Dual-luciferase Reporter Assay Research Articles

Immune infiltration related circular RNA, circGLIS2, facilitated progression of endometriosis through miR-4731-5p/IL-1β axis

Aberrantly expressed circRNAs affects various anti-tumour immune responses and immune cell regulation. However, the exact function of circGLIS2 on the pathogenesis of EMs remains unclear. In this study, we found that circGLIS2 was upregulated in EMs tissues and intimately related to clinicopathologic characteristics of EMs patients. Functionally, IHC and mouse model of EMs proved that circGLIS2 recruited immune cells infiltrated into ectopic endometrial microenvironment. RNA-seq, ELISA, RT-qPCR, and western blot results indicated that circGLIS2 influenced immune infiltration by regulating IL-1β. Besides, expression of circGLIS2 and IL-1β was also closely correlated in 284 human endometrium tissues. Mechanistically, RNA sequencing and intermolecular interaction prediction established “circGLIS2-miR-4731-5p-IL-1β” network. RNA pull down, RIP, dual luciferase reporter assay and expression regulation analysis confirmed the interaction among “circGLIS2-miR-4731-5p-IL-1β” axis. In summary, our findings demonstrated that circGLIS2 facilitated EMs progression by increasing immune cell infiltration via miR-4731-5p/IL-1β axis.

EIF4G2 Promotes Hepatocellular Carcinoma Progression via IRES-dependent PLEKHA1 Translation Regulation.

Hepatocellular carcinoma (HCC) is a highly lethal cancer, and proteomic studies have shown increased protein diversity and abundance in HCC tissues, whereas the role of protein translation has not been extensively explored in HCC. Our research focused on key molecules in the translation process to identify a potential contributor in HCC. We discovered that EIF4G2, a crucial translation initiation factor, is significantly upregulated in HCC tissues and associated with poor prognosis. This study uniquely highlights the impact of EIF4G2 deletion, which suppresses tumor growth and metastasis both in vitro and in vivo. Furthermore, polysome analysis and nascent protein synthesis assays revealed EIF4G2's role in regulating protein translation, specifically identifying PLEKHA1 as a key translational product. This represents a novel mechanistic insight into HCC malignancy. RNA immunoprecipitation (RIP) and Dual-luciferase reporter assays further revealed that EIF4G2 facilitates PLEKHA1 translation via an IRES-dependent manner. Importantly, the synergistic effects of EIF4G2 depletion and PLEKHA1 reduction in inhibiting cell migration and invasion underscore the therapeutic potential of targeting this axis. This study not only advances our understanding of translational regulation in HCC but also identifies the EIF4G2-PLEKHA1 axis as a promising therapeutic target, offering new avenues for intervention in HCC treatment.

Schistosoma japonicum infection-mediated downregulation of lncRNA Malat1 contributes to schistosomiasis hepatic fibrosis by the Malat1/miR-96/Smad7 pathway

BackgroundSchistosoma japonicum infection causes hepatic fibrosis, a primary cause of morbidity and mortality associated with the disease, and effective treatments are still lacking. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenic process of various tissue fibroses. However, the role of lncRNAs in schistosomiasis hepatic fibrosis (HF) is poorly understood. Understanding the role of lncRNAs in schistosomiasis HF will enhance knowledge of disease processes and aid in the discovery of therapeutic targets and diagnostic biomarkers.MethodsDifferentially expressed lncRNA profiles in primary hepatic stellate cells (HSCs) of mice infected with S. japonicum were identified using high-throughput lncRNA sequencing. Primary HSCs were isolated from infected mice using collagenase digestion and density-gradient centrifugation, cultured in DMEM with 10% fetal bovine serum. Dual-luciferase reporter assays, nuclear cytoplasm fractionation and RIP assays were employed to assess the relationship between Malat1 and miRNA-96. Malat1 lentivirus and ASO-Malat1 were constructed for forced expression and downregulated expression of Malat1. The Malat1-KO mouse was constructed by CRISPR/Cas9 technology. Pathological features of the liver were evaluated by hematoxylin-eosin (HE), Masson’s trichrome staining and immunohistochemistry (IHC). The expression levels of fibrosis-related genes were determined by quantitative real-time PCR (qRT-PCR) and Western blot.ResultsA total of 1561 differentially expressed lncRNAs were identified between infected and uninfected primary HSCs. Among the top altered lncRNAs, the downregulated Malat1 was observed in infected HSCs and verified by qPCR. Treatment of infected mice with praziquantel (PZQ) significantly increased the Malat1 expression. Elevated Malat1 expression in infected primary HSC reduced the expressions of profibrogenic genes, whereas Malat1 knockdown had the opposite effect. Moreover, Malat1 was found to interact with miR-96, a profibrotic miRNA, by targeting Smad7. Forced Malat1 expression reduced miR-96 levels in infected primary HSCs, attenuating fibrogenesis and showing negative correlation between Malat1 expression and the expression levels of miR-96 and profibrogenic genes α-SMA and Col1α1. Notably, in Malat1-KO mice, knockout of Malat1 aggravates schistosomiasis HF, while restored Malat1 expression in the infected HSCs reduced the expression of profibrogenic genes.ConclusionsWe demonstrate that lncRNA is involved in regulation of schistosomiasis HF. Elevated lncRNA Malat1 expression in infected HSCs reduces fibrosis via the Malat1/miR-96/Smad7 pathway, thus providing a novel therapeutic target for schistosomiasis HF. Furthermore, Malat1 expression is sensitive to PZQ treatment, thus offering a potential biomarker for assessing the response to chemotherapy.Graphical abstract

