Luciferase Reporter Assay Research Articles

MiR-875-5p suppresses Gli1 to alter the hedgehog signaling pathway, which in turn has hepatocellular cancer-related tumor suppressing properties

BackgroundOne of the most prevalent cancers worldwide is HCC, which has put patient health at risk. Increasing evidence indicated that messenger RNAs (mRNAs) played significant roles in modulating tumorigenesis. It has been established that Gli1 acts as an oncogene in a number of malignancies. However, more research was necessary to understand the Gli1 regulation mechanism in HCC. MethodsMicroarray technology was used to evaluate the expression of mRNAs. RT-qPCR was utilized to evaluate Gli1 and miR-875-5p expression. To investigate the role of Gli1, tests using CCK-8, EdU, transwell, immunofluorescence, and Western blot analysis was performed. RIP, RNA pull down, and luciferase reporter assays were employed to verify the interaction between Gli1 and miR-875-5p. ResultsIn tissues and cells of HCC, Gli1 expression appeared to be upregulated, especially in metastatic samples and advanced stages of the disease. A worse outcome was predicted by elevated Gli1 expression. Additionally, in HCC, Gli1 inhibition impeded the growth, migration, and development of the EMT. Since miR-875-5p was shown to have a molecular target in Gli1, miR-875-5p mediated the negative regulation of Gli1. In HCC tissues, its expression pattern was less prominent. In HCC tissues, there was an inverse relationship between Gli1 expression and miR-875-5p expression. Overexpressing Gli1 helped to partially counteract the suppression of HCC migration, proliferation, and EMT formation by miR-875-5p overexpression. ConclusionsMiR-875-5p in HCC suppresses tumors by downregulating Gli1, which supplies a novel treatment for HCC patients.

LINC00665 aggravates the malignant phenotypes in chondrosarcoma cells through miR-665/FGF9 pathway

Long non-coding RNAs (lncRNAs) have been demonstrated to participate in a variety of physiological and pathological processes, including tumor initiation and development. Nevertheless, few of them have been investigated in chondrosarcoma. Here, we were intended to unveil the role of long intergenic non-protein coding RNA 665 (LINC00665) in chondrosarcoma. RT-qPCR was adopted for gene expression detection. The biological processes in chondrosarcoma cells were detected by CCK-8, EdU, TUNEL, Transwell and wound healing assays. The relationships between genes in chondrosarcoma cells were evaluated by a series of mechanism experiments including RIP, luciferase reporter assays and so on.LINC00665 expressed at a high level in chondrosarcoma cell lines. LINC00665 interference suppressed cell proliferation, migration and invasion in chondrosarcoma. Besides, LINC00665 interacted with microRNA-665 (miR-665), which was then verified to be down-regulated in chondrosarcoma cells. Additionally, LINC00665 and miR-665 were mutually inhibited by each other in chondrosarcoma cells. Importantly, LINC00665 stimulated fibroblast growth factor 9 (FGF9) expression in chondrosarcoma cells via sponging miR-665. Furthermore, FGF9 participated in the regulation of LINC00665-promoted chondrosarcoma development. ConclusionLINC00665 facilitates chondrosarcoma progression via miR-665/FGF9 axis, which might indicate a new path for the treatment of chondrosarcoma.

A SAR Study on a Class of 6-(Trifluoromethyl)-pyridine Derivatives as RORγt Inverse Agonists

Background: The nuclear retinoic acid-related orphan receptor γt (RORγt) is an important transcription factor in immune cells. Functionally, RORγt plays an important role in promoting the differentiation of T helper 17 cells and regulating the expression of proinflammatory factors, such as interleukin 17. Therefore, RORγt is considered a promising target for the treatment of the autoimmune disorder. Currently, 21 RORγt inverse agonists with various scaffolds have entered clinical trials. Objective: To discover novel and potent RORγt inverse agonists, a series of novel 6-(trifluoromethyl) pyridine derivatives were designed and synthesized. Methods: We designed and synthesized a series of potent RORγt inverse agonists W1~W16 based on VTP-43742. Molecular docking, molecular dynamics (MD) simulation, and MM/GBSA were used to study the structure-activity relationship (SAR) of the derivatives. Results: The biological activity evaluation indicated that the target compounds showed potent RORγt inhibitory activity. The most active compound, W14, exhibited low nanomolar inhibitory activity (IC50 = 7.5 nM) in the luciferase reporter assay, which was superior to the clinical compound VTP-43742. Analysis of the binding mode of W14 demonstrated that the interaction of -CF3 with Leu324, Leu396, and His479 has an important contribution to the binding. Furthermore, W14 broke the H-bond formed by His479 and Tyr502 via a “push-pull” mechanism. Conclusion: Compound W14 could be used as a potential RORγt inverse agonist for further modification.

