were significantly decreased in the high-glucose group (50 mM, HG) compared with those in the normal glucose group (15 mM, CON) in primary hepatocytes of turbot. By further exploring the function of FXR in turbot, the full length of FXR in turbot was cloned, and its nuclear localization function was characterized by subcellular localization. The results revealed that the FXR had the highest expression in the liver, and its capability to activate SHP expression through heterodimer formation with retinoid X receptor (RXR) was proved, which proved RXR could bind to 15 binding sites of FXR by forming hydrogen bonds. Activation of FXR in both the CON and HG groups significantly increased the expression of glucokinase (gk) and pyruvate kinase (pk), while it decreased the expression of cytosolic phosphoenolpyruvate carboxykinase (cpepck), mitochondrial phosphoenolpyruvate carboxykinase (mpepck), glucose-6-phosphatase 1 (g6pase1) and glucose-6-phosphatase 2 (g6pase2), and caused no significant different in glycogen synthetase (gs). ELISA experiments further demonstrated that under the condition of high glucose with activated FXR, it could significantly decrease the activity of PEPCK and G6PASE in hepatocytes. In a dual-luciferase reporter assay, the FXR could significantly inhibit the activity of G6PASE2 and cPEPCK promoters by binding to the binding site ‘ATGACCT’. Knockdown of SHP after activation of FXR reduced the inhibitory effect on gluconeogenesis. In summary, FXR can bind to the mpepck and g6pase2 promoters to inhibit their expression, thereby directly inhibiting the gluconeogenesis pathway. FXR can also indirectly inhibit the gluconeogenesis pathway by activating shp. These findings suggest the possibility of FXR as a molecular target to regulate glucose homeostasis in turbot.

Hsa_circ_0072732 enhances sunitinib resistance of renal cell carcinoma by inhibiting ferroptosis

BackgroundRenal cell carcinoma (RCC) is one of the most diagnosed urological malignancies with high mortality and increasing incidence. What’s more, the sunitinib resistance undoubtedly increased the difficulties in RCC therapy. Circular RNAs (circRNAs) are a newly found type of non-coding RNAs with a special circular structure, and are found to participate in the occurrence development, chemoresistance, and prognosis of cancers. Ferroptosis regulates disease progression mainly via polyunsaturated fatty acid metabolism and glutamine catabolic pathways. The mechanism of circRNAs contributed to sunitinib resistance through ferroptosis has not been elucidated clearly.Materials and methodsIn our research, we identified a novel circRNA Hsa_circ_0072732 from circRNA datasets (GSE108735 and GSE100186). RNase R and Actinomycin D assays were used to detect the loop structure and stability of circRNAs. qRT-PCR and western blot were used for the detection of RNA and protein levels. CCK8 assays were used to detect proliferation and cell viability. Lipid peroxidation (MDA), and reactive oxygen species (ROS) were detected by indicted kits. Dual-luciferase reporter and RNA pull-down assays were used to detect the RNA interactions.ResultsOur results showed that Hsa_circ_0072732 was highly expressed in RCC cells. Further investigations showed that the silence of Hsa_circ_0072732 could increase RCC sensitivity to sunitinib. Hsa_circ_0072732 contributed to sunitinib chemoresistance by impairing ferroptosis. Hsa_circ_0072732 exerts its function mainly by acting as sponges for miR-548b-3p and regulating the expression SLC7A11. Our research suggests that ferroptosis is involved in sunitinib resistance, and targeting ferroptosis is a promising way for RCC treatment.ConclusionOur research suggests Hsa_circ_0072732 enhanced renal cell carcinoma sunitinib resistance by inhibiting ferroptosis through miR-548b-3p/SLC7A11.

Intestinal FXR deficiency induces dysregulation of xanthine oxidase and accounts for sex difference in hyperuricemia

Overproduction of uric acid caused by increased expression and/or enhanced activity of xanthine oxidase (XO) is one of the major etiologies of hyperuricemia, which had a significant sex differences. As an important enzyme involved in production of reactive oxygen species and uric acid, activity of XO is highly correlated with hyperuricemia and its complications. However, the mechanisms underlying XO dysregulation remain unclear, and sex difference in the prevalence of hyperuricemia has been well known. To explore the potential role of intestinal farnesoid X receptor (FXR) on XO regulation and production, and the mechanisms of sex differences in this pathological process. Two hundred and sixty-one dyslipidemia participants and intestine-specific FXR-knockout mice were used to study the relationship between the intestinal FXR and the serum uric acid level. Western blotting, quantitative real-time PCR, and dual-luciferase reporter assay, were applied to clarify the regulatory role of FXR deficiency on XO. Special inhibitors, agonists, siRNA, sex hormones were used to investigate the mechanism of sex difference in FXR deficiency induced hyperuricemia in cell and animal model. Serum fibroblast growth factor 19 (FGF19) levels were lower in hyperuricemia patients in a sex difference manner. Increased local TNFα level driven by intestinal FXR deficiency/inhibition induced overexpression and hyperactivity of intestinal XO, leading to elevated intestinal uric acid synthesis, and subsequently resulting in hyperuricemia. We found that estrogens inhibited XO expression and activity, whereas androgens enhanced XO activity, leading to the sex difference in FXR deficiency induced hyperuricemia. Infliximab treatment eliminated the sex difference in uric acid levels in intestinal FXR-knockout mice. This study demonstrated the role of intestinal FXR in the pathogenesis of hyperuricemia, and partially elucidated the mechanisms underlying the sex differences of hyperuricemia.

