IRE1α regulates macrophage polarization in type 2 diabetic periodontitis through promoting endoplasmic reticulum stress.

OBJECTIVES: The aim of this study was to investigate the effect of 4µ8c, an inhibitor targeting the endoplasmic reticulum stress-associated factor IRE1α, on macrophage polarization in an experimental model of diabetic periodontitis through ex vivo experiments. MATERIALS AND METHODS: Local alveolar bone parameters were evaluated using Micro-CT following intraperitoneal administration of 4µ8c in mice with experimental diabetic periodontitis. Surface markers indicating macrophage polarization were identified using immunofluorescence. In vitro experiments were performed employing bone marrow-derived macrophages and gingival fibroblasts. Macrophage polarization was determined using flow cytometry. Principal impacted signaling pathways were identified through Western blot analysis. RESULTS: Results from both in vitro and in vivo experiments demonstrated that 4µ8c mitigated alveolar bone resorption and inflammation in mice with diabetic periodontitis. Furthermore, it modulated macrophage polarization towards the M2 phenotype and augmented M2 macrophage polarization through the MAPK signaling pathway. CONCLUSIONS: These findings suggest that inhibiting IRE1α can modulate macrophage polarization and alleviate ligature-induced diabetic periodontitis via the MAPK signaling pathway. This unveils a novel mechanism, offering a scientific foundation for the treatment of experimental diabetic periodontitis.

G-quadruplexes promote the motility in MAZ phase-separated condensates to activate CCND1 expression and contribute to hepatocarcinogenesis.

G-quadruplexes (G4s) can recruit transcription factors to activate gene expression, but detailed mechanisms remain enigmatic. Here, we demonstrate that G4s in the CCND1 promoter propel the motility in MAZ phase-separated condensates and subsequently activate CCND1 transcription. Zinc finger (ZF) 2 of MAZ is a responsible for G4 binding, while ZF3-5, but not a highly disordered region, is critical for MAZ condensation. MAZ nuclear puncta overlaps with signals of G4s and various coactivators including BRD4, MED1, CDK9 and active RNA polymerase II, as well as gene activation histone markers. MAZ mutants lacking either G4 binding or phase separation ability did not form nuclear puncta, and showed deficiencies in promoting hepatocellular carcinoma cell proliferation and xenograft tumor formation. Overall, we unveiled that G4s recruit MAZ to the CCND1 promoter and facilitate the motility in MAZ condensates that compartmentalize coactivators to activate CCND1 expression and subsequently exacerbate hepatocarcinogenesis.

Advanced glycation end products impair bone marrow mesenchymal stem cells osteogenesis in periodontitis with diabetes via FTO-mediated N6-methyladenosine modification of sclerostin.

BACKGROUND: Diabetes mellitus (DM) and periodontitis are two prevalent diseases with mutual influence. Accumulation of advanced glycation end products (AGEs) in hyperglycemia may impair cell function and worsen periodontal conditions. N6-methyladenosine (m6A) is an important post-transcriptional modification in RNAs that regulates cell fate determinant and progression of diseases. However, whether m6A methylation participates in the process of periodontitis with diabetes is unclear. Thus, we aimed to investigate the effects of AGEs on bone marrow mesenchymal stem cells (BMSCs), elucidate the m6A modification mechanism in diabetes-associated periodontitis. METHODS: Periodontitis with diabetes were established by high-fat diet/streptozotocin injection and silk ligation. M6A modifications in alveolar bone were demonstrated by RNA immunoprecipitation sequence. BMSCs treated with AGEs, fat mass and obesity associated (FTO) protein knockdown and sclerostin (SOST) interference were evaluated by quantitative polymerase chain reaction, western blot, immunofluorescence, alkaline phosphatase and Alizarin red S staining. RESULTS: Diabetes damaged alveolar bone regeneration was validated in vivo. In vitro experiments showed AGEs inhibited BMSCs osteogenesis and influenced the FTO expression and m6A level in total RNA. FTO knockdown increased the m6A levels and reversed the AGE-induced inhibition of BMSCs differentiation. Mechanically, FTO regulated m6A modification on SOST transcripts, and AGEs affected the binding of FTO to SOST transcripts. FTO knockdown accelerated the degradation of SOST mRNA in presence of AGEs. Interference with SOST expression in AGE-treated BMSCs partially rescued the osteogenesis by activating Wnt Signaling. CONCLUSIONS: AGEs impaired BMSCs osteogenesis by regulating SOST in an m6A-dependent manner, presenting a promising method for bone regeneration treatment of periodontitis with diabetes.