Knockdown of LncRNA-HAGLR restrains the viability and motility of pancreatic cancer via miR-625-5p/TAF15 axis in vitro and invivo

Dysregulation of long non-coding RNAs (lncRNAs) has been strongly involved to the development of pancreatic cancer (PC). However, the potential mechanisms by which lncRNA regulate PC development still need to be further explored. We attempted to elucidate the functional role and regulatory mechanism of lncRNA HAGLR on PC progression in vitro and vivo. RT-qPCR, Western blot, RNA pull-down, luciferase reporter assay, RNA immunoprecipitation assay, CCK-8 assay, EdU assay, flow cytometry, transwell assay and xenograft tumor experiment were performed in this study. We found that the expressions of HAGLR and TAF15 were increased in PC tissues and cells. HAGLR silencing restrained the PC cell growth and invasion, but induced cell apoptosis. Moreover, HAGLR targeted miR-625-5p to modulate the expression of TAF15. HAGLR overexpression partially eliminated the suppressive effect of TAF15 depletion on PC cell growth and the stimulative effect on apoptosis. In vivo assays showed that HAGLR knockdown inhibited PC cell growth by regulating the TAF15 expression. These findings suggest HAGLR could facilitate PC cell malignant behaviors through regulating the TAF15 expression, demonstrating that HAGLR might be a valuable target for the PC treatment.

Sinensetin Inhibits Angiogenesis in Lung Adenocarcinoma via the miR-374c-5p/VEGF-A/VEGFR-2/AKT Axis.

Sinensetin is a product isolated from Orthosiphon aristatus, and its antitumor activities have been well established. This study focused on the role and mechanism of sinensetin in lung adenocarcinoma (LUAD). LUAD cells were treated with various concentrations of sinensetin. The proliferation, migration, invasion, and angiogenesis of LUAD cells were detected using colony formation, transwell, and tube formation assays, respectively. The protein levels of VEGF-A, VEGFR-2, and phosphorylated AKT (ser473) were measured by western blotting. The targeted relationship between VEGF-A and miR-374c-5p was verified by luciferase reporter assay. BALB/c nude mice inoculated with A549 cells were treated with sinensetin (40 mg/kg/day) by gavage for 21 days to investigate the effect of sinensetin on tumor growth and angiogenesis in vivo. We found that sinensetin reduced proliferation, migration, invasion, angiogenesis, and cancer stem characteristics of LUAD cells. Sinensetin also suppressed LUAD tumor growth and angiogenesis in vivo. Sinensetin downregulated VEGF-A expression in LUAD cells by enhancing miR-374c-5p expression. MiR-374c-5p inhibited the VEGF-A/VEGFR-2/AKT pathway in LUAD cells. The antitumor effect of sinensetin was reversed by overexpression of VEGF-A or inhibition of miR-374c-5p. Overall, sinensetin upregulates miR-374c-5p to inhibit the VEGF-A/VEGFR-2/AKT pathway, thereby exerting antitumor effect on LUAD.

Haploinsufficiency of intraflagellar transport protein 172 causes autism-like behavioral phenotypes in mice through BDNF

