Luciferase Reporter Assay Research Articles

Qingshen Granules alleviates renal fibrosis in mice by regulating exosomes, miR-330-3p, and CREBBP expression

To explore the effects of Qingshen Granules (QSG) on adenine-induced renal fibrosis in mice and in uric acid (UA)-stimulated NRK-49F cells and its mechanism for regulating exosomes, miR-330-3p and CREBBP. A mouse model of adenine-induced renal fibrosis were treated daily with QSG at 8.0 g·kg-1·d-1 via gavage for 12 weeks. An adenoassociated virus vector was injected into the tail vein, and renal tissues of the mice were collected for analyzing exosomal marker proteins CD9, Hsp70, and TSG101 and expressions of Col-III, α-SMA, FN, and E-cad using Western blotting and immunofluorescence and for observing pathological changes using HE and Masson staining. In the cell experiment, NRK-49F cells were stimulated with uric acid (400 μmol/L) followed by treatment with QSG-medicated serum from SD rats, and the changes in expressions of the exosomal markers and Col-III, α-SMA, FN, and E-cad were analyzed. Dual luciferase reporter assay was employed to examine the targeting relationship between miR-330-3p and CREBBP, whose expressions were detected by RT-qPCR and Western blotting in treated NRK-49F cells. The mouse models of adenine-induced renal fibrosis showed significantly increased levels of CD9, Hsp70, and TSG101, which were decreased by treatment with QSG. The expressions of Col-III, α-SMA, and FN increased and Ecad decreased in the mouse models but these changes were reversed by QSG treatment. QSG treatment obviously alleviated renal fibrosis in the mouse models. Intravenous injection of adeno-associated viral vector obviously inhibited miR-330-3p, increased CREBBP levels, and reduced fibrosis in the mouse models. Dual luciferase assay confirmed CREBBP as a target of miR-330-3p, which was consistent with the results of the cell experiments. QSG inhibits renal fibrosis in mice by regulating the exosomes, reducing miR-330-3p levels, and increasing CREBBP expression.

A specific GAGTT insertion/deletion variation in the IL-10 gene promoter alters the disease resistance of grass carp

IL-10, an anti-inflammatory cytokine, plays crucial roles in the context of infectious inflammatory diseases, whose gene variations and expression are tightly linked to disease resistance in animals. However, there is limited research on the genetic variations governing IL-10 expression and the underlying effects in fish. In this study, we identified a GAGTT insertion/deletion (Ins/Del) variation within the IL-10 gene promoter and analyzed the effects of this variation on IL-10 expression, inflammatory response and its association with disease resistance in grass carp. With dual luciferase reporter assays, we demonstrated that the GAGTT Ins/Del variation significantly altered the IL-10 promoter activity in grass carp. DNA pull-down combined with mass spectrometry revealed that the spectrum of transcription factors that bind to IL-10 promoter variants caused by GAGTT Ins/Del variation was quite different. Moreover, three representative transcription factors, JunD/AP-1, p50/NF-κB, and HMGB1, differentially transactivated grass carp IL-10 promoter variants, as confirmed by western blotting and dual luciferase reporter assays. Using the GAGTT/− genotype of the heterozygous grass carp parents, we obtained full-sib family offspring of the GAGTT/GAGTT, GAGTT/−, and −/− genotypes. During grass carp reovirus (GCRV) infection, these three IL-10 genotypes of grass carp offspring displayed different survival rates. Importantly, grass carp with the GAGTT/− genotype had greater survival than those with the other two genotypes after GCRV infection. Furthermore, the results indicated that the improved intestinal inflammation at 1 d post-GCRV infection and enhanced immunocompetence in the spleen at 5 d post-GCRV infection may contribute to the disease resistance of grass carp with the GAGTT/− genotype. Overall, this study offers new insight into the inflammatory and immune regulatory mechanisms specific to IL-10 from a genetic perspective in fish and may provide a valuable molecular marker for breeding disease-resistant grass carp.

Positive cooperativity in synergistic activation of Wnt proteins

BackgroundWnt proteins are crucial for embryonic development, stem cell growth, and tissue regeneration. Wnt signaling pathway is activated when Wnt proteins bind to cell membrane receptors.Methods and resultsWe employed a luciferase reporter assay in HEK293STF cells to measure Wnt protein-induced signaling. We observed that Wnt3a uniquely promotes the Wnt/β-catenin pathway through positive cooperativity. Additionally, MFH-ND, a molecular mimic of Wnt ligands, markedly increased Wnt3a-induced signaling in a dose-responsive manner. This suggests that various Wnt ligands can synergistically enhance Wnt pathway activation.ConclusionsThe study suggests the likelihood of various Wnt ligands coexisting in a single signalosome on the cell membrane, providing new insights into the complexities of Wnt signaling mechanisms.

