mRNA Microarray Research Articles

LncRNA RP4-639F20.1 interacts with THRAP3 to attenuate atherosclerosis by regulating c-FOS in vascular smooth muscle cells proliferation and migration

Background and aimsThe aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) play an essential role in the pathogenesis of atherosclerosis (AS). Long noncoding RNAs (lncRNAs) have been reported as important regulators in a number of diseases. However, very little is known regarding the functional role of lncRNAs in governing proliferation and migration of VSMCs and AS development. MethodsBoth in vitro and in vivo assays were performed to investigate the role of lncRNA in the pathophysiology of AS. Our previous lncRNA arrays revealed that lncRNA RP4-639F20.1 was significantly decreased in atherosclerotic plaques. Lentivirus overexpressing RP4-639F20.1 and lncRNA RP4-639F20.1 silencing vectors (Si-lnc-RP4-639F20.1) were constructed and transfected in VSMCs. The in vitro functions of lncRNA were analyzed by CCK-8 assays, EdU assays, scratch wound assays, transwell assays, qRT-PCR and Western blot analyses. RNA fluorescence in situ hybridization, immunoprecipitation and mRNA microarrays were used to explore the underlying mechanism. Adeno-associated-virus-9 (AAV9) overexpressing RP4-639F20.1 was constructed and injected intravenously into ApoE−/− mice to explore the role of lncRNA in vivo. ResultsIn vitro experiments showed that lncRNA RP4-639F20.1 interacted with THRAP3 and downregulated c-FOS expression. Both increase of lncRNA RP4-639F20.1 expression and knockdown of c-FOS inhibited the expression of MMP10 and VEGF-α in VSMCs and suppressed VSMCs proliferation and migration. In vivo experiments using ApoE−/− mice fed a high-fat diet demonstrated that lncRNA RP4-639F20.1 overexpression deterred atherosclerosis and decreased lipid levels in atherosclerotic lesions. Patients with coronary artery disease were found to have higher c-FOS levels than healthy individuals and c-FOS expression was positively correlated with the SYNTAX score of patients. ConclusionsOverall, these data indicated that lncRNA RP4-639F20.1/THRAP3/c-FOS pathway protects against the development of atherosclerosis by suppressing VSMCs proliferation and migration. LncRNA RP4-639F20.1 and c-FOS could represent potential therapeutic targets to ameliorate atherosclerosis-related diseases.

Bioinformatics analysis and identification of hub genes of neutrophils in Kawasaki disease: a pivotal study.

Kawasaki disease (KD) is considered the main contributor to acquired heart diseases in developed countries. However, the precise pathogenesis of KD remains unclear. Neutrophils play roles in KD. This study aimed to select hub genes in neutrophils in acute KD. mRNA microarray of neutrophils from four acute KD patients and three healthy controls was performed to screen differentially expressed mRNAs (DE-mRNAs). DE-mRNAs were analyzed and predicted by Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways, and protein-protein interaction networks. Real time-PCR was finally conducted to confirm the reliability and validity of the expression level of DE-mRNAs from blood samples of healthy controls and KD patients in both acute and convalescent stage. A total of 1950 DE-mRNAs including 1287 upregulated and 663 downregulated mRNAs were identified. GO and KEGG analyses revealed the DE-mRNAs were mainly enriched in the regulation of transcription from RNA polymerase II promoter, apoptotic process, intracellular signal transduction, protein phosphorylation, protein transport, metabolic pathways, carbon metabolism, lysosome, apoptosis, pyrimidine metabolism, alzheimer disease, prion disease, sphingolipid metabolism, huntington disease, glucagon signaling pathway, non-alcoholic fatty liver disease, pyruvate metabolism, sphingolipid signaling pathway, and peroxisome. Twenty hub DE-mRNAs were selected including GAPDH, GNB2L1, PTPRC, GART, HIST2H2AC, ACTG1, H2AFX, CREB1, ATP5A1, ENO1, RAC2, PKM, BCL2L1, ATP5B, MRPL13, SDHA, TLR4, RUVBL2, TXNRD1, and ITGAM. The real-time PCR results showed that BCL2L1 and ITGAM mRNA were upregulated in acute KD and were normalized in the convalescent stage. These findings may improve our understanding of neutrophils in KD. Key Points • Neutrophilic BCL2L1 and ITGAM mRNA were first reported to be correlated with the pathogenic mechanism of KD.

Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia

ObjectiveJiedu Recipe (JR), a Chinese herbal remedy, has been shown to prolong overall survival time and decrease recurrence and metastasis rates in patients with hepatocellular carcinoma (HCC). This work investigated the mechanism of JR in HCC treatment. MethodsThe chemical constituents of JR were detected using liquid chromatography-mass spectrometry. The potential anti-HCC mechanism of JR was screened using network pharmacology and messenger ribonucleic acid (mRNA) microarray chip assay, followed by experimental validation in human HCC cells (SMMC-7721 and Huh7) in vitro and a nude mouse subcutaneous transplantation model of HCC in vivo. HCC cell characteristics of proliferation, migration and invasion under hypoxic setting were investigated using thiazolyl blue tetrazolium bromide, wound healing and Transwell assays, respectively. Image-iT™ Hypoxia Reagent was added to reveal hypoxic conditions. Stem cell sphere formation assay was used to detect the stemness. Epithelial-mesenchymal transition (EMT) markers like E-cadherin, vimentin and α-smooth muscle actin, and pluripotent transcription factors including nanog homeobox, octamer-binding transcription factor 4, and sex-determining region Y box protein 2 were analyzed using Western blotting and real-time polymerase chain reaction. Western blot was performed to ascertain the anti-HCC effect of JR under hypoxia involving the Wnt/β-catenin pathway. ResultsAccording to network pharmacology and mRNA microarray chip analysis, JR may potentially act on hypoxia and inhibit the Wnt/β-catenin pathway. In vitro and in vivo experiments showed that JR significantly decreased hypoxia, and suppressed HCC cell features of proliferation, migration and invasion; furthermore, the hypoxia-induced increases in EMT and stemness marker expression in HCC cells were inhibited by JR. Results based on the co-administration of JR and an agonist (LiCl) or inhibitor (IWR-1-endo) verified that JR suppressed HCC cancer stem-like properties under hypoxia by blocking the Wnt/β-catenin pathway. ConclusionJR exerts potent anti-HCC effects by inhibiting cancer stemness via abating the Wnt/β-catenin pathway under hypoxic conditions.Please cite this article as: Guo BJ, Ruan Y, Wang YJ, Xiao CL, Zhong ZP, Cheng BB, Du J, Li B, Gu W, Yin ZF. Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia. J Integr Med. 2023; 21(5): 474–486.

LncRNA NEAT1: A novel regulator associated with the inflammatory response in acute respiratory distress syndrome

BackgroundAcute respiratory distress syndrome (ARDS) is a life-threatening condition with an unfavorable prognosis. As the pathogenesis of ARDS remains unclear, we aimed to identify the core genes associated with ARDS and the mechanisms by which competing endogenous RNAs (ceRNAs) regulate the disease’s progression. MethodsThree mRNA microarray datasets (GSE17355, GSE48787, and GSE130936), derived from the Gene Expression Omnibus (GEO) database, were selected. Common differentially expressed genes (DEGs) related to acute lung injury (ALI) were identified and subjected to enrichment analysis. Then, hub genes were figured out through the protein–protein interaction (PPI) network and functional analysis, and targeted miRNAs and lncRNAs were predicted. Finally, the ceRNA networks associated with ALI were constructed and validated experimentally. ResultsA total of 155 upregulated and 93 downregulated DEGs were identified in the three datasets. The TNF signaling pathway and IL-17 signaling pathway were the most enriched pathways. Then, eleven DEGs enriched in the IL-17 signaling pathway were selected as the hub genes. Three miRNAs (mmu-mir-155-5p, mmu-mir-21a-5p, and mmu-mir-122-5p), which were located in the lung tissue and predicted to bind the hub genes at the same time, and two lncRNAs (Neat1 and Tug1), which have binding sites for the aforementioned miRNAs, were filtered. With qPCR verification, we identified a ceRNA network composed of NEAT1, miR-21-5p, MMP9, and CXCL5. NEAT1 knockdown promoted the migration and reduced the expression of pro-inflammatory factor and reactive oxygen species (ROS) in lung epithelial cells. We eventually confirmed that NEAT1/miR-21-5p/CXCL5/MMP9 played a pivotal role in regulating the inflammatory response in ALI. ConclusionThe IL-17 signaling pathway is of great importance in the pathogenesis of ARDS. NEAT1/miR-21-5p is involved in the inflammation of ALI by regulating CXCL5 and MMP9.