IntroductionPrimary cilia are hair-like solitary organelles growing on most mammalian cells that play fundamental roles in embryonic patterning and organogenesis. Defective cilia often cause a suite of inherited diseases called ciliopathies with multifaceted manifestations. Intraflagellar transport (IFT), a bidirectional protein trafficking along the cilium, actively facilitates the formation and absorption of primary cilia. IFT172 is the largest component of the IFT-B complex, and its roles in Bardet-Biedl Syndrome (BBS) have been appreciated with unclear mechanisms. ObjectivesWe performed a battery of behavioral tests with Ift172 haploinsufficiency (Ift172+/-) and WT littermates. We use RNA sequencing to identify the genes and signaling pathways that are differentially expressed and enriched in the hippocampus of Ift172+/- mice. Using AAV-mediated sparse labeling, electron microscopic examination, patch clamp and local field potential recording, western blot, luciferase reporter assay, chromatin immunoprecipitation, and neuropharmacological approach, we investigated the underlying mechanisms for the aberrant phenotypes presented by Ift172+/- mice. ResultsIft172+/- mice displayed excessive self-grooming, elevated anxiety, and impaired cognition. RNA sequencing revealed enrichment of differentially expressed genes in pathways relevant to axonogenesis and synaptic plasticity, which were further confirmed by less spine density and synaptic number. Ift172+/- mice demonstrated fewer parvalbumin-expressing neurons, decreased inhibitory synaptic transmission, augmented theta oscillation, and sharp-wave ripples in the CA1 region. Moreover, Ift172 haploinsufficiency caused less BDNF production and less activated BDNF-TrkB signaling pathway through transcription factor Gli3. Application of 7,8-Dihydroxyflavone, a potent small molecular TrkB agonist, fully restored BDNF-TrkB signaling activity and abnormal behavioral phenotypes presented by Ift172+/- mice. With luciferase and chip assays, we provided further evidence that Gli3 may physically interact with BDNF promoter I and regulate BDNF expression. ConclusionsOur data suggest that Ift172 per se drives neurotrophic effects and, when defective, could cause neurodevelopmental disorders reminiscent of autism-like disorders.

ArNAC148 induces Acer rubrum leaf senescence by activating the transcription of the ABA receptor gene ArPYR13

Acer rubrum, an ornamental tree known for its stunning autumn colors, has an elusive molecular mechanism that governs its leaf senescence. We performed the genome-wide analysis of NAC transcription factor genes and PYRABACTIN RESISTANCE1-LIKE (PYLs) and found that ArNAC148 and ArPYL13 were significantly upregulated in senescing leaves as compared to mature leaves. Subcellular localization studies confirmed the nuclear localization of ArNAC148 and the cytoplasmic localization of ArPYL13. Electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that ArNAC148 directly binds to the promoter of ArPYL13. Luciferase reporter assays further showed that ArNAC148 activates the transcription of ArPYL13. The transient expression of ArNAC148 and ArPYL13 in tobacco leaves promoted chlorophyll degradation, increased H2O2 level, MDA contents, and electrolyte leakage in response to abscisic acid (ABA). Moreover, the virus-induced gene silencing of ArNAC148 and ArPYL13 in A. rubrum produced results that were opposite to those observed in transient expression experiments. Our findings suggest that ArNAC148 induces leaf senescence by directly activating the transcription of ArPYL13, providing insights into the ABA-mediated regulatory mechanisms governing leaf senescence in A. rubrum. This study offers new perspectives for researchers to explore the roles of NAC and PYL genes in regulating leaf senescence in woody ornamental plants.

Identification of Noncoding Functional Regulatory Variants of STIM1 Gene in Idiopathic Pulmonary Arterial Hypertension.

STIM1 (stromal interaction molecule 1) regulates store-operated calcium entry and is involved in pulmonary artery vasoconstriction and pulmonary artery smooth muscle cell proliferation, leading to pulmonary arterial hypertension (PAH). Bioinformatics analysis and a 2-stage matched case-control study were conducted to screen for noncoding variants that may potentially affect STIM1 transcriptional regulation in 242 patients with idiopathic PAH and 414 healthy controls. Luciferase reporter assay, real-time quantitative polymerase chain reaction, western blot, 5-ethynyl-2'-deoxyuridine (EdU) assay, and intracellular Ca2+ measurement were performed to study the mechanistic roles of those STIM1 noncoding variants in PAH. Five noncoding variants (rs3794050, rs7934581, rs3750996, rs1561876, and rs3750994) were identified and genotyped using Sanger sequencing. Rs3794050, rs7934581, and rs1561876 were associated with idiopathic PAH (recessive model, all P<0.05). Bioinformatics analysis showed that these 3 noncoding variants possibly affect the enhancer function of STIM1 or the microRNA (miRNA) binding to STIM1. Functional validation performed in HEK293 and pulmonary artery smooth muscle cells demonstrated that the noncoding variant rs1561876-G (STIM1 mutant) had significantly stronger transcriptional activity than the wild-type counterpart, rs1561876-A, by affecting the transcriptional regulatory function of both hsa-miRNA-3140-5p and hsa-miRNA-4766-5p. rs1561876-G enhanced intracellular Ca2+ signaling in human pulmonary artery smooth muscle cells secondary to calcium-sensing receptor activation and promoted proliferation of pulmonary artery smooth muscle cells under both normoxia and hypoxia conditions, suggesting a possible contribution to PAH development. The potential clinical implications of the 3 noncoding variants of STIM1, rs3794050, rs7934581, and rs1561876, are 2-fold, as they may help predict the risk and prognosis of idiopathic PAH and guide investigations on novel therapeutic pathway(s).