HNRNPD/MAD2L2 axis facilitates lung adenocarcinoma progression and is a potential prognostic biomarker

BackgroundAlthough progress has been made in the treatment of LAUD, the survival rate for patients remains poor. An in-depth grasp of the molecular pathways implicated in LUAD progression is vital for improving diagnosis and treatment strategies. This study aims to explore novel molecular mechanisms driving LUAD progression and identify new potential prognostic biomarkers for LAUD patients. MethodsBased on mass spectrometry analysis of human LUAD tissues, HNRNPD and MAD2L2 were identified as potential key proteins involved in LUAD progression. Subsequently, the interplay between HNRNPD and MAD2L2 was examined through dual-luciferase reporter assays, RNA-seq analysis, and various molecular biology techniques. Ultimately, the role of the HNRNPD/MAD2L2 axis in LUAD advancement and its potential as a prognostic indicator were investigated utilizing LUAD specimens, cell lines, and xenograft mouse models. ResultsIn human LAUD tissues and cell lines, elevated levels of HNRNPD and MAD2L2 proteins were discovered. It was determined that HNRNPD binds to the MAD2L2 promoter, forming a regulatory axis at the transcriptional level. Subsequently, both in vitro and in vivo data demonstrated that the downregulation of the HNRNPD/MAD2L2 axis inhibited LUAD progression, while this effect could be rescued by MAD2L2 upregulation. Conversely, the upregulation of the HNRNPD/MAD2L2 axis facilitated LUAD progression, and this outcome could be reversed by MAD2L2 knockdown. Mechanistically, the downregulation of HNRNPD suppressed the promoter activity and transcription of MAD2L2, thus inhibiting the PI3K/HIF1α/ANGPTL4 pathway and tumor angiogenesis. Finally, it was confirmed that LUAD patients with high levels of both HNRNPD and MAD2L2 exhibited the poorest prognosis. Therefore, the HNRNPD/MAD2L2 axis has been identified as a potential predictive indicator for LUAD patients. ConclusionsThe HNRNPD/MAD2L2 axis facilitates LUAD progression and serves as a potential prognostic biomarker.

SlSTOP1-regulated SlHAK5 expression confers Al tolerance in tomato by facilitating citrate secretion from roots.

SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) is a core transcription factor that regulates the expression of aluminum (Al) resistance genes to manage Al toxicity in plants. However, the genome-wide roles of SlSTOP1 in the Al stress response of tomato (Solanum lycopersicum) remain largely unknown. Here, we report that SlSTOP1 is crucial for Al tolerance in tomato, as loss-of-function mutants of SlSTOP1 displayed hypersensitivity to Al stress. Aluminum stress had no effect on SlSTOP1 mRNA expression, but promoted accumulation of SlSTOP1 protein in the nucleus. Through integrated DNA affinity purification sequencing and RNA sequencing analysis, we identified 39 SlSTOP1-targeted Al-responsive genes, some of which are homologous to known Al resistance genes in other plant species, suggesting that these SlSTOP1-targeted genes play essential roles in Al resistance in tomato. Furthermore, using peak enrichment analysis of SlSTOP1-targeted sequences, we identified a cis-acting element bound by SlSTOP1 and validated this finding via dual-luciferase reporter and electrophoretic mobility shift assay (EMSA). Additionally, we demonstrated SlHAK5 is one of direct targets of SlSTOP1 and functionally characterized it in terms of Al stress tolerance. Compared with wild-type plants, Slhak5 mutants developed by CRISPR/Cas9 technology presented increased sensitivity to Al stress, which was associated with reduced citrate secretion from the roots. Together, our findings demonstrate that SlSTOP1 directly interacts with cis-acting elements located in the promoters of target genes involved in diverse pathways contributing to Al resistance in tomato.