LncRNA TUG1 Knockdown Reduces Cardiomyocyte Damage in Viral Myocarditis by Targeting the miR-140-3p/CXCL8 Axis.

The purpose of this study was to probe the specific role of long noncoding RNA taurine upregulation 1 (LncRNA TUG1) in viral myocarditis (VMC). The mouse model of VMC was induced by Coxsackievirus type B3 (CVB3). LncRNA TUG1 was subsequently silenced, and micro-140-3p (miR-140-3p) was overexpressed in VMC mice. GenePharma synthesized wild-type and mutant LncRNA TUG1 or CXCL8 (C-X-C Motif Chemokine Ligand 8, Interleukin-8) fragments containing the miR-140-3p binding site and cloned them into the pmirGLO luciferase reporter vector. Dual luciferase reporter assays were performed to test the activity of LncRNA TUG1 or CXCL8 fragments containing miR-140-3p mimic and mimic NC. The effects of silencing LncRNA TUG1 on cell proliferation, apoptosis, and inflammation in the VMC mouse model and in vitro were investigated by flow cytometry, enzyme linked immunosorbent assay, and western blot. In the VMC mouse model, LncRNA TUG1 and CXCL8 were upregulated, while miR-140-3p was downregulated. Suppressing LncRNA TUG1 led to inhibition of CXCL8 by promoting miR-140-3p. Suppressing LncRNA TUG1 or CXCL8 or restoring miR-140-3p were observed to increase cell viability and decrease apoptosis rate of cardiomyocytes. LncRNA TUG1 knockdown suppresses inflammation and damage of VMC cardiomyocytes via the miR-140-3p/CXCL8 axis.

Exosomal LncRNA and CircRNA Regulate Peripheral Blood Mononuclear Cell Function through a Competitive Endogenous RNA Mechanism in Allergic Rhinitis.

Peripheral blood mononuclear cells (PBMCs) dysfunction is involved in the pathogenesis and progression of allergic rhinitis (AR). This study aims to investigate the competing endogenous RNA (ceRNA) networks in PBMCs influenced by differentially expressed long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) found in plasma exosomes induced by AR. All subjects were from the Affiliated Taizhou People's Hospital of Nanjing Medical University. Differential expression of mRNAs in PBMCs and lncRNAs/circRNAs in plasma exosomes was analyzed using high-throughput sequencing. Differentially expressed lncRNAs and circRNAs that target mRNAs were identified using bioinformatics methods. The predicted target mRNAs were intersected with the differentially expressed mRNAs in PBMCs to construct ceRNA networks. The subcellular localizations of lncRNAs and circRNAs within the ceRNA networks were determined using RNA fluorescence in situ hybridization or bioinformatics methods. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of differentially expressed mRNAs in PBMCs were conducted using the clusterProfiler R package. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the expression levels of each molecule within the constructed ceRNA networks in clinical samples, along with receiver operating characteristic (ROC) curve analysis to assess diagnostic value. Further validation was performed using in vitro cultured PBMCs and dual-luciferase reporter assays. Five differentially expressed circRNAs and 31 differentially expressed lncRNAs were identified in exosomes. In PBMCs, 130 differentially expressed mRNAs were identified. Six ceRNA networks were constructed, affecting PBMCs chemorepellent activity, JAK-STAT signaling pathway, and other functions or pathways. The expression level of ENST00000650850 in plasma exosomes was significantly lower in AR patients, suggesting its potential diagnostic value. The expression level of ENST00000650850 in plasma exosomes was positively correlated with the expression levels of ENST00000650850 and IL6 mRNA in PBMCs. PBMCs from healthy individuals were stimulated with plasma exosomes isolated from AR patients, leading to a reduction in IL6R mRNA expression levels in the PBMCs. Differentially expressed lncRNA (ENST00000650850) in plasma-derived exosomes of AR patients may regulate IL6R mRNA expression in PBMCs via miR-6747-3p, thereby influencing PBMC function and contributing to the pathogenesis and progression of AR.