MiRNA-133a-3p Attenuates Renal Tubular Epithelial Cell Injury via Targeting MALM1 and Suppressing the Notch Signaling Pathway in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes characterized by structural and functional changes of kidneys. Human renal tubular epithelial (HK-2) cells are important for kidney recovery post injury and usually used for establishment of DN cell models. The study explored the role of microRNA (miR)-133a-3p in DN cell model and animal model. A cell model for DN was established via high glucose (HG) stimulation to HK-2 cells. Cell viability and apoptotic rate were measured by cell counting kit 8 and flow cytometry. Polymerase chain reaction was performed to quantify levels of miR-133a-3p and targets. Luciferase reporter assay was conducted to verify the binding of miR-133a-3p and MAML1. After establishment of a mouse model of DN, levels of renal function indicators were measured by biochemical analysis. Hematoxylin-eosin and periodic acid-schiff staining of kidney samples were performed to analyze histological changes. Western blotting was conducted to quantify levels of apoptotic markers, MAML1, and factors related to Notch signaling. Results showed that HG induced HK-2 cell apoptosis and the reduction of cell viability. MiR-133a-3p was lowly expressed in HG-stimulated HK-2 cells. Overexpressed miR-133a-3p improved HK-2 cell injury by increasing cell viability and hampering apoptosis under HG condition. In addition, miR-133a-3p directly targets MAML1 3'-untranslated region. MAML1 overexpression countervailed the repressive impact of miR-133a-3p on cell apoptosis in the context of HG. Moreover, miR-133a-3p inhibited the activity of Notch pathway by downregulating MAML1. MiR-133a-3p inhibits DN progression in mice, as evidenced by reduced fasting blood glucose level, improved levels of renal function parameters, and alleviation of kidney atrophy. In conclusion, miR-133a-3p improves HG-induced HK-2 cell injury and inhibits DN progression by targeting MAML1 and inactivating Notch signaling.

The vitamin D analog EB1089 sensitizes triple-negative breast cancer cells to the antiproliferative effects of antiestrogens

PurposePatients bearing estrogen receptor (ER)α-negative breast cancer tumors confront poor prognosis and are typically unresponsive to hormone therapy. Previous studies have shown that calcitriol, the active vitamin D metabolite, can induce ERα expression in ERα-negative cells. EB1089, a calcitriol analog with reduced calcemic effects, exhibits greater potency than calcitriol in inhibiting cancer cell growth. However, the impact of EB1089 on ERα expression in triple-negative breast cancer (TNBC) cells remains unexplored. This study aims to investigate whether EB1089 could induce functional ERα expression in TNBC cell lines, potentially enabling the antiproliferative effects of antiestrogens. Materials and methodsTNBC cell lines HCC1806 and HCC1937 were treated with EB1089, and ERα expression was analyzed using real-time PCR and Western blots. The transcriptional activity of induced ERα was evaluated through a luciferase reporter assay. The antiproliferative effects of tamoxifen and fulvestrant antiestrogens were assessed using the sulforhodamine B assay in the EB1089-treated cells. ResultsOur findings indicated that EB1089 significantly induced ERα mRNA and protein expression in TNBC cells. Moreover, EB1089-induced ERα exhibited transcriptional activity and effectively restored the inhibitory effects of antiestrogens, thereby suppressing cell proliferation in TNBC cells. ConclusionEB1089 induced the expression of functional ERα in TNBC cells, restoring the antiproliferative effects of antiestrogens. These results highlight the potential of using EB1089 as a promising strategy for re-establishment of the antiproliferative effect of antiestrogens as a possible management for TNBC. This research lays the foundation for potential advancements in TNBC treatment, offering new avenues for targeted and effective interventions.