Identification of fabclavine derivatives, Fcl-7 and Fcl-8, from Xenorhabdus budapestensis as major antifungal natural products against Rhizoctonia solani.

AIMS: Black scurf disease, caused by Rhizoctonia solani, is a severe soil-borne and tuber-borne disease, which occurs and spreads in potato growing areas worldwide and poses a serious threat to potato production. New biofungicide is highly desirable for addressing the issue, and natural products (NPs) from Xenorhabdus spp. provide prolific resources for biofungicide development. In this study, we aim to identify antifungal NPs from Xenorhabdus spp. for the management of this disease. METHODS AND RESULTS: Out of the 22 Xenorhabdus strains investigated, Xenorhabdus budapestensis 8 (XBD8) was determined to be the most promising candidate with the measured IC50 value of its cell-free supernatant against R. solani as low as 0.19 ml l-1. The major antifungal compound in XBD8 started to be synthesized in the middle logarithmic phase and reached a stable level at stationary phase. Core gene deletion coupled with high-resolution mass spectrometry analysis determined the major antifungal NPs as fabclavine derivatives, Fcl-7 and 8, which showed broad-spectrum bioactivity against important pathogenic fungi. Impressively, the identified fabclavine derivatives effectively controlled black scurf disease in both greenhouse and field experiments, significantly improving tuber quality and increasing with marketable tuber yield from 29 300 to 35 494 kg ha-1, comparable with chemical fungicide fludioxonil. CONCLUSIONS: The fabclavine derivatives Fcl-7 and 8 were determined as the major antifungal NPs in XBD8, which demonstrated a bright prospect for the management of black scurf disease.

Macrocycle Self-Assembly Hydrogel for High-Efficient Oil-Water Separation.

Supramolecular hydrogels involved macrocycles have been explored widely in recent years, but it remains challenging to develop hydrogel based on solitary macrocycle with super gelation capability. Here, the construction of lantern[33 ]arene-based hydrogel with low critical gelation concentration (0.05 wt%), which can be used for efficient oil-water separation, is reported. The lantern[33 ]arenes self-assemble into hydrogen-bonded organic nanoribbons, which intertwine into entangled fibers to form hydrogel. This hydrogel which exhibits reversible pH-responsiveness characteristics can be coated on stainless-steel mesh by in situ sol-gel transformation. The resultant mesh exhibits excellent oil-water separation efficiency (>99%) and flux (>6 × 104 L m-2 h-1 ). This lantern[33 ]arene-based hydrogel not only sheds additional light on the gelation mechanisms for supramolecular hydrogels, but also extends the application of macrocycle-based hydrogels as functional interfacial materials.

Circular RNAs with protein-coding ability in oncogenesis.

As ubiquitously expressed transcripts in eukaryotes, circular RNAs (circRNAs) are covalently closed and lack a 5'-cap and 3'-polyadenylation (poly (A)) tail. Initially, circRNAs were considered non-coding RNA (ncRNA), and their roles as sponging molecules to adsorb microRNAs have been extensively reported. However, in recent years, accumulating evidence has demonstrated that circRNAs could encode functional polypeptides through the initiation of translation mediated by internal ribosomal entry sites (IRESs) or N6-methyladenosine (m6A). In this review, we collectively discuss the biogenesis, cognate mRNA products, regulatory mechanisms, aberrant expression and biological phenotypes or clinical relevance of all currently reported, cancer-relevant protein-coding circRNAs. Overall, we provide a comprehensive overview of circRNA-encoded proteins and their physiological and pathological functions.