Huangqi-Danshen decoction improves heart failure by regulating pericardial adipose tissue derived extracellular vesicular miR-27a-3p to activate AMPKα2 mediated mitophagy

BackgroundHuangqi-Danshen decoction (HDD) is a classic traditional Chinese medicine for treating heart failure. Pericardial adipose tissue (PAT) has recently gained increasing attention in cardiovascular diseases. PurposeThis study aimed to investigate the effect of pericardial adipose tissue-derived extracellular vesicles on heart failure, the protective effect of HDD on myocardial remodel in heart failure rats, and identify the potential molecular mechanisms involved. MethodsUPLC-MS/MS identified active components of HDD. Extracellular vesicles (EVs) from pericardial adipose tissue of sham-operated and HF rats were identified through transmission electron microscopy, nanoparticle tracking analysis and western blot. EVs were co-cultured with H9c2 cardiomyocytes in order to examine their uptake and effects. MicroRNA sequencing, dual-luciferase reporter assay and PCR were conducted for exploring specific mechanisms of EVs on hypertrophic cardiomyocytes. In vivo, heart failure was modeled in rats via transverse aortic constriction (TAC). In vitro, the hypertrophic cardiomyocyte model were established using Ang II-induced H9c2 cardiomyocytes. ResultsUPLC-MS/MS identified 11 active components in serum of HDD administrated rats. Echocardiography showed HDD improved cardiac function in TAC model rats. HE and Masson staining indicated HDD ameliorated myocardial hypertrophy and fibrosis. MicroRNA sequencing found that HDD treatment resulted in 37 differentially expressed miRNAs (DMEs) (P < 0.05 and |log2FC| ≥ 1). KEGG analysis revealed that DEMs were enriched in the AMPK signaling pathway. PCR identified miR-27a-3p with the greatest difference in AMPK-related DMEs. Dual-luciferase reporter assay and Targetscan website were utilized to identify the target relationship between miR-27a-3p and PRKAA2 (AMPKα2). The miR-27a-3p negatively regulated AMPKα2 to inhibit mitophagy mediated by PINK1/Parkin pathway. HDD inhibited miR-27a-3p secretion from failing heart pericardial adipose tissue-derived extracellular vesicles, thereby improving inflammation, cardiac function, and myocardial remodeling through above pathways. ConclusionHDD inhibited the PAT-derived extracellular vesicular miR-27a-3p in failing hearts to activate AMPK/PINK1/Parkin signaling-mediated mitophagy, which improved cardiomyocyte energy metabolism, myocardial remodeling and heart failure.

Mesenchymal Stem Cells-Derived Exosomal miR-223-3p Alleviates Ocular Surface Damage and Inflammation by Downregulating Fbxw7 in Dry Eye Models.

Our previous study indicated that exosomes derived from mouse adipose-derived mesenchymal stem cells (mADSC-Exos) alleviated the benzalkonium chloride (BAC)-induced mouse dry eye model. However, the specific active molecules in mADSC-Exos that contribute to anti-dry eye therapy remain unidentified. In this study, we aimed to investigate the efficacy and mechanisms of miR-223-3p derived from mADSC-Exos in dry eye models. Enzyme-linked immunosorbent assay (ELISA) experiments were conducted to determine miR-223-3p derived from mADSC-Exos that exerted anti-inflammatory effects on hyperosmolarity-induced mouse corneal epithelial cells (MCECs). The therapeutic efficacy of miR-223-3p was evaluated in mice with dry eye induced by either BAC or scopolamine (Scop). Mice were randomly assigned to 5 groups: sham, model, miR-223-3p overexpression, miR-223-3p knockdown, and 0.1% pranoprofen (positive group). Post-treatment, the severity of dry eye symptoms, and the pro-inflammatory cytokine levels were assessed. The effect of miR-223-3p on silencing the target gene was verified using ELISA and dual luciferase reporter assays. The mADSC-Exos that knocked out miR-223-3p did not reduce interleukin (IL)-6 content. Supplementing with miR-223-3p could restore the reduction of IL-6. The miR-223-3p effectively ameliorated ocular surface damage and decreased pro-inflammatory cytokines or chemokines in both BAC- and Scop-induced mouse dry eye models. Furthermore, miR-223-3p inhibited cell apoptosis. F-box and WD repeat domain-containing 7 (Fbxw7) was the potential direct target of miR-223-3p. The miR-223-3p suppressed the 3'-untranslated region of Fbxw7. The Fbxw7 knockdown suppressed hyperosmolarity-induced inflammation in MCECs. The mADSC-derived exosomal miR-223-3p mitigates ocular surface damage and inflammation, indicating its potential as a promising treatment option for dry eye.