The Impact of miR-34a on Endothelial Cell Viability and Apoptosis in Ischemic Stroke: Unraveling the MTHFR-Homocysteine Pathway.

Ischemic stroke (IS) is a global health concern, often tied to dyslipidemia and vascular endothelial dysfunction. MicroRNA-34a (miR-34a) was reported to be up-regulated in the blood samples of patients with IS, but the specific role of miR-34a and methylenetetrahydrofolate reductase (MTHFR) in IS remains to be elucidated. We studied 143 subjects: 71 IS patients, and 72 healthy controls. Human umbilical vein endothelial cells (HUVECs) were cultured and transfected with a miR-34a mimic, inhibitor, or negative control. The miR-34a expression in serum and HUVECs was quantified via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Viability and apoptosis of HUVECs were assessed using CCK-8 assay and flow cytometry. The expression levels of bcl-2, bax, cyt-c, cleaved caspase 3, MTHFR, and homocysteine were measured by Western blot or enzyme-linked immunosorbent assay (ELISA). The relationship between miR-34a and MTHFR was verified by luciferase reporter assay. The levels of MTHFR and homocysteine in serum were examined by ELISA. MiR-34a expression was increased in IS patients and inhibited viability of HUVECs while promoting their apoptosis. Overexpression of miR-34a up-regulated pro-apoptotic proteins (bax, cyt-c and cleaved caspase 3) and down-regulated anti-apoptotic protein bcl-2 in HUVECs. MTHFR was identified as the downstream target of miR-34a and its expression was reduced by miR-34a overexpression, while homocysteine levels increased. Consistently, MTHFR levels were lower and homocysteine levels were higher in IS patients compared with controls. Our results suggest that up-regulated miR-34a plays a role in the pathogenesis of IS, potentially through inhibiting MTHFR expression and increasing homocysteine in endothelial cells. Therefore, miR-34a might be a therapeutic target for IS.

Development of 4′-Oxo-MLN4924 as potential neddylation inhibitors: Design, synthesis, anti-HCMV evaluation, and molecular docking

MLN4924 (1), a neddylation inhibitor, has shown promising anticancer and antiviral properties. In this study, we designed and synthesized new derivatives of 4′-oxo-MLN4924, inspired by the structures of MLN4924 and another known neddylation inhibitor, referred to as compound 2. Like compound 2, we used a specific type of sugar molecule but incorporated a modified building block known as 7-deazapurine for the nucleobase part. Additionally, we arranged the sulfamate and 2′-hydroxyl as key functional groups. We found that the 2′-OH group is essential for activity against human cytomegalovirus (HCMV) using luciferase reporter assay. Molecular docking and molecular dynamics (MD) studies revealed that the conformation of the sugar moiety plays a crucial role in protein–ligand interactions. These studies suggest that derivatives with a bulky aromatic ring at the 6-position of the nucleobase are likely to have enhanced binding, which is supported by experimental findings.

Gardenin B, A Natural Inhibitor for USP7: In vitro Evaluation and In silico Identification

Background: Ubiquitin-specific protease 7 (USP7) is one of the most widely studied deubiquitin enzymes (DUBs). The protein level of USP7 is highly expressed in a variety of malignant cancers, which suggests that it is a prognostic marker of cancers and a potential drug target for oncotherapy. Objective: The aim of this study was to identify natural and effective USP7 inhibitors, in order to understand the activation of the USP7/p53 pathway by natural inhibitors. Methods: In this study, USP7 enzyme activity screening assay system and p53 luciferase reporter assay system have been applied for the discovery of natural USP7 inhibitors targeting the catalytic active site. Molecular docking and molecular dynamics (MD) simulation revealed the combined mechanism between USP7 with gardenin B. Results: The gardenin B was screened from our home-lab natural products (160 flavonoids) and had cytotoxicity in HCT116 cells (IC50 = 46.28 ± 2.16 μM). Preliminary in vitro studies disclosed its antiproliferative activity and activated p53 signaling pathway in HCT116 cells. We found that the complex formed by gardenin B and 5vsk (Ledock score = -6.86, MM/GBSA score = -53.35) had the optimal binding conformation. Moreover, the MD simulation showed that the π-π interactions between gardenin B with His461 and Phe409, and the hydrogen bonds interaction between gardenin B with Leu406 played an important role in maintaining the close binding of the complexes. Conclusion: In conclusion, gardenin B could be used as a natural product inhibitor of USP7 for further optimized design and development potential.