Molecular classification of muscle-invasive bladder cancer based on a simplified immunohistochemical panel using GATA3, CK5/6 and p16.

The choice of therapy for muscle-invasive bladder cancer (MIBC) could be influenced by the tumor's molecular subtype. Currently, well-defined consensus subtypes are based on tumor microarray mRNA data. Clearly defined and easy-to-use surrogate molecular subtypes, based on immunohistochemistry (IHC) performed on whole slides, are needed to make subtyping cost-effective and useful in routine work and future research. To aid in the development of a simple immunohistochemical classifier, a retrospective single-center series of 92 cases of localized bladder cancer was identified. Routine IHC for GATA3, cytokeratins 5 and 6 (CK5/6), and p16 was performed on whole tissue blocks containing muscle-invasive disease. Electronic medical records were retrieved and searched for clinical variables, treatment, and survival data. The mean age was 69.6 years, and 73% were males. Conservative treatment was used in 55% of cases, while cystectomy with chemotherapy was used in 45%. GATA3 and CK5/6 expression divided cases into broad luminal and basal subtypes, respectively, while p16 expression was used to subclassify luminal cases into luminal papillary and luminal unstable types according to the consensus molecular classification. When subtyped in this way, GATA3 and CK5/6 negative cases showed worse overall survival. Molecular subtyping of MIBC on whole slides containing muscle-invasive tumor using only three commonly used, consensus-based antibodies, is a feasible and cost-effective method for detecting subtypes of invasive bladder cancer. Future work combining morphological analysis and IHC is needed to fully translate the consensus molecular classification into a comprehensive, cost-effective subtyping strategy.

Fructo-oligosaccharide-mediated alteration in claudin expression in small intestinal absorptive Caco-2 cells is positively associated with the induction of inflammatory genes and the glucan receptor gene CLEC7A

ObjectivesIndigestible carbohydrates may strengthen tight junctions (TJs) independently of intestinal bacteria. This study investigated whether indigestible carbohydrates (i.e., fructo-oligosaccharides [FOS]) promote TJs directly to intestinal absorptive Caco-2 cells and examined the association between the expression of genes constructing TJs and other genes using mRNA microarray analysis. MethodsCaco-2 cells at 1.0 × 105/mL were seeded in a type I collagen plate and cultured in high-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS); the cells reached confluence at 7 d after seeding. Ten days after the cells reached confluency, they were cultured for 24 h in 10% FCS-containing DMEM medium supplemented with 0%, 5%, or 10% FOS. We performed mRNA microarray to identify the genes whose expression was altered by FOS. Subsequently, quantitative reverse transcription polymerase chain reaction was performed for these altered genes, including CLEC7A encoding the glucan receptor, and for the claudin (CLDN) family genes. The expression of CLDN2, CLDN4, and CLEC7A proteins was assessed using western blot analysis. ResultsFOS decreased the mRNA and protein expression of CLDN2, which weakens TJs, and increased those of CLDN4, which strengthens TJs, in Caco-2 cells. FOS treatment (10%) reduced the mRNA expression of antioxidative genes and induced the expression of immune response–related genes, including CLEC7A, CCL2, and ITGA2. Furthermore, the expression of CLEC7A protein was enhanced by FOS. ConclusionsInduction of TJ-strengthening CLDN4 and reduction of TJ-weakening CLDN2 by FOS treatment in small intestinal absorptive Caco-2 cells is positively associated with the induction of inflammatory genes, including CLEC7A.