Ethyl Acetate Fractions of Salvia miltiorrhiza Bunge (Danshen) Crude Extract Modulate Fibrotic Signals to Ameliorate Diabetic Kidney Injury.

Diabetic nephropathy, a leading cause of end-stage renal disease, accounts for significant morbidity and mortality. It is characterized by microinflammation in the glomeruli and myofibroblast activation in the tubulointerstitium. Salvia miltiorrhiza Bunge, a traditional Chinese medicine, is shown to possess anti-inflammatory and anti-fibrotic properties, implying its renal-protective potential. This study investigates which type of component can reduce the damage caused by diabetic nephropathy in a single setting. The ethyl acetate (EtOAc) layer was demonstrated to provoke peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ activities in renal mesangial cells by dual luciferase reporter assay. In a high glucose (HG)-cultured mesangial cell model, the EtOAc layer substantially inhibited HG-induced elevations of interleukin-1β, transforming growth factor-β1 (TGF-β1), and fibronectin, whereas down-regulated PPAR-γ was restored. In addition, among the extracts of S. miltiorrhiza, the EtOAc layer effectively mitigated TGF-β1-stimulated myofibroblast activation. The EtOAc layer also showed a potent ability to attenuate renal hypertrophy, proteinuria, and fibrotic severity by repressing diabetes-induced proinflammatory factor, extracellular matrix accumulation, and PPAR-γ reduction in the STZ-induced diabetes mouse model. Our findings, both in vitro and in vivo, indicate the potential of the EtOAc layer from S. miltiorrhiza for future drug development targeting diabetic nephropathy.

LncRNA MALAT1 Knockdown Alleviates Fibrogenic Response in Human Endometrial Stromal Cells Via the miR-22-3p/TGFβR1/Smad2/3 Pathway.

Intrauterine adhesion (IUA) resulting from irreversible fibrotic repair of endometrium is the main cause of secondary infertility in women, and current therapeutic approaches to IUA are limited. Increasing evidence has suggested the important role of competitive endogenous RNA (ceRNA) in IUA pathologies. This study aimed to investigate the long noncoding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1)-associated ceRNA in IUA development. We harvested endometrial tissues from patients with or without IUA and extracted endometrial stromal cells (ESCs) from normal endometrial tissues. Transforming growth factor β1 (TGF-β1) was used to induce fibrosis in ESCs. The expression of transforming growth factor β receptor 1 (TGFβR1), α-smooth muscle actin, phosphorylated suppressor of mother against decapentaplegic (p-Smad)2/3, collagen type I alpha 1, MALAT1, and microRNA (miR)-22-3p in endometrial tissues and ESCs was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) or western blotting. Pearson's correlation analysis was conducted to assess the correlation between miR-22-3p expression or TGFβR1 and MALAT1 expression in endometrial tissues. The expression of TGFβR1 in ESCs was also evaluated by immunofluorescence staining. The location of MALAT1 was examined by fluorescence in situ hybridization. Luciferase reporter assays were performed to verify the binding relationship between MALAT1 or TGFβR1 and miR-22-3p. Cell viability was assessed via cell counting kit-8 assays. Our findings revealed that lncRNA MALAT1 and TGFβR1 were upregulated while miR-22-3p was downregulated in IUA endometrial tissues or TGF-β1-stimulated ESCs, and lncRNA MALAT1 expression was negatively correlated with miR-22-3p expression while being positively correlated with TGFβR1 expression in IUA endometrial tissues. Additionally, lncRNA MALAT1 was mainly located in the cytoplasm of ESCs and directly targeted miR-22-3p to regulate TGFβR1 expression. Moreover, knockdown of lncRNA MALAT1 exerted anti-fibrotic effects on ESCs by targeting miR-22-3p, and miR-22-3p overexpression inhibited the fibrosis of ESCs by binding to TGFβR1 3'untranslated region. Collectively, lncRNA MALAT1 promotes endometrial fibrosis by sponging miR-22-3p to regulate TGFβR1 and Smad2/3, and inhibition of MALAT1 may represent a promising therapeutic option for suppressing endometrial fibrosis.