Ischemic-Preconditioning Induced Serum Exosomal miR-133a-3p Improved Post-Myocardial Infarction Repair via Targeting LTBP1 and PPP2CA.

Ischemic preconditioning-induced serum exosomes (IPC-exo) protected rat heart against myocardial ischemia/reperfusion injury. However, whether IPC-exo regulate replacement fibrosis after myocardial infarction (MI) and the underlying mechanisms remain unclear. MicroRNAs (miRs) are important cargos of exosomes and play an essential role in cardioprotection. We aim to investigate whether IPC-exo regulate post-MI replacement fibrosis by transferring cardioprotective miRs and its action mechanism. Exosomes obtained from serum of adult rats in control (Con-exo) and IPC groups were identified and analyzed, subsequently intracardially injected into MI rats following ligation. Their miRs profiles were identified using high-throughput miR sequencing to identify target miRs for bioinformatics analysis. Luciferase reporter assays confirmed target genes of selected miRs. IPC-exo transfected with selected miRs antagomir or NC were intracardially administered to MI rats post-ligation. Cardiac function and degree of replacement fibrosis were detected 4 weeks post-MI. IPC-exo exerted cardioprotective effects against excessive replacement fibrosis. MiR sequencing and RT-qPCR identified miR-133a-3p as most significantly different between IPC-exo and Con-exo. MiR-133a-3p directly targeted latent transforming growth factor beta binding protein 1 (LTBP1) and protein phosphatase 2, catalytic subunit, alpha isozyme (PPP2CA). KEGG analysis showed that transforming growth factor-β (TGF-β) was one of the most enriched signaling pathways with miR-133a-3p. Comparing to injection of IPC-exo transfected with miR-133a-3p antagomir NC, injecting IPC-exo transfected with miR-133a-3p antagomir abolished protective effects of IPC-exo on declining excessive replacement fibrosis and cardiac function enhancement, while increasing the messenger RNA and protein expression of LTBP1, PPP2CA, and TGF-β1in MI rats. IPC-exo inhibit excessive replacement fibrosis and improve cardiac function post-MI by transferring miR-133a-3p, the mechanism is associated with directly targeting LTBP1 and PPP2CA, and indirectly regulating TGF-β pathway in rats. Our finding provides potential therapeutic effect of IPC-induced exosomal miR-133a-3p for cardiac repair.

Relationship between COVID-19 and orofacial clefts.

MicroRNAs (miRNAs) are essential modulators of gene expression and are associated with various pathological processes, including spinal cord injury (SCI). This investigation aimed to elucidate miR-10a activity in SCI and its potential interaction with sirtuin 1 (SIRT1). The SCI rat model was established to assess hind limb movement, measure levels of miR-10a, SIRT1, neuronal survival, and inflammatory factors. An in-vitro SCI cell model was also developed to evaluate cell viability and inflammatory factor levels. The interaction between miR10a and SIRT1 was verified. Upregulated miR-10a and downregulated SIRT1 expression were found in the tissues of SCI rats. miR-10a knockdown in SCI rats enhanced the recovery of motor function, increased neuronal survival, and reduced the levels of inflammatory cytokines. Luciferase reporter assays confirmed that miR-10a targeted SIRT1 directly. In PC12 cells, downregulation of miR-10a increased SIRT1 expression, enhanced cell viability, and reduced inflammatory factor levels after LPS stimulation. Conversely, SIRT1 knockdown inhibited the protective effects of downregulated miR-10a on cell viability and inflammatory responses. The results suggest that miR-10a downregulation protects against SCI by upregulating SIRT1 expression, improving functional recovery, and reducing inflammation. Targeting the miR-10a/SIRT1 axis is a promising strategy for SCI treatment.