Role of miR-455-3p in the alleviation of LPS-induced acute lung injury by allicin

Acute lung injury (ALI) is a type of diffuse lung injury that seriously affects the survival of critically ill patients. MicroRNAs (miRNAs) can serve as promising therapeutic targets or offer insights for the development of potential therapeutic strategies against ALI. In our previous study, we demonstrated the protective effect of allicin in ALI, but the role of miRNAs in the alleviation of ALI by allicin remains unclear. This study aimed to investigate whether miRNAs mediate the effects of allicin on ALI. Cell viability and proliferation were determined using CCK-8 and EdU assays, respectively, while cellular apoptosis was analyzed by flow cytometry. The claudin-4 protein was detected by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting. The binding of miR-455 with claudin-4 was determined by bioinformatics analysis and validated by dual luciferase reporter assays. The lung wet/dry ratio of lipopolysaccharide (LPS)-treated rats was determined by hematoxylin and eosin (HE) and TUNEL staining of the pulmonary tissues. The levels of myeloperoxidase (MPO), interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α were determined by enzyme-linked immunosorbent assay (ELISA). We observed that allicin alleviated LPS-induced injury in A549 cells, and claudin-4 knockdown reversed the protective effect of allicin in ALI. Claudin-4 is a direct target of miR-455-3p, and miR-455-3p overexpression partially reversed the protective effect of allicin in LPS-treated A549 cells. Subsequent in vivo experiments confirmed that allicin protects against LPS-induced ALI by regulating the miR-455-3p/claudin-4 axis. The study revealed that the protective effect of allicin in ALI is mediated via miR-455-3p, which suppresses the expression of claudin-4.

The transcription factor MYC2 positively regulates terpene trilactone biosynthesis through activating GbGGPPS expression in Ginkgo biloba.

Terpene trilactones (TTLs) have important medicinal value, but their low content in Ginkgo biloba leaves makes their exploitation extremely costly, thereby limiting the development of TTL-related industries. It was found that exogenous methyl jasmonate (MeJA) treatment increased the accumulation of TTLs, but the molecular mechanism is still unclear. Here, we identified two bHLH transcription factors in G. biloba, with the protein subcellular localizations in the nucleus. Expression of GbMYC2s was strongly induced by MeJA treatment, and the interactions between GbJAZs and GbMYC2s were demonstrated by yeast two-hybrid and bimolecular fluorescence complementation experiments. Overexpression of GbMYC2_4 and GbMYC2_5 enhanced Arabidopsis root sensitivity and significantly increased TTL content. In addition, GbGGPPS was found to be a common target of GbMYC2_4 and GbMYC2_5 by yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase reporter assays and DAP-seq, and they achieved regulation of GbGGPPS by binding to the G-box. Further findings revealed that GbMYC2_4 and GbMYC2_5 bind the G-box not universally but selectively. Our study revealed that jasmonic acid signaling mediates TTL biosynthesis through the GbJAZ-GbMYC2-GbGGPPS module, which enriches the terpenoid biosynthesis regulatory networks and provides a research basis and target genes for enhancing TTL content through genetic engineering.

Hsa_circ_0048764 facilitates the progression of non-small cell lung cancer by targeting miR-1178-3p/HMGA1 axis

Non-small cell lung cancer (NSCLC) remains a highly lethal disease, with a lack of fully established biomarkers and therapies. Circular RNAs (circRNAs) have emerged as powerful regulators of gene expression in multiple cancers. The role of circRNAs in NSCLC progression is still not well understood. In this study, GEO database analysis and qRT-PCR results revealed that hsa_circ_0048764 (circ_0048764) was overexpressed in NSCLC tissues and associated with poorer overall survival in patients with NSCLC. Functional assays demonstrated that silencing circ_0048764 inhibited NSCLC cell proliferation and metastasis. Bioinformatics analysis identified miR-1178-3p as having complementary binding sites with circ_0048764, a finding further validated by the dual-luciferase reporter assay. Additionally, predictions from the Starbase3.0 database, along with cellular experiments, revealed that miR-1178-3p regulates HMGA1 expression in NSCLC. Taken together, our findings suggest that circ_0048764 promotes NSCLC progression by enhancing HMGA1 expression through sponging miR-1178-3p, offering potential therapeutic targets for NSCLC treatment.

Low-energy red light-emitting diode irradiation enhances osteogenic differentiation of periodontal ligament stem cells by regulating miR-146a-5p.