The feedback loop between MTA1 and MTA3/TRIM21 modulates stemness of breast cancer in response to estrogen

The metastasis-associated protein (MTA) family plays a crucial role in the development of breast cancer, a common malignancy with a high incidence rate among women. However, the mechanism by which each member of the MTA family contributes to breast cancer progression is poorly understood. In this study, we aimed to investigate the roles of MTA1, MTA3, and tripartite motif-containing 21 (TRIM21) in the proliferation, invasion, epithelial-mesenchymal transition (EMT), and stem cell-like properties of breast cancer cells in vivo and in vitro. The molecular mechanisms of the feedback loop between MTA1 and MTA3/TRIM21 regulated by estrogen were explored using Chromatin immunoprecipitation (ChIP), luciferase reporter, immunoprecipitation (IP), and ubiquitination assays. These findings demonstrated that MTA1 acts as a driver to promote the progression of breast cancer by repressing the transcription of tumor suppressor genes, including TRIM21 and MTA3. Conversely, MTA3 inhibited MTA1 transcription and TRIM21 regulated MTA1 protein stability in breast cancer. Estrogen disrupted the balance between MTA1 and MTA3, as well as between MTA1 and TRIM21, thereby affecting stemness and the EMT processes in breast cancer. These findings suggest that MTA1 plays a vital role in stem cell fate and the hierarchical regulatory network of EMT through negative feedback loops with MTA3 or TRIM21 in response to estrogen, supporting MTA1, MTA3, and TRIM21 as potential prognostic biomarkers and MTA1 as a treatment target for future breast cancer therapies.

MiR-185–5p rewires cisplatin resistance by restoring miR-203a-3p expression via downregulation of SOX9

Chemotherapeutic drug resistance is a challenge for the effective treatment of OSCC. There are a couple of studies on the involvement of microRNAs (miRNAs) in chemoresistance of oral squamous cell carcinoma (OSCC), but the exact molecular events in many cases are not clearly understood. In this work, we intend to track down key miRNA(s) and unveil their regulatory molecular mechanisms in imparting chemoresistance in this lethal cancer. We analyzed gene and miRNA array profiles of drug-resistant OSCC cells, predicted miRNA targets, performed enrichment analysis, and validated our findings in cisplatin-sensitive and cisplatin-resistant SCC9 and H357 OSCC cells. We evaluated the anticancer and chemosensitivity roles of selected miRNA by adopting several molecular assays like qRT-PCR, MTT assay, wound healing assay, fluorescence imaging by DCFHDA, AO/EB staining, DAPI, and γ-H2AX accumulation assay. We also validated the miRNA-target binding by qRT-PCR and luciferase reporter assay. Among the enriched miRNAs, we found miR-185–5p downregulated in cisplatin-resistant OSCC cells as a signature miRNA modulating chemoresistance. The upregulation of miR-185–5p by mimic transfection restores cisplatin sensitivity by decreasing cell viability in a dose-dependent manner and increasing ROS-induced DNA damage and apoptosis. miR-185–5p overexpression increases miR-203a-3p expression through negative regulation of SOX9. siRNA-mediated silencing of the SOX9 also shows similar results. Mechanistically, miR-185–5p dependent miR-203a-3p expression decreases cisplatin efflux and cisplatin-induced DNA damage repair by regulating ABCC1, ABCB1, RRM2, and RAN. This study will pave the way for employing this miR-185–5p as a combination therapeutic strategy to combat cisplatin resistance in oral cancer.

HSP70 promotes pancreatic cancer cell epithelial-mesenchymal transformation and growth via the NF-κB signaling pathway.