Molecular characterization and transcriptional regulation between PPAR and CPT1 in freshwater bivalve Hyriopsis cumingii

Peroxisome proliferator activated receptors (PPARs) exert their roles in lipid metabolism and adaptive immunity by transactivating carnitine palmitoyltransferase 1 (CPT1). However, it remains unclear whether the PPAR-CPT1 signaling pathway exists in mollusks that only carry out innate immunity. This study cloned and characterized PPAR and CPT1 genes from Hyriopsis cumingii for the first time, designated as HcPPARs and HcCPT1s, respectively. The bioinformatics analysis revealed conservative molecular characteristics of these genes across species. Real-time quantitative PCR results indicated that higher expression levels of HcPPARs and HcCPT1s in the blood, mantle, and intestine suggested their potential involvement in lipid metabolism and innate immunity of mollusks. Treatments with agonists and inhibitors demonstrated a correlation in the expression of HcPPARs and HcCPT1s. Dual luciferase reporter assay identified regions with high transcriptional activities on promoters of HcCPT1s and potential binding sites for HcPPARs through prediction and mutation sites. These results suggested that the PPAR-CPT1 signaling might exist in H. cumingii. This research provides a necessary foundation for exploring the role of the PPAR-CPT1 signaling in innate immunity, and offers new theoretical evidence for the molecular regulatory mechanism of mollusks and the treatment of metabolic disorders and inflammatory diseases.

Has_circ_0002360 promotes the progression of lung adenocarcinoma by activating miR-762 and regulating PODXL expression.

Circular RNAs (circRNAs) have been found to be linked to cancer progression and metastasis, but there is not much known about their connection to lung adenocarcinoma (LAC). In the previous study reported by our group, has_circ_0002360 was highly expressed in LAC tissues. The goal of this study was to investigate the potential impact of has_circ_0002360 in LAC. Bioinformatics software, TargetScan, and miRanda were used to study the interactions of RNAs. Luciferase reporter assays further confirmed their relationship. The relative expression of has_circ_0002360 in 122 patients and four cell lines of the lung were obtained using real-time qualitative polymerase chain reaction (qRT-PCR). The target gene podocalyxin-like (PODXL) expression was confirmed by immunohistochemistry (IHC) in ten pairs of clinical samples. Then, cell counting kit-8 (CCK8), wound healing, and transwell experiments were applied to examine cell growth, migration, and infection-induced cell invasion. LAC cell lines were infected, and the process was monitored by examination of the related epithelial-mesenchymal transition (EMT) proteins. The resulting data indicated that has_circ_0002360 and PODXL were overexpressed in LAC tissues, whereas miR-762 expression was repressed. The reduction of has_circ_0002360 or upregulation of miR-762 mitigated the proliferation, migration, invasion of LAC cells. Mechanistically, has_circ_0002360 upregulated PODXL expressions by targeting miR-762 to promote LAC progression. In general, the has_circ_0002360/miR-762/PODXL axis affected the progress of LAC. The results of our study identified has_circ_0002360 as a novel oncogenic RNA in LAC.

The sEVs miR-487a/Notch2/GATA3 axis promotes osteosarcoma lung metastasis by inducing macrophage polarization toward the M2-subtype

Small extracellular vesicles (sEVs) are important mediators of intercellular communication between tumor cells and their surrounding environment. Furthermore, the mechanisms by which miRNAs carried in tumor sEVs regulate macrophage polarization remain largely unknown. To concentrate sEVs, we used the traditional ultracentrifugation method. Western blot, NanoSight, and transmission electron microscopy were used to identify sEVs. To determine the function of sEVs-miR-487a, we conducted in vivo and in vitro investigations. The intercellular communication mechanism between osteosarcoma cells and M2 macrophages, mediated by sEVs carrying miR-487a, was validated using luciferase reporter assays, transwell assays, and Western blot analysis. In vitro, sEVs enriched in miR-487a and delivered miR-487a to macrophages, promoting macrophage polarization toward an M2-like type, which promotes proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of osteosarcoma cells. In vivo, sEVs enriched in miR-487a facilitate lung metastasis of osteosarcoma. Moreover, plasma miR-487a in sEVs was shown to be a potential biomarker applicable for osteosarcoma diagnosis. In summary, miR-487a derived from osteosarcoma cells can be transferred to macrophages via sEVs, then promote macrophage polarization towards an M2-like type by targeting Notch2 and activating the GATA3 pathway. In a feedback loop, the activation of macrophages accelerates epithelial-mesenchymal transition (EMT), which in turn promotes the migration, invasion, and lung metastasis of osteosarcoma cells. This reciprocal interaction between activated macrophages and osteosarcoma cells contributes to the progression of the disease. Our data demonstrate a new mechanism that osteosarcoma tumor cells derived exosomal-miR-487a which is involved in osteosarcoma development by regulating macrophage polarization in tumor microenvironment (TME).