SPARC accelerates biliary tract cancer progression through CTGF-mediated tumor-stroma interactions: SPARC as a prognostic marker of survival after neoadjuvant therapy.

In biliary tract cancer (BTC), malignancy is strongest at the invasion front. To improve the BTC prognosis, the invasion front should be controlled. We evaluated tumor-stroma crosstalk at the tumor center and at the invasion front of BTC lesions. We investigated the expression of SPARC, a marker of cancer-associated fibroblasts, and determined its ability to predict BTC prognosis after neoadjuvant chemoradiotherapy (NAC-RT). We performed immunohistochemistry to evaluate SPARC expression in resected specimens from patients that underwent BTC surgery. We established highly invasive (HI) clones in two BTC cell lines (NOZ, CCLP1), and performed mRNA microarrays to compare gene expression in parental and HI cells. Among 92 specimens, stromal SPARC expression was higher at the invasion front than at the lesion center (p = 0.014). Among 50 specimens from patients treated with surgery alone, high stromal SPARC expression at the invasion front was associated with a poor prognosis (recurrence-free survival: p = 0.033; overall survival: p = 0.017). Coculturing fibroblasts with NOZ-HI cells upregulated fibroblast SPARC expression. mRNA microarrays showed that connective tissue growth factor (CTGF) was upregulated in NOZ-HI and CCLP1-HI cells. A CTGF knockdown suppressed cell invasion in NOZ-HI cells. Exogeneous CTGF upregulated SPARC expression in fibroblasts. SPARC expression at the invasion front was significantly lower after NAC-RT, compared to surgery alone (p = 0.003). CTGF was associated with tumor-stroma crosstalk in BTC. CTGF activated stromal SPARC expression, which promoted tumor progression, particularly at the invasion front. SPARC expression at the invasion front after NAC-RT may serve as a prognosis predictor.

Agomir miRNA-150-5p alleviates pristane-induced lupus by suppressing myeloid dendritic cells activation and inflammation via TREM-1 axis.

Triggering receptors expressed on myeloid cells-1 (TREM-1) has been shown to participate in inflammatory autoimmune diseases. Nevertheless, the detailed underlying mechanisms and therapeutic benefits by targeting TREM-1 remain elusive, especially in myeloid dendritic cells (mDCs) and systemic lupus erythematosus (SLE). Disorders of epigenetic processes including non-coding RNAs give rise to SLE, resulting in complicated syndromes. Here, we aim to address this issue and explore the miRNA to inhibit the activation of mDCs and alleviate the progress of SLE by targeting TREM-1 signal axis. Bioinformatics methods were used to analyze the differentially expressed genes (DEGs) between patients with SLE and healthy individuals by four mRNA microarray datasets from Gene Expression Omnibus (GEO). Then we identified the expression of TREM-1 and its soluble form (sTREM-1) in clinical samples by ELISA, quantitative real-time PCR and Western blot. Phenotypic and functional changes of mDCs elicited by TREM-1 agonist were determined. Three databases of miRNAs target prediction and a dual-luciferase reporter assay were used to screen and verify miRNAs that can directly inhibit TREM-1 expression in vitro. Moreover, pristane-induced lupus mice were injected with miR-150-5p agomir to evaluate the effects of miR-150-5p on mDCs in lymphatic organs and disease activity in vivo. We screened TREM-1 as one of the hub genes closely correlated with the progression of SLE and identified sTREM-1 in serum as a valuable diagnostic biomarker for SLE. Moreover, activation of TREM-1 by its agonist promoted activation and chemotaxis of mDCs and increased the production of inflammatory cytokines and chemokines, showing higher expression of IL-6, TNF-α, and MCP-1. We showed that lupus mice displayed a unique miRNA signature in spleen, among which miR-150 was the most significantly expressed miRNA that targeting TREM-1 compared with wild type group. Transfection of miRNA-150-5p mimics directly suppressed the expression of TREM-1 by binding to its 3' UTR. Our in vivo experiments first indicated that administration of miR-150-5p agomir effectively ameliorated lupus symptoms. Intriguingly, miR-150 inhibited the over activation of mDCs through TREM-1 signal pathway in lymphatic organs and renal tissues. TREM-1 represents a potentially novel therapeutic target and we identify miR-150-5p as one of the mechanisms to alleviate lupus disease, which is attributable for inhibiting mDCs activation through TREM-1 signaling pathway.