To study the effects of HSP70 on proliferation, migration, invasion, and epithelial-mesenchymal transformation (EMT) of pancreatic cancer cells and explore its underlying mechanisms. Pancreatic cancer cell models with either reduced HSP70 or increased HSP70 expression were established. RT-qPCR and Western blot assays were used to determine mRNA and protein levels of HSP70, IKK/IκBa/NF-κB signaling pathway-related genes, and EMT markers. The CCK-8 and cell cloning assays were used to evaluate cell proliferation and cloning abilities. Transwell and wound healing assays were used to assess the invasive and migratory properties of the cells. Effects of NF-κB signaling modulation were explored using an IKK inhibitor (BAY11-7082) and an IKK overexpression vector (pCMV-IKK). Electrophoretic mobility shift assay (EMSA) and luciferase reporter assays were conducted to analyze NF-κB's promoter binding and transcriptional activities. HSP70 knockdown inhibited p-p65 nuclear translocation and reduced the expression of p-p65, p-IKKα/β, p-IκBα, N-cadherin, Vimentin, and Twist. It also decreased NF-κB's promoter binding and transcriptional activities, increased E-cadherin levels, and suppressed pancreatic cancer cell proliferation, cloning, migration, and invasion. In contrast, HSP70 overexpression led to increased expression of p-p65, p-IKKα/β, p-IκBα, N-cadherin, Vimentin, and Twist, decreased E-cadherin levels, and enhanced cell proliferation, cloning, migration, and invasion capabilities. NF-κB signaling pathway modulation reversed EMT changes induced by altered HSP70 expression levels. rhHSP70 also increased p-IKKα/β and p-IκBα protein levels. HSP70 promotes the EMT and enhances pancreatic cancer cell proliferation, migration, and invasion by activating the NF-κB pathway.

Vorinostat Treatment of Gastric Cancer Cells Leads to ROS-Induced Cell Inhibition and a Complex Pattern of Molecular Alterations in Nrf2-Dependent Genes.

Histone deacetylase inhibitors (HDACi) show high antineoplastic potential in preclinical studies in various solid tumors, including gastric carcinoma; however, their use in clinical studies has not yet yielded convincing efficacies. Thus, further studies on cellular/molecular effects of HDACi are needed, for improving clinical efficacy and identifying suitable combination partners. Here, we investigated the role of oxidative stress in gastric cancer cells upon treatment with HDACi. A particular focus was laid on the role of the Nrf2 pathway, which can mediate resistance to cell-inhibitory effects of reactive oxidative species (ROS). Using fluorescence-based ROS sensors, oxidative stress was measured in human gastric cancer cell lines. Activation of the Nrf2 pathway was monitored in luciferase reporter assays as well as by mRNA and proteomic expression analyses of Nrf2 regulators and Nrf2-induced genes. Furthermore, the effects of ROS scavenger N-acetyl-L-cysteine (NAC) and Nrf2-knockdown on HDACi-dependent antiproliferative effects were investigated in colorimetric formazan-based and clonogenic survival assays. HDACi treatment led to increased oxidative stress levels and consequently, treatment with NAC reduced cytotoxicity of HDACi. In addition, vorinostat treatment stimulated expression of a luciferase reporter under the control of an antioxidative response element, indicating activation of the Nrf2 system. This Nrf2 activation was only partially reversible by treatment with NAC, suggesting ROS independent pathways to contribute to HDACi-promoted Nrf2 activation. In line with its cytoprotective role, Nrf2 knockdown led to a sensitization against HDACi. Accordingly, the expression of antioxidant and detoxifying Nrf2 target genes was upregulated upon HDACi treatment. In conclusion, oxidative stress induction upon HDAC inhibition contributes to the antitumor effects of HDAC inhibitors, and activation of Nrf2 represents a potentially important adaptive response of gastric cancer cells in this context.

C-Myc-XRCC2-FOS axis promotes the proliferation and the resistance to Doxorubicin of NSCLC

Lung cancer represents one of the most prevalent malignant neoplasms, commanding an alarming incidence and mortality rate globally. Non-small cell lung cancer (NSCLC), constituting approximately 80 %-90 % of all lung cancer cases, is the predominant pathological manifestation of this disease, with a disconcerting 5-year survival rate scarcely reaching 10 %. Extensive prior investigations have elucidated that the aberrant expression of X-ray repair cross-complementing gene 2 (XRCC2), a critical meiotic gene intricately involved in the DNA damage repair process, is intimately associated with tumorigenesis. Nevertheless, the precise roles and underlying mechanistic pathways of XRCC2 in NSCLC remain largely elusive. In the present study, we discerned an overexpression of XRCC2 within NSCLC patient tissues, particularly in high-grade samples, when juxtaposed with normal tissues. Targeted knockdown of XRCC2 notably impeded the proliferation of NSCLC both in vitro and in vivo. Comprehensive RNA sequencing and flow rescue assays unveiled that XRCC2 augments the proliferation of NSCLC cells through the down-regulation of FOS expression. Moreover, the c-Myc gene was definitively identified as an XRCC2 transcriptional factor by means of chromatin immunoprecipitation (ChIP) and luciferase reporter assays, whereby pharmacological attenuation of c-Myc expression, in conjunction with Doxorubicin, synergistically curtailed NSCLC cell growth both in vitro and in vivo. Collectively, our findings proffer critical insights into the novel c-Myc-XRCC2-FOS axis in promoting both proliferation and resistance to Doxorubicin in NSCLC cells, thereby extending a promising avenue for potential new diagnostic strategies and therapeutic interventions in NSCLC.