Overcoming chemoresistance in non-angiogenic colorectal cancer by metformin via inhibiting endothelial apoptosis and vascular immaturity.

The development of chemoresistance which results in a poor prognosis often renders current treatments for colorectal cancer (CRC). In this study, we identified reduced microvessel density (MVD) and vascular immaturity resulting from endothelial apoptosis as therapeutic targets for overcoming chemoresistance. We focused on the effect of metformin on MVD, vascular maturity, and endothelial apoptosis of CRCs with a non-angiogenic phenotype, and further investigated its effect in overcoming chemoresistance. In situ transplanted cancer models were established to compare MVD, endothelial apoptosis and vascular maturity, and function in tumors from metformin- and vehicle-treated mice. An in vitro co-culture system was used to observe the effects of metformin on tumor cell-induced endothelial apoptosis. Transcriptome sequencing was performed for genetic screening. Non-angiogenic CRC developed independently of angiogenesis and was characterized by vascular leakage, immaturity, reduced MVD, and non-hypoxia. This phenomenon had also been observed in human CRC. Furthermore, non-angiogenic CRCs showed a worse response to chemotherapeutic drugs in vivo than in vitro. By suppressing endothelial apoptosis, metformin sensitized non-angiogenic CRCs to chemo-drugs via elevation of MVD and improvement of vascular maturity. Further results showed that endothelial apoptosis was induced by tumor cells via activation of caspase signaling, which was abrogated by metformin administration. These findings provide pre-clinical evidence for the involvement of endothelial apoptosis and subsequent vascular immaturity in the chemoresistance of non-angiogenic CRC. By suppressing endothelial apoptosis, metformin restores vascular maturity and function and sensitizes CRC to chemotherapeutic drugs via a vascular mechanism.

Metformin enhances T lymphocyte anti-tumor immunity by increasing the infiltration via vessel normalization.

Abnormal tumor vasculature blocks the extravasation of T lymphocytes into the tumor, thereby suppressing anti-tumor immunity. Recently, metformin has been shown to affect tumor vasculature and enhance T lymphocyte anti-tumor immunity. However, whether or how metformin affects T lymphocyte anti-tumor immunity via a vascular mechanism remains poorly understood. Herein, we show that a large number of CD8+ lymphocytes gathered in the peri-tumoral region, while very few infiltrated the tumor. Metformin administration increased the expression of anti-tumor immunity-associated genes and the number of tumor-infiltrating CD8+ lymphocytes. Injection of CD8 but not CD4 neutralization antibody into tumor-bearing mice significantly abrogated the anti-tumor effect of metformin. Critically, CD8+ lymphocytes were found to pass through the wall of perfused vessel. Further results of immunofluorescent staining showed that metformin greatly elevated tumor perfusion, which was accompanied by increased vascular maturity in the intratumoral region (ITR) but not peritumoral region (PTR). These findings provide evidence for the vascular mechanism involved in metformin-induced enhancement of T lymphocyte anti-tumor immunity. By remodeling the abnormal tumor vasculature, also called vessel normalization metformin increases vascular maturity and tumor perfusion, thus allowing more CD8+ lymphocytes to infiltrate the tumor.

Polydatin Inhibits Hepatocellular Carcinoma Cell Proliferation and Sensitizes Doxorubicin and Cisplatin through Targeting Cell Mitotic Machinery.

Polydatin (PD) is a natural compound with anticancer activities, but the underlying mechanisms remain largely unclear. To understand how PD inhibited hepatocellular carcinoma (HCC), we studied PD treatments in HCC HepG2 and SK-HEP1 cells, and normal liver HL-7702 cells. PD selectively blocked the proliferation of HCC cells but showed low toxicity in normal cells, while the effects of doxorubicin (DOX) and cisplatin (DDP) on HCC and normal liver cells were opposite. In the cotreatment studies, PD synergistically improved the inhibitory activities of DOX and DDP in HCC cells but alleviated their toxicity in HL-7702 cells. Furthermore, RNA-seq studies of PD-treated HepG2 cells revealed multiple altered signaling pathways. We identified 1679 Differentially Expressed Genes (DEGs) with over a 2.0-fold change in response to PD treatment. Integrative analyses using the DEGs in PD-treated HepG2 cells and DEGs in a TCGA dataset of HCC patients revealed five PD-repressed DEGs regulating mitotic spindle midzone formation. The expression of these genes showed significantly positive correlation with poor clinical outcomes of HCC patients, suggesting that mitotic machinery was likely a primary target of PD. Our findings improve the understanding of PD's anticancer mechanisms and provide insights into developing effective clinical approaches in HCC therapies.