The study aimed to investigate the role of miR-146a-5p in osteogenesis of hPDLSCs irradiated with low-energy red LEDs. After irradiation with 5 J/cm2 red LED, miR-146a-5p expression was detected by real-time quantitative polymerase chain reaction (RT-qPCR), and osteogenic markers expression was determined by RT-qPCR and Western blotting. Alkaline phosphatase (ALP) activity was assessed by ALP staining, and mineralization was assessed by Alizarin Red staining, respectively. Lentiviral vectors were designed to regulate miR-146a-5p expression. Dual-luciferase reporter assay was performed to confirm the targeted relationship between miR-146a-5p and MAPK1. Short hairpin RNA (shRNA) was used to regulate MAPK1 expression. RT-qPCR and western blotting revealed that 5 J/cm2 irradiation elevated the levels of the osteogenic markers osterix (OSX) and bone sialoprotein (BSP) in hPDLSCs. miR-146a-5p is downregulated in hPDLSCs under the low-energy red LED light irradiation. miR-146a-5p underexpression markedly promoted the osteogenic potential of hPDLSCs. miR-146a-5p targeted MAPK1. 5 J/cm2 red LED irradiation rescued the inhibitory effects of upregulated miR-146a-5p on osteogenic differentiation, and the positive influence of red LED irradiation could be reversed by downregulated MAPK1. These findings confirm that miR-146a-5p is involved in the effect of LED irradiation on the osteogenic differentiation of hPDLSCs by targeting MAPK1. Red LED irradiation may be a potential clinical adjunct therapy for periodontal regeneration.

Tanshinone T1/T2A inhibits non-small cell lung cancer through Lin28B-let-7-BORA/MYC regulatory network

BackgroundLung cancer is the leading cause of cancer-related deaths worldwide. Tanshinones are a group of compounds in Salvia miltiorrhiza. Although the effects of tanshinone I (T1) and tanshinone IIA (T2A) are widely concerned, the mechanisms of T1 and T2A in lung cancer is rarely studied. Experimental procedureXenograft tumor growth was performed to detect the role of T1/T2A in vivo. Next-generation sequencing of miRNA expression profiles in T1/T2A-treated A549 cells showed that T1/T2A upregulated the expression of the let-7 family. Then, let-7a-5p and its downstream target gene BORA were identified as the research objects in this paper. Mechanistically, we examined the interplay between miR-let-7 and BORA through the dual-luciferase reporter assay. Finally, the potential regulatory role of T1/T2A on Lin28B and MYC was explored. ResultsThis study found that the let-7 family was significantly up-regulated via “Next-generation” sequencing (NGS) in the T1/T2A-treated A549 cell line, while BORA was downregulated. BORA was confirmed as a direct target of let-7. LncRNA MYCLo-5 was up-regulated after treatment with tanshinones. Knockdown of MYCLo-5 promoted the cell cycle and proliferation of non-small cell lung cancer (NSCLC) cells. ConclusionsThis study explored the effects of tanshinone T1 and T2A on NSCLC in vitro and in vivo, revealing the T1/T2A-let-7/BORA/MYCLo-5 regulatory pathway, which provided new insights for lung cancer treatment.

Differential miRNA Profiling Reveals miR-4433a-5p as a Key Regulator of Chronic Obstructive Pulmonary Disease Progression via PIK3R2- mediated Phenotypic Modulation.

In this study, a high-throughput sequencing technology was used to screen the differentially expressed miRNA in the patients with "fast" and "slow" progression of chronic obstructive pulmonary disease (COPD). Moreover, the possible mechanism affecting the progression of COPD was preliminarily analyzed based on the target genes of candidate miRNAs. The "fast" progressive COPD group included 6 cases, "slow" and Normal progressive COPD groups included 5 cases each, and COPD group included 3 cases. The peripheral blood samples were taken from the participants, followed by total RNA extraction and high throughput miRNA sequencing. The differentially expressed miRNAs among the progressive COPD groups were identified using bioinformatics analysis. Then, the candidate miRNAs were externally verified. In addition, the target gene of this miRNA was identified, and its effects on cell activity, cell cycle, apoptosis, and other biological phenotypes of COPD were analyzed. Compared to the Normal group, a total of 35, 16, and 7 differentially expressed miRNAs were identified in the "fast" progressive COPD, "slow" progressive COPD group, and COPD group, respectively. The results were further confirmed using dual-luciferase reporter assay and transfection tests with phosphoinositide- 3-kinase, regulatory subunit 2 (PIK3R2) as a target gene of miR-4433a-5p; the result showed a negative regulatory correlation between the miRNA and its target gene. The phenotype detection showed that the activation of the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway might participate in the progression of COPD by promoting the proliferation of inflammatory A549 cells and inhibiting cellular apoptosis. MiR-4433a-5p can be used as a marker and potential therapeutic target for the progression of COPD. As a target gene of miR-4433a-5p, PIK3R2 can affect the progression of COPD by regulating phenotypes, such as cellular proliferation and apoptosis.