MicroRNA-124-3p targets Sp1 transcription factor to regulate glioma progression in rats

Gliomas are the most prevalent malignancies of the central nervous system (CNS). Downregulation of microRNA‑124 (miR‑124) has been identified in glioma; however, its biological functions in glioma are not yet fully understood. Specificity protein 1 (SP1) is a type of transcription factor that is involved in cancer progression. In this study, we examined the targeting of Sp1 mRNA by miR-124-3p in a rat glioma model. After confirming and selecting the binding of Sp1 to miR-124 with the help of bioinformatics methods, adult male Wistar rats were used to induce glioma by microinjection of 1 × 106 C6 cells into the striatum area of brain. The rats were divided into 3 groups; intact, sham and glioma groups. The presence of glioma was confirmed 21 days after implantation through histological analysis. The expression levels of miR-124 and SP1 genes in the experimental groups were examined using quantitative real-time polymerase chain reaction (qRT-PCR). Our data showed that the expression of miR-124 was significantly downregulated in glioma group compared to the sham and intact group, while the expression of SP1 was significantly upregulated. We found that the expression levels of miR-124 and Sp1 were decreased and increased in C6 cell line compared to the normal brain tissue cell line, respectively. The results indicated that Sp1 was identified as a direct target of miR‑124 through luciferase reporter assays. In summary, this study demonstrated for the first time that miR-124 expression is downregulated and Sp1 expression is upregulated in an animal model of glioma, which, in turn, may be involved in the development of glioma brain cancer.

Restoration of miR-299-3p promotes efferocytosis and ameliorates atherosclerosis via repressing CD47 in mice.

Atherosclerotic plaque formation is largely attributed to the impaired efferocytosis, which is known to be associated with the pathologic upregulation of cluster of differentiation 47 (CD47), a key antiphagocytic molecule. By gene expression omnibus (GEO) datasets analysis, we identified that four miRNAs are aberrantly downregulated in atherosclerosis, coronary artery disease, and obesity. Of them, hsa-miR-299-3p (miR-299-3p) was predicted to target the 3'UTR of human CD47 mRNA by bioinformatics analysis. Further, we demonstrated that miR-299-3p negatively regulates CD47 expression by binding to the target sequence "CCCACAU" in the 3'UTR of CD47 mRNA through luciferase reporter assay and site-directed mutagenesis. Additionally, we found that miR-299-3p was downregulated by ~32% in foam cells in response to oxidized low-density lipoprotein (ox-LDL) stimulation, thus upregulating CD47 and contributing to the impaired efferocytosis. Whereas, restoration of miR-299-3p reversed the ox-LDL-induced upregulation of CD47, thereby facilitating efferocytosis. In high-fat diet (HFD) fed ApoE-/- mice, we discovered that miR-299-3p was downregulated thus leading to upregulation of CD47 in abdominal aorta. Conversely, miR-299-3p restoration potently suppressed HFD-induced upregulation of CD47 and promoted phagocytosis of foam cells by macrophages in atherosclerotic plaques, thereby reducing necrotic core, increasing plaque stability, and mitigating atherosclerosis. Conclusively, we identify miR-299-3p as a negative regulator of CD47, and reveal a molecular mechanism whereby the ox-LDL-induced downregulation of miR-299-3p leads to the upregulation of CD47 in foam cells thus contributing to the impaired efferocytosis in atherosclerosis, and propose miR-299-3p can potentially serve as an inhibitor of CD47 to promote efferocytosis and ameliorate atherosclerosis.

Large-Scale Alternative Polyadenylation-Wide Association Studies to Identify Putative Cancer Susceptibility Genes.