Recognition elements based on the molecular biological techniques for detecting pesticides in food: A review.

Excessive use of pesticides can cause contamination of the environment and agricultural products that are directly threatening human life and health. Therefore, in the process of food safety supervision, it is crucial to conduct sensitive and rapid detection of pesticide residues. The recognition element is the vital component of sensors and methods for fast testing pesticide residues in food. Improper recognition elements may lead to defects of testing methods, such as poor stability, low sensitivity, high economic costs, and waste of time. We can use the molecular biological technique to address these challenges as a good strategy for recognition element production and modification. Herein, we review the molecular biological methods of five specific recognition elements, including aptamers, genetic engineering antibodies, DNAzymes, genetically engineered enzymes, and whole-cell-based biosensors. In addition, the application of these identification elements combined with biosensor and immunoassay methods in actual detection was also discussed. The purpose of this review was to provide a valuable reference for further development of rapid detection methods for pesticide residues.

Irisin Promotes Osteogenesis by Modulating Oxidative Stress and Mitophagy through SIRT3 Signaling under Diabetic Conditions.

Advanced glycation end products (AGEs) accumulate in the bone tissue of patients with diabetes mellitus, resulting in oxidative stress, poor bone healing, or regeneration. Irisin, a novel exercise-induced myokine, is involved in the regulation of bone metabolism. However, the effects of irisin on adipose-derived stem cell (ASC) osteogenic differentiation and bone healing under diabetic conditions remain poorly understood. ASCs were obtained from inguinal fat of Sprague-Dawley rats and treated with different concentrations of AGEs and irisin. Cell proliferation, apoptosis, and osteogenic differentiation abilities of ASCs were detected. To explore the regulatory role of sirtuin 3 (SIRT3), ASCs were transfected with lentivirus-mediated SIRT3 overexpression or knockdown vectors. Next, we investigated mitochondrial functions, mitophagy, and mitochondrial biogenesis in different groups. Moreover, SOD2 acetylation and potential signaling pathways were assessed. Additionally, a diabetic rat model was used to evaluate the effect of irisin on bone healing in calvarial critical-sized defects (CSDs) in vivo. Our results showed that irisin incubation mitigated the inhibitory effects of AGEs on ASCs by increasing cell viability and promoting osteogenesis. Moreover, irisin modulated mitochondrial membrane potential, intracellular ROS levels, mitochondrial O2 ·- status, ATP generation, complex I and IV activities, mitophagy, and mitochondrial biogenesis via a SIRT3-mediated pathway under AGEs exposure. Furthermore, in calvarial CSDs of diabetic rats, transplantation of gels encapsulating irisin-pretreated ASCs along with irisin largely enhanced bone healing. These findings suggest that irisin attenuates AGE-induced ASC dysfunction through SIRT3-mediated maintenance of oxidative stress homeostasis and regulation of mitophagy and mitochondrial biogenesis. Thus, our studies shed new light on the role of irisin in promoting the ASC osteogenesis and targeting SIRT3 as a novel therapeutic intervention strategy for bone regeneration under diabetic conditions.

C-reactive protein perturbs alveolar bone homeostasis: An experimental study of periodontitis and diabetes in the rat.