G-quadruplex forming regions in GCK and TM6SF2 are targets for differential DNA methylation in metabolic disease and hepatocellular carcinoma patients

The alarming increase in global rates of metabolic diseases (MetDs) and their association with cancer risk renders them a considerable burden on our society. The interplay of environmental and genetic factors in causing MetDs may be reflected in DNA methylation patterns, particularly at non-canonical (non-B) DNA structures, such as G-quadruplexes (G4s) or R-loops. To gain insight into the mechanisms of MetD progression, we focused on DNA methylation and functional analyses on intragenic regions of two MetD risk genes, the glucokinase (GCK) exon 7 and the transmembrane 6 superfamily 2 (TM6SF2) intron 2-exon 3 boundary, which harbor non-B DNA motifs for G4s and R-loops.Pyrosequencing of 148 blood samples from a nested cohort study revealed significant differential methylation in GCK and TM6SF2 in MetD patients versus healthy controls. Furthermore, these regions harbor hypervariable and differentially methylated CpGs also in hepatocellular carcinoma versus normal tissue samples from The Cancer Genome Atlas (TCGA). Permanganate/S1 nuclease footprinting with direct adapter ligation (PDAL-Seq), native polyacrylamide DNA gel electrophoresis and circular dichroism (CD) spectroscopy revealed the formation of G4 structures in these regions and demonstrated that their topology and stability is affected by DNA methylation. Detailed analyses including histone marks, chromatin conformation capture data, and luciferase reporter assays, highlighted the cell-type specific regulatory function of the target regions. Based on our analyses, we hypothesize that changes in DNA methylation lead to topological changes, especially in GCK exon 7, and cause the activation of alternative regulatory elements or potentially play a role in alternative splicing.Our analyses provide a new view on the mechanisms underlying the progression of MetDs and their link to hepatocellular carcinomas, unveiling non-B DNA structures as important key players already in early disease stages.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Alleviate Nuclear Pulposus Cells Degeneration Through the miR-145a-5p/USP31/HIF-1α Signaling Pathway.

Bone marrow mesenchymal stem cell (BMSC)-derived exosomes possess therapeutic potential against degenerative diseases. This study aimed to investigate the effects of BMSC-derived exosomes on intervertebral disc degeneration (IVDD) and explore the underlying molecular mechanisms. Through transcriptome sequencing and histological analysis, we observed a significant increase in HIF-1α expression in degenerative nucleus pulposus (NP) tissues. The addition of HIF-1α resulted in elevated expression of inflammatory factors IL-1β and IL-6, higher levels of matrix-degrading enzyme MMP13, and lower expression of aggrecan in NP cells. Co-culturing with BMSCs diminished the expression of HIF-1α, MMP13, IL-1β, and IL-6 in degenerative NP cells induced by overload pressure. miRNA chip analysis and PCR validation revealed that miR-145a-5p was the primary miRNA carried by BMSC-derived exosomes. Overexpression of miR-145a-5p was effective in minimizing the expression of HIF-1α, MMP13, IL-1β, and IL-6 in degenerative NP cells. Luciferase reporter assays confirmed USP31 as the target gene of miR-145a-5p, and the regulation of NP cells by BMSC-derived exosomes via miR-145a-5p was dependent on USP31. In conclusion, BMSC-derived exosomes alleviated IVDD through the miR-145a-5p/USP31/HIF-1α signaling pathway, providing valuable insights into the treatment of IVDD.