Alternative polyadenylation (APA) modulates mRNA processing in the 3'-untranslated regions (3' UTR), affecting mRNA stability and translation efficiency. Research into genetically regulated APA has the potential to provide insights into cancer risk. In this study, we conducted large APA-wide association studies to investigate associations between APA levels and cancer risk. Genetic models were built to predict APA levels in multiple tissues using genotype and RNA sequencing data from 1,337 samples from the Genotype-Tissue Expression project. Associations of genetically predicted APA levels with cancer risk were assessed by applying the prediction models to data from large genome-wide association studies of six common cancers among European ancestry populations: breast, ovarian, prostate, colorectal, lung, and pancreatic cancers. A total of 58 risk genes (corresponding to 76 APA sites) were associated with at least one type of cancer, including 25 genes previously not linked to cancer susceptibility. Of the identified risk APAs, 97.4% and 26.3% were supported by 3'-UTR APA quantitative trait loci and colocalization analyses, respectively. Luciferase reporter assays for four selected putative regulatory 3'-UTR variants demonstrated that the risk alleles of 3'-UTR variants, rs324015 (STAT6), rs2280503 (DIP2B), rs1128450 (FBXO38), and rs145220637 (LDHA), significantly increased the posttranscriptional activities of their target genes compared with reference alleles. Furthermore, knockdown of the target genes confirmed their ability to promote proliferation and migration. Overall, this study provides insights into the role of APA in the genetic susceptibility to common cancers. Significance: Systematic evaluation of associations of alternative polyadenylation with cancer risk reveals 58 putative susceptibility genes, highlighting the contribution of genetically regulated alternative polyadenylation of 3'UTRs to genetic susceptibility to cancer.

Nilotinib boosts the efficacy of anti-PDL1 therapy in colorectal cancer by restoring the expression of MHC-I

BackgroundAlthough immune checkpoint inhibitors (ICIs) have revolutionized the landscape of cancer treatment, only a minority of colorectal cancer (CRC) patients respond to them. Enhancing tumor immunogenicity by increasing major histocompatibility complex I (MHC-I) surface expression is a promising strategy to boost the antitumor efficacy of ICIs.MethodsDual luciferase reporter assays were performed to find drug candidates that can increase MHC-I expression. The effect of nilotinib on MHC-I expression was verified by dual luciferase reporter assays, qRT-PCR, flow cytometry and western blotting. The biological functions of nilotinib were evaluated through a series of in vitro and in vivo experiments. Using RNA-seq analysis, immunofluorescence assays, western blotting, flow cytometry, rescue experiments and microarray chip assays, the underlying molecular mechanisms were investigated.ResultsNilotinib induces MHC-I expression in CRC cells, enhances CD8+ T-cell cytotoxicity and subsequently enhances the antitumor effects of anti-PDL1 in both microsatellite instability and microsatellite stable models. Mechanistically, nilotinib promotes MHC-I mRNA expression via the cGAS-STING-NF-κB pathway and reduces MHC-I degradation by suppressing PCSK9 expression in CRC cells. PCSK9 may serve as a potential therapeutic target for CRC, with nilotinib potentially targeting PCSK9 to exert anti-CRC effects.ConclusionThis study reveals a previously unknown role of nilotinib in antitumor immunity by inducing MHC-I expression in CRC cells. Our findings suggest that combining nilotinib with anti-PDL1 therapy may be an effective strategy for the treatment of CRC.

METTL3-mediated m6A modification of circGLIS3 promotes prostate cancer progression and represents a potential target for ARSI therapy

BackgroundCircular RNAs (circRNAs) have been shown to be involved in tumorigenesis and progression. However, the role of circGLIS3 (hsa_circ_0002874) in prostate cancer (PCa) has yet not been reported.MethodsCandidate circRNA were determined through comprehensive analysis of public datasets, PCa cell lines, and tissues data. A series of cellular functional assays, including CCK-8, colony formation, wound healing, and transwell assays were performed. Subsequently, RNA sequencing, RNA immunoprecipitation, methylated RNA immunoprecipitation, microRNA pulldown, luciferase reporter assay, and western blot were used to explore the underlying molecular mechanisms. Moreover, the xenograft tumor mouse model was established to elucidate the function of circGLIS3.ResultsCircGLIS3, derived from exon 2 of the parental GLIS3 gene, was identified as a novel oncogenic circRNA in PCa that was closely associated with the biochemical recurrence. Its expression levels were upregulated in PCa tissues and cell lines as well as enzalutamide high-resistant cells. The cellular functional assays revealed that circGLIS3 promoted PCa cell proliferation, migration, and invasion. METTL3-mediated N6-methyladenosine (m6A) modification maintained its upregulation by enhancing its stability. Mechanically, CircGLIS3 sponged miR-661 to upregulate MDM2, thus regulating the p53 signaling pathway to promote cell proliferation, migration, and invasion. Furthermore, in vitro and in vivo experiments, the knockdown of circGLIS3 improved the response of PCa cells to ARSI therapies such as enzalutamide.ConclusionsMETTL3-mediated m6A modification of circGLIS3 regulates the p53 signaling pathway via the miR-661/MDM2 axis, thereby facilitating PCa progression. Meanwhile, this study unveils a promising potential target for ARSI therapy for PCa.