AIM: To explore the role of C-reactive protein (CRP) in periodontitis and diabetes and its mechanism in alveolar bone homeostasis. MATERIALS AND METHODS: In vivo, normal, and Crp knockout (KO) rats were randomly divided into control, diabetes, periodontitis, and diabetes and periodontitis groups, respectively. The diabetes model was established using a high-fat diet combined with streptozotocin injection. The periodontitis model was established by ligature combined with lipopolysaccharide (LPS) injection. Alveolar bones were analysed using micro-computed tomography, histology, and immunohistochemistry. In vitro, human periodontal ligament cells (hPDLCs) were treated with LPS and high glucose. CRP knockdown lentivirus or CRP overexpression adenovirus combined with a PI3K/AKT signalling inhibitor or agonist were used to explore the regulatory mechanism of CRP in osteogenesis and osteoclastogenesis of hPDLCs, as evidenced by alkaline phosphatase staining, Western blot, and quantitative polymerase chain reaction. RESULTS: In periodontitis and diabetes, CRP KO decreased the alveolar bone loss and the expression levels of osteoclastogenic markers, while increasing the expression levels of osteogenic markers. CRP constrained osteogenesis while promoting the osteoclastogenesis of hPDLCs via PI3K/AKT signalling under high glucose and pro-inflammatory conditions. CONCLUSIONS: CRP inhibits osteogenesis and promotes osteoclastogenesis via PI3K/AKT signalling under diabetic and pro-inflammatory conditions, thus perturbing alveolar bone homeostasis.

A histidine cluster determines YY1-compartmentalized coactivators and chromatin elements in phase-separated enhancer clusters.

As an oncogenic transcription factor, Yin Yang 1 (YY1) regulates enhancer and promoter connection. However, gaps still exist in understanding how YY1 coordinates coactivators and chromatin enhancer elements to assemble enhancers and super-enhancers. Here, we demonstrate that a histidine cluster in YY1's transactivation domain is essential for its formation of phase separation condensates, which can be extended to additional proteins. The histidine cluster is also required for YY1-promoted cell proliferation, migration, clonogenicity and tumor growth. YY1-rich nuclear puncta contain coactivators EP300, BRD4, MED1 and active RNA polymerase II, and colocalize with histone markers of gene activation, but not that of repression. Furthermore, YY1 binds to the consensus motifs in the FOXM1 promoter to activate its expression. Wild-type YY1, but not its phase separation defective mutant, connects multiple enhancer elements and the FOXM1 promoter to form an enhancer cluster. Consistently, fluorescent in situ hybridization (FISH) assays reveal the colocalization of YY1 puncta with both the FOXM1 gene locus and its nascent RNA transcript. Overall, this study demonstrates that YY1 activates target gene expression through forming liquid-liquid phase separation condensates to compartmentalize both coactivators and enhancer elements, and the histidine cluster of YY1 plays a determinant role in this regulatory mechanism.

Ethylene-responsive factor ERF114 mediates fungal pathogen effector PevD1-induced disease resistance in Arabidopsis thaliana.

APETALA2/ethylene-responsive factor (AP2/ERF) family transcription factors are well-documented in plant responses to a wide range of biotic and abiotic stresses, but their roles in mediating elicitor-induced disease resistance remains largely unexplored. PevD1 is a Verticillium dahliae secretory effector that can induce disease resistance in cotton and tobacco plants. In our previous work, Nicotiana benthamiana ERF114 (NbERF114) was identified in a screen of genes differentially expressed in response to PevD1 infiltration. Here, we found that the ortholog of NbERF114 in Arabidopsis thaliana (ERF114) also strongly responded to PevD1 treatment and transcripts were induced by Pseudomonas syringae pv. tomato (Pst) DC3000 infection. Loss of ERF114 function caused impaired disease resistance, while overexpressing ERF114 (OE-ERF114) enhanced resistance to Pst DC3000. Moreover, ERF114 mediated PevD1-induced disease resistance. RNA-sequencing analysis revealed that the transcript level of phenylalanine ammonia-lyase1 (PAL1) and its downstream genes were significantly suppressed in erf114 mutants compared with A. thaliana Col-0. Reverse transcription-quantitative PCR (RT-qPCR) analysis further confirmed that the PAL1 mRNA level was significantly elevated in overexpressing OE-ERF114 plants but reduced in erf114 mutants compared with Col-0. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) and electrophoretic mobility shift assay verified that ERF114 directly bound to the promoter of PAL1. The gene expression profiles of ERF114 and PAL1 in oestradiol-inducible transgenic plants confirmed ERF114 could activate PAL1 transcriptional expression. Further investigation revealed that ERF114 positively modulated PevD1-induced lignin and salicylic acid accumulation, probably by activating PAL1 transcription.