Molecular mechanisms of transmitted endoplasmic reticulum stress mediating immune escape of gastric cancer via PVR overexpression in TAMs

Gastric cancer (GC) is the fourth leading cause of cancer death worldwide. Due to the complex tumor microenvironment (TME), the efficacy of immunotherapy in GC has not met expectations. Malignant changes in the TME induce endoplasmic reticulum stress (ERS). ERS can be transmitted between tumor cells and tumor-associated macrophages (TAMs), promoting tumor immune escape, but the specific mechanism in GC remains unclear. We established a TAM model of transmitted ERS (TERS), and iTRAQ proteomic analysis identified overexpressed proteins. The overexpression of poliovirus receptor (PVR) was screened while flow cytometry and ELISA showed that PVR mediated the immunosuppressive function of TAMs by downregulating the proliferative activity and cytotoxicity of cocultured CD8+ T lymphocytes. With EMSA and dual-luciferase reporter assays, we confirmed that erythropoietin-producing hepatocellular receptor A2 (EphA2) affected PVR expression by increasing the transcriptional activity of activator protein-1 (AP-1). MFC cells were mixed with EphA2 knockdown or control RAW264.7 cells to establish subcutaneous tumor models with or without tunicamycin treatment in vivo. The vivo experiments revealed that ERS promoted subcutaneous xenograft growth, which was reversed by EphA2 knockdown. Clinically, GC patients with high expression of PVR and EphA2 tended to have an immunosuppressive TME, which were determined by immunohistochemical and immunofluorescence analyses. In conclusion, the transcriptional activity of AP-1 is upregulated in ERS-transmitted TAMs through EphA2 to increase PVR expression, which promotes immune escape in GC. Our study provides a new perspective on the role of ERS in tumor immunity.

Unveiling the molecular symphony: microRNA160a-Auxin Response Factor 18 module orchestrates low potassium tolerance in banana (Musa acuminata L.)

Potassium (K) is an essential nutrient for the growth and development of most plants. In banana (Musa acuminata L.), microRNA160a (miR160a) is suggested to potentially contribute to the response to low K+ stress by modulating the auxin signaling pathway. However, further investigation is required to elucidate its specific regulatory mechanism. This study presents evidence highlighting the critical role of the miR160a-Auxin Response Factor 18 (ARF18) module in conferring low K+ tolerance in banana. Both miR160a and its predicted target gene ARF18 displayed elevated expression levels in banana roots, with their expression profiles significantly altered under low K+ stress. The inhibitory effect of mac-miR160a on the expression of MaARF18-like-2 was confirmed through tobacco transient transformation and dual-Luciferase reporter assay. Surprisingly, Arabidopsis lines overexpressing mac-miR160a (mac-miR160a OE) exhibited enhanced tolerance to low K+ stress. Conversely, Arabidopsis lines overexpressing MaARF18-like-2 (MaARF18-like-2 OE) displayed increased sensitivity to K+ deficiency. Additionally, RNA sequencing (RNA-seq) analysis revealed that MaARF18-like-2 mediates the response of Arabidopsis to low K+ stress by influencing the expression of genes associated with Ca2+, ion transport, and reactive oxygen species (ROS) signaling. In conclusion, our study provides novel insights into the molecular mechanism of the miR160a-ARF18-like-2 module in the plant response to low K+ stress.

Overexpression of NOP58 Facilitates Proliferation, Migration, Invasion, and Stemness of Non-small Cell Lung Cancer by Stabilizing hsa_circ_0001550.

NOP58 ribonucleoprotein (NOP58) is associated with the recurrence of lung adenocarcinoma. Few investigations concentrate on the role of NOP58 in non-small cell lung cancer (NSCLC), which is the focus of our current study. Following transfection, the proliferation, migration, and invasion of NSCLC cells were assessed by 5- ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assays. The percentage of CD9+ cells was evaluated by flow cytometry assay. Based on target genes and binding sites predicted through bioinformatics analysis, a dual-luciferase reporter assay was performed to verify the targeting relationship between hsa_circ_0001550 and NOP58. The effect of NOP58 overexpression on hsa_circ_0001550 stability was gauged using Actinomycin D. The hsa_circ_0001550 and NOP58 expression levels, as well as protein expressions of CD44, CD133, OCT4, and SOX2 in NSCLC cells were determined by quantitative real-time PCR and Western blot, respectively. Hsa_circ_0001550 was remarkably up-regulated in NSCLC cell lines A549 and PC9, silencing of which weakened cell abilities to proliferate, migrate and invade, decreased CD9+ cell ratio, and diminished protein expressions of CD44, CD133, OCT4, and SOX2. NOP58 could bind to hsa_circ_0001550 and stabilize its expression, and NOP58 overexpression partially abrogated hsa_circ_0001550 knockdown-inhibited NSCLC cell proliferation, migration, invasion and stemness. Overexpression of NOP58 facilitates proliferation, migration, invasion, and stemness of NSCLC cells by stabilizing hsa_circ_0001550, hinting that NOP58 is a novel molecular target for NSCLC therapy.