GDH1 exacerbates renal fibrosis by inhibiting the transcriptional activity of peroxisome proliferator-activated receptor gamma.

Renal fibrosis is the common outcome of practically all progressive forms of chronic kidney disease (CKD), a significant societal health concern. Glutamate dehydrogenase (GDH) 1 is one of key enzymes in glutamine metabolism to catalyze the reversible conversion of glutamate to α-ketoglutarate and ammonia. However, its function in renal fibrosis has not yet been proven. In this study, GDH1 expression was significantly downregulated in kidney tissues of both children with kidney disease and animal models of CKD. In vivo, the use of R162 (a GDH1 inhibitor) significantly improved renal fibrosis, as indicated by Sirius red and Masson trichrome staining. These findings are consistent with the impaired expression of fibrosis indicators in kidneys from both the unilateral ureteral obstruction (UUO) and 5/6 nephrectomy (5/6 Nx) models. In vitro, silencing GDH1 or pretreatment with R162 inhibited the induction of fibrosis indicators in tissue kidney proximal tubular cells (TKPTS) treated with Transforming growth factor Beta 1 (TGF-β1), whereas activating GDH1 worsened TGF-β1's induction impact. Using RNA-sequence, luciferase reporter assays and Biacore analysis, we demonstrated that GDH1 interacts with Peroxisome proliferator-activated receptor gamma (PPARγ) and blocks its transcriptional activity, independent of the protein's expression. Additionally, R162 treatment boosted PPARγ transcriptional activity, and blocking of this signaling pathway reversed R162's protective effect. Finally, we discovered that R162 treatment or silencing GDH1 greatly lowered reactive oxygen species (ROS) and lipid accumulation. These findings concluded that suppressing GDH1 or R162 treatment could prevent renal fibrosis by augmenting PPARγ transcriptional activity to control lipid accumulation and redox balance.

WTAP promotes the progression of ulcerative colitis by silencing the expression of CES2 through m6A modification

ObjectiveThis study will explore the function of WTAP, the critical segment of m6A methyltransferase complex, in UC and its regulation on immune response. MethodsThe expression levels of key proteins were detected in colon tissues which were derived from UC patients and mice. Macrophage polarization and CD4+ T cell infiltration were detected by flow cytometry and IF staining. ELISA assay was utilized to analyze the level of the inflammatory cytokines. m6A-RIP-PCR, actinomycin D test, and RIP assays were utilized to detect the m6A level, stability, and bound proteins of CES2 mRNA. A dual luciferase reporter assay was conducted to confirm the transcriptional interactions between genes. A co-culture system of intestinal epithelium-like organs was constructed to detect the primary mouse intestinal epithelial cells (PMIEC) differentiation. The interaction between proteins was detected via Co-IP assay. ResultsThe expression of WTAP and CES2 in UC tissues was increased and decreased, respectively. Knockdown of WTAP inhibited the progression of UC in mice by inhibiting M1 macrophage polarization and CD4+ T cell infiltration. WTAP combined YTHDF2 to promote the m6A modification of CES2 mRNA and inhibited its expression. CES2 co-expressed with EPHX2 and overexpression of CES2 promoted the differentiation of PMIEC. The inhibitory effect of WTAP knockdown on the progress of UC was partially abrogated by CES2 knockdown. ConclusionWTAP/YTHDF2 silences CES2 by promoting its m6A modification and then promotes the progression of UC. WTAP could be a promoting therapy target of UC.