BCAS2 is involved in alternative splicing and mouse oocyte development.

Alternative splicing (AS) is an important mechanism to regulate organogenesis and fertility. Breast carcinoma amplified sequence 2 (BCAS2) is one of the core components of the PRP19 complex, a multiple function complex including splicing, and it is involved in the initiation of meiosis through regulating AS in male mice. However, the role of BCAS2 in mouse oogenesis remains largely unknown. In this study, we found that BCAS2 was highly expressed in the oocytes of primordial follicles. Vasa-Cre-mediated deletion of Bcas2 caused poor oocyte quality, abnormal oogenesis and follicular development. The deletion of Bcas2 in mouse oocytes caused alteration in 991 AS events that corresponded to 706 genes, including Pabpc1l, Nobox, Zfp207, Mybl2, Prc1, and Spc25, which were associated with oogenesis and spindle assembly. Moreover, the disruption of BCAS2 led to degradation of PRP19 core proteins in mouse oocytes. These results suggested that BCAS2 was involved in the AS of functional genes through PRP19 complex during mouse oocyte development.

The Regulatory Network of Sturgeon Chondroitin Sulfate on Colorectal Cancer Inhibition by Transcriptomic and Proteomic Analysis.

Chondroitin sulfate (CS) is a food-derived bioactive substance with multiple biological functions, which exists in animal cartilage and/or bone. Sturgeon, a type of cartilaginous fish, is rich in CS. Our recent study demonstrated the effect of sturgeon chondroitin sulfate (SCS) on reducing colorectal cancer cell proliferation and tumor formation. However, the molecular mechanisms of its anticancer activity remain unknown. In this study, the cell proliferation assay and flow cytometric analysis were used to examine the cell viability and apoptosis of colon cancer cell HT-29 cells and normal colonic epithelial cell NCM460 cells. Transcriptomic and proteomic studies were used to identify the main targets of SCS. SCS showed little effect on the genes/proteins expression profile of NCM460 cells but more sensitive to HT-29, in which 188 genes and 10 proteins were differentially expressed after SCS treatment. Enrichment analysis of those genes/proteins showed that the majority of them are involved in DNA replication, cell cycle progression and apoptosis. Quantitative RT-PCR and Western blot were used to determine essential genes/proteins and networks targeted by SCS to exert inhibiting the development of colorectal cancer function. This study provided great insights into developing food-derived novel therapeutics for colorectal cancer treatment.

SRSF5 regulates alternative splicing of DMTF1 pre-mRNA through modulating SF1 binding.

ABBREVIATIONS: ARF: alternative reading frame, that is, p14ARF, or CDKN2A (cyclin-dependent kinase inhibitor 2A); ß-gal: ß-galactosidase; CLIP-seq: crosslinking and immunoprecipitation-sequencing; DMTF1: the cyclin D binding myb-like transcription factor 1; ESS/ESE: exonic splicing silencer/enhancer; Ex: exon; FBS: fetal bovine serum; Gluc: Gaussia luciferase; hnRNPs: heterogeneous nuclear ribonucleoproteins; In: intron; ISS/ISE: intronic splicing silencer/enhancer; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PSI: percent-splice-in; qPCR: quantitative real-time PCR; RIP: RNA immunoprecipitation; RNAseq: RNA sequencing; RT: reverse transcription; SF1: splicing factor 1; SR: serine/arginine-rich proteins; SRSF5: serine and arginine-rich splicing factor 5; TCGA: the cancer genome atlas; UCSC: University of California, Santa Cruz. WT: Wild type.

Disruption of YY1-EZH2 Interaction Using Synthetic Peptides Inhibits Breast Cancer Development.