ZBED3 exacerbates hyperglycemia by promoting hepatic gluconeogenesis through CREB signaling

BackgroundElevated hepatic glucose production (HGP) is a prominent manifestation of impaired hepatic glucose metabolism in individuals with diabetes. Increased hepatic gluconeogenesis plays a pivotal role in the dysregulation of hepatic glucose metabolism and contributes significantly to fasting hyperglycemia in diabetes. Previous studies have identified zinc-finger BED domain-containing 3 (ZBED3) as a risk gene for type 2 diabetes (T2DM), and its single nucleotide polymorphism (SNPs) is closely associated with the fasting blood glucose level, suggesting a potential correlation between ZBED3 and the onset of diabetes. This study primarily explores the effect of ZBED3 on hepatic gluconeogenesis and analyzes the relevant signaling pathways that regulate hepatic gluconeogenesis. MethodsThe expression level of ZBED3 was assessed in the liver of insulin-resistant (IR)-related disease. RNA-seq and bioinformatics analyses were employed to examine the ZBED3-related pathway that modulated HGP. To investigate the role of ZBED3 in hepatic gluconeogenesis, the expression of ZBED3 was manipulated by upregulation or silencing using adeno-associated virus (AAV) in mouse primary hepatocytes (MPHs) and HHL-5 cells. In vivo, hepatocyte-specific ZBED3 knockout mice were generated. Moreover, AAV8 was employed to achieve hepatocyte-specific overexpression and knockdown of ZBED3 in C57BL/6 and db/db mice. Immunoprecipitation and mass spectrometry (IP-MS) analyses were employed to identify proteins that interacted with ZBED3. Co-immunoprecipitation (co-IP), glutathione S-transferase (GST) - pulldown, and dual-luciferase reporter assays were conducted to further elucidate the underlying mechanism of ZBED3 in regulating hepatic gluconeogenesis. ResultsThe expression of ZBED3 in the liver of IR-related disease models was found to be increased. Under the stimulation of glucagon, ZBED3 promoted the expression of hepatic gluconeogenesis-related genes PGC1A, PCK1, G6PC, thereby increasing HGP. Consistently, the rate of hepatic gluconeogenesis was found to be elevated in mice with hepatocyte-specific overexpression of ZBED3 and decreased in those with ZBED3 knockout. Additionally, the knockdown of ZBED3 in the liver of db/db mice resulted in a reduction in hepatic gluconeogenesis. Moreover, the study revealed that ZBED3 facilitated the nuclear translocation of protein arginine methyltransferases 5 (PRMT5) to influence the regulation of PRMT5-mediated symmetrical dimethylation of arginine (s-DMA) of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), which in turn affects the phosphorylation of CREB and ultimately promotes HGP. ConclusionsThis study indicates that ZBED3 promotes hepatic gluconeogenesis and serves as a critical regulator of the progression of diabetes.

LINC00668 silencing retards tumorigenesis via sponging miR-518c-3p to regulating WDR1 in triple negative breast cancer

The emergence of long noncoding RNAs (lncRNAs) was proved to be crucial to the aggravation of triple negative breast cancer (TNBC), a fatal female malignancy. LINC00668 was unveiled as an overexpressed lncRNA in TNBC previously. However, its exact function and whether it functioned in TNBC development needs to be ascertained. To explore this, qRT-PCR was used to detect its dysregulation in TNBC cells. Biological functions of LINC00668 were determined through loss-of-function experiments. Bioinformatics analysis was utilized to predict the downstream modulatory genes of LINC00668. Dual-luciferase reporter assay plus RNA immunoprecipitation analysis, quantitative PCR analysis, and rescue assays were employed for the exploration of potential action of mode in competitive endogenous RNA (ceRNA) network. It was revealed that LINC00668 was upregulated and its depletion resulted in impeded proliferation and migration of TNBC cells. Bioinformatics analysis and mechanical assays uncovered that LINC00668 sponged miR-518c-3p to facilitate WDR1 level in TNBC. Furthermore, rescue experiments demonstrated that LINC00668/miR-518c-3p pathway contributed to TNBC cell proliferation and migration in the form of WDR1 dependency. Overall our study might discover a vital clue for the cure of lncRNA-directed treatment for TNBC patients.

Long Non-Coding RNA PVT1 Facilitates Radiation-Induced Vascular Endothelial Cell Injury through Sponging MicroRNA-9-5p.