The MYC transcription factor PbrMYC8 negatively regulates PbrMSL5 expression to promote pollen germination in Pyrus

The successful germination of pollen is essential for double fertilization in flowering plants. Mechanosensitive channels of small conductance (MscS-like, MSL) inhibit pollen germination and maintains cellular integrity of pollen during this process. Therefore, it is vital to carefully regulate the expression of MSL to promote successful pollen germination. Despite its importance, the molecular mechanisms governing MSL expression in plants remain poorly understood. Here, we had identified 15 MSL genes in the pear, among which PbrMSL5 was expressed in pollen development. Subcellular localization experiments revealed that PbrMSL5 was located in both plasma membrane and cytoplasm. Functional investigations, including complementation experiments using the atmsl8 mutant background, demonstrated the involvement of PbrMSL5 in preserving pollen cell integrity and inhibiting germination. Antisense oligonucleotide experiments further confirmed that PbrMSL5 suppressed pear pollen germination by reducing osmotic pressure and Cl− content. Yeast one-hybrid, electrophoretic mobility shift assays, and dual luciferase reporter assay elucidated that PbrMYC8 interacts directly with the N-box element, leading to the suppression of PbrMSL5 expression and promoted pollen germination. These results represented a significant advancement in unraveling the molecular mechanisms controlling plant MSL expression. This study showed valuable contribution to advancing our comprehension of the mechanism underlying pollen germination.

Long noncoding RNA MALAT1 as a ceRNA drives mouse fibroblast activation via the miR-335-3p/P2ry2 axis.

Fibrosis is a complex pathological process that can lead to the permanent loss of biological function, with P2ry2 playing a crucial role in this process. Long non-coding RNAs (lncRNAs) have been reported to play an critically important role in the fibrotic process. However, it remains unclear whether lncRNAs can regulate fibrosis through P2ry2. In this study, we detected the expression of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (lnc-MALAT1). We investigated the expression patterns of lnc-MALAT1 and P2ry2 in denervated skeletal muscle, a classical model of fibrosis. Additionally, we utilized a TGF-β-mediated fibrosis model in NIH/3T3 cells to examine the effects of lnc-MALAT1 and P2ry2 on fibroblast activation and the underlying regulatory mechanisms in vitro. Our results demonstrated that the expression levels of lnc-MALAT1 and P2ry2 were consistently elevated in denervated skeletal muscle, correlating with the degree of fibrosis. In vitro experiments confirmed the regulatory effect of lnc-MALAT1 on P2ry2. Furthermore, we identified miR-335-3p as a potential key molecule in the regulatory relationship of lnc-MALAT1/P2ry2. Dual luciferase reporter assays and AGO2-RIP verified the molecular sponging effect of lnc-MALAT1 on miR-335-3p. Additionally, we validated the regulation of the lnc-MALAT1/miR-335-3p/P2ry2 axis through experimental approaches. In conclusion, our study identified a crucial role of lnc-MALAT1/miR-335-3p/P2ry2 axis in fibroblast activation, providing a promising treatment option against the fibrosis.

Adipocytes promote metastasis of breast cancer by attenuating the FOXO1 effects and regulating copper homeostasis

BackgroundObesity and the forkhead box O1(FOXO1) affect the survival of breast cancer patients, but the underlying mechanism remains unclear. We aimed to investigate the role of FOXO1 in obesity-associated-breast cancer.MethodsWe screened 383 breast disease patients from the first affiliated hospital with Nanjing Medical University in 2020. We performed wound healing, transwell, matrigel assays to assess the metastatic ability of cancer cells. We adopted mRNAs sequencing to select the differentially expressed transcripts in breast cancer. We applied immunohistochemistry, western blot, tissue microarrays to assess the level of FOXO1 and epithelial-mesenchymal transition (EMT) pathways. We conducted bioinformatic analysis to investigate interactions between FOXO1 and miR-135b. We used fluorescence in situ hybridization, RT-qPCR to confirm the characteristics of circCNIH4. We conducted luciferase reporter assay, rescue experiments to investigate interactions between circCNIH4 and miR-135b.ResultsObesity was positively correlated with the incidence and progression of breast cancer. Adipocytes enhanced the migration of breast cancer and attenuated the effects of FOXO1. MiR-135b was a binding gene of FOXO1 and was regulated by circCNIH4. CircCNIH4 exhibited antitumor activity in vitro and in vivo.ConclusionAdipocytes might accelerate the progression of breast cancer by modulating FOXO1/miR-135b/ circCNIH4 /EMT axis and regulating copper homeostasis.