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase to mediate lysine 27 trimethylation in histone H3 (i.e., H3K27me3) and repress gene expression. In solid tumors, EZH2 promotes oncogenesis and is considered a therapeutic target. As a transcription factor, Yin Yang 1 (YY1) recruits EZH2 through its oncoprotein binding (OPB) domain to establish gene repression. In this study, we mapped the YY1 protein binding (YPB) domain on EZH2 to a region of 27 amino acids. Both YPB and OPB domain synthetic peptides could disrupt YY1EZH2 interaction, markedly reduce breast cancer cell viability, and efficiently inhibit tumor growth in a xenograft mouse model. We analyzed MDA-MB-231 cells treated with YPB, OPB, and control peptides by chromatin immunoprecipitation DNA sequencing (ChIP-seq) using an antibody against H3K27me3. YPB and OPB treatments altered H3K27me3 on 465 and 1137 genes, respectively, compared to the control. Of these genes, 145 overlapped between the two peptides. Among them, PTENP1, the PTEN pseudogene, showed reduced H3K27me3 signal when treated by either YPB or OPB peptide. Consistently, the two peptides enhanced both PTENP1 and PTEN expression with concomitantly reduced AKT activation. Further studies validated PTENP1's contribution to the anticancer activity of YPB and OPB peptides.

AKT1 is positively regulated by G-quadruplexes in its promoter and 3'-UTR.

AKT1 plays a key role in cell growth and survival, and its activation in cancers is mediated by different mechanisms. In this study, we investigated the potential of G-quadruplex (G4) formation by multiple consecutive G-tracts in the AKT1 promoter and its 3'-UTR. In circular dichroism analyses, synthetic oligonucleotides based on these G-tract regions showed molar ellipticity peaks at specific wavelengths of G4 structures. We verified G4 forming potential of these oligonucleotides using dimethyl sulfate footprinting, gel-shift and immunostaining assays. In reporter assays, mutations of the G-tracts in either the promoter or the 3'-UTR of AKT1 reduced expression mediated by these G-rich regions, suggesting positive regulation of AKT1 gene expression by these G4 structures. Furthermore, SP1 bound to its consensus sites regardless of the presence of G4 motifs in the AKT1 promoter, and both the G4 motifs and SP1 binding sites were needed to reach the strongest promoter strength.

Transcriptome analysis of signaling pathways targeted by Ellagic acid in hepatocellular carcinoma cells.

BACKGROUND: Ellagic acid (EA) possesses prominent inhibitory activities against various cancers, including hepatocellular carcinoma (HCC). Our recent study demonstrated EA's activities in reducing HCC cell proliferation and tumor formation. However, the mechanisms of EA to exert its anticancer activities and its primary targets in cancer cells have not been systematically explored. METHODS: Cell proliferation assay and flow cytometric analysis were used to examine the effects of EA treatment on viability and apoptosis, respectively, of HepG2 cells. RNA-seq studies and associated pathway analyses by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed to determine EA's primary targets. Differentially expressed genes (DEG) in EA-treated HepG2 cells were verified by RT-qPCR and Western blot. Integrative analyses of the RNA-seq dataset with a TCGA dataset derived from HCC patients were conducted to verify EA-targeted genes and signaling pathways. Interaction network analysis of the DEGs, shRNA-mediated knockdown, cell viability assay, and colony formation assay were used to validate EA's primary targets. RESULTS: EA reduced cell viability, caused DNA damage, and induced cell cycle arrest at G1 phase of HepG2 cells. We identified 5765 DEGs encoding proteins with over 2.0-fold changes in EA-treated HepG2 cells by DESeq2. These DEGs showed significant enrichment in the pathways regulating DNA replication and cell cycle progression. As primary targets, p21 was significantly upregulated, while MCM2-7 were uniformly downregulated in response to EA treatment. Consistently, p21 knockdown desensitized liver cells to EA in cell viability and colony formation assays. CONCLUSION: EA induced G1 phase arrest and promoted apoptosis of HCC cells through activating the p21 gene and downregulating the MCM2-7 genes, respectively. GENERAL SIGNIFICANCE: The discoveries in this study provide helpful insights into developing novel strategies in the therapeutic treatment of HCC patients.