Radiotherapy is a common therapeutic strategy for various solid tumors, with vascular endothelial injury being a common side effect. The study aimed to examine the effect of long non-coding RNA PVT1 on radiation-induced vascular endothelial cell injury, and explore the possible underlying mechanism. Human umbilical vein endothelial cells (HUVECs) were exposed to different doses of X ray to mimic radiation. LncRNA and miRNA levels were detected via qRT-PCR. Interaction between lncRNA and miRNAs was determined through dual-luciferase reporter assay. Statistical processing was conducted using student's t test between two groups and one-way ANOVA among multiple groups, and P < 0.05 means a significant difference. GO and KEGG were performed for the function and pathway enrichment analysis. LncRNA PVT1 elevated along with the increase of radiation dose in HUVECs. Poorly expressed lncRNA PVT1 promotes cell viability and inhibits oxidative stress. PVT1 serves as a competitive endogenous RNA (ceRNA) of miR-9-5p. miR-9-5p inhibitor inverted the influence of PVT1 knockdown on radiation-stimulated cell apoptosis and oxidative stress in HUVECs. KEGG analysis identified significant enrichment of the MAPK signaling pathway among overlapping target genes of miR-9-5p. LncRNA PVT1 knockdown alleviated radiation-induced vascular endothelial injury via sponging miR-9-5p. The underlying mechanism might be probably MAPK signaling-related.

Fam3a-mediated prohormone convertase switch in α-cells regulates pancreatic GLP-1 production in an Nr4a2-Foxa2-dependent manner

BackgroundFam3a has been demonstrated to regulate pancreatic β-cell function and glucose homeostasis. However, the role and mechanism of Fam3a in regulating α-cell function remain unexplored. MethodsGlucagon and glucagon-like peptide-1 (GLP-1) levels in pancreas and plasma were measured in global Fam3a knockout (Fam3a−/−) mice. Human islet single-cell RNA sequencing (scRNA-seq) datasets were utilized to analyze gene expression correlations between FAM3A and PCSK1 (encoding PC1/3, which processes proglucagon into GLP-1). Mouse pancreatic α-cell line αTC1.9 cells were transfected with Fam3a siRNA or plasmid for Fam3a knockdown or overexpression to explore the effects of Fam3a on PC1/3 expression and GLP-1 production. The downstream mediator (including Nr4a2) was identified by transcriptomic analysis, and its role was confirmed by Fam3a knockdown or overexpression in αTC1.9 cells. Based on the interacted protein of Nr4a2 and the direct binding to Pcsk1 promoter, the transcription factor Foxa2 was selected for further verification. Nuclear translocation assay and dual-luciferase reporter assay were used to clarify the involvement of Fam3a-Nr4a2-Foxa2 pathway in PC1/3 expression and GLP-1 production. Moreover, α-cell-specific Fam3a knockout (Fam3aα−/−) mice were constructed to evaluate the metabolic variables and hormone levels under normoglycemic, high-fat diet (HFD)-fed and streptozotocin (STZ)-induced diabetic conditions. Exendin 9–39 (Ex9), a GLP-1 receptor antagonist, was used to investigate GLP-1 paracrine effects in Fam3aα−/− mice and in their primary islets. ResultsCompared with wild-type mice, pancreatic and plasma active GLP-1 levels were increased in Fam3a−/− mice. Analysis of human islet scRNA-seq datasets showed a significant negative correction between FAM3A and PCSK1 in α-cells. Fam3a knockdown upregulated PC1/3 expression and GLP-1 production in αTC1.9 cells, while Fam3a overexpression displayed inverse effects. Transcriptomic analysis identified Nr4a2 as a key downstream mediator of Fam3a, and Nr4a2 expression in αTC1.9 cells was downregulated and upregulated by Fam3a knockdown and overexpression, respectively. Nr4a2 silencing increased PC1/3 expression, albeit Nr4a2 did not directly bind to Pcsk1 promoter. Instead, Nr4a2 formed a complex with Foxa2 to facilitate Fam3a-mediated Foxa2 nuclear translocation. Foxa2 negatively regulated PC1/3 expression and GLP-1 production. Besides, Foxa2 inhibited the transcriptional activity of Pcsk1 promoter at specific binding sites 10 and 6, and this inhibition was intensified by Nr4a2 in αTC1.9 cells. Compared with Flox/cre littermates, improved glucose tolerance, increased active GLP-1 level in pancreas and plasma, upregulated plasma insulin level in response to glucose, and decreased plasma glucagon level were observed in Fam3aα−/− mice. Primary islets isolated from Fam3aα−/− mice also showed an increase in active GLP-1 and insulin release. In addition, the insulinotropic effect of intra-islet GLP-1 was blocked by Ex9 in Fam3aα−/− mice and in their primary islets. Similarly, HFD-fed Fam3aα−/− mice also exhibited an improved glucose tolerance. Both HFD-fed and STZ-induced diabetic Fam3aα−/− mice showed an increased pancreatic active GLP-1 level, an elevated plasma insulin level and a reduced plasma glucagon level. ConclusionsFam3a deficiency in α-cells enhances pancreatic GLP-1 production to improve β-cell function via paracrine signaling in an Nr4a2-Foxa2-PC1/3-dependent manner. Our study unveils a novel strategy for reprogramming α-cell proglucagon processing output from glucagon to GLP-1 and deepen the understanding of crosstalk between α-cells and β-cells.