Random forest selected transcripts from multiple neurodegenerative diseases reveal common affected cellular processes

AbstractBackgroundNeurodegenerative diseases such as Alzheimer’s disease are associated with changes in specific molecular pathways involving metabolomic and biological processes active in both the central nervous system and vascular peripheral system. Each disease results in neurodegeneration in vulnerable regions and cell types specific to that disease and are expressed in whole blood as disease progresses. Machine learning can be applied to the entire blood transcriptome of neurodegenerative diseases providing information for a pathophysiological relationship and gives us the advantage of using the vast knowledge of gene expression to analyze pathways affected thereby guiding insight into possible treatment strategies. We applied our unique machine learning algorithm to public data sets for selection of transcripts that guide our discovery of affected pathways.MethodsWe used large public blood microarray mRNA expression datasets, with platforms of more than 30,000 probes covering 10,000 genes, to discover disease transcripts using a novel Random Forest algorithm which selected supervised predictors for six different neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), behavioral variant frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and Friedreich’s ataxia (FRDA).ResultsOur machine learning selection of transcript disease classifiers reveals that neurodegenerative diseases have common themes as well as unique individual differences. Molecular components which overlap across diseases might be considered neurodegeneration generalities or a result of mixed pathology, while differential components identified by selected feature transcripts may represent unique dysfunctions.ConclusionOur Random Forest algorithm selected important features hidden in mRNA expression values from blood of affected individuals of six neurodegenerative diseases compared to healthy controls. For each of the six diseases studied here, the selected features were categorized by pathway analysis into eight main functional groups. The dysfunction of any or multiple functional groups combined can lead to synapse and cell loss in these neurodegenerative diseases each with specific vulnerabilities in particular tissue and cell types. Vulnerability is usually associated with those cells vulnerable to the disease pathology and are often first to exhibit cell death from cytotoxic events such as neuroinflammation, mitochondrial dysfunction, transcription alterations, apoptosis, protein synthesis dysfunction, cytoskeletal changes, and ubiquitylation/proteasome stalls.

MRNA Expression Profiles and Molecular Biomarkers Involved in Inflammatory Response for Essential Hypertension in Kazakh Population From Xinjiang

Abstract Background To identify hypertensive inflammation-related mRNA in peripheral blood mononuclear cells (PBMCs) from patients with essential hypertension (EH) of Kazakh Chinese population in Xinjiang, and offer theoretical foundation for further understanding the immune mechanisms that contribute to EH in Kazakh and provide specific molecular biomarkers for clinical diagnosis and targeted-specific treatments of this disorder. Methods Thirty Kazakh patients with EH and 30 age-matched healthy Kazakh people were divided into EH group and control group. After performing blood pressure measurement and determination of serum lipids and blood glucose, total RNA was isolated from PBMCs of hypertensive Kazakh patients and healthy Kazakh subjects, and differentially expressed mRNA in 6 randomly selected samples from each group were identified with human lncRNA/mRNA V4.0 microarray. Gene set enrichment analysis (GSEA), disease process, the Gene Ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of differentially expressed mRNA were carried out using R language and ClusterProfiler software package. Reverse transcription quantitative real-time PCR (qRT-PCR) was used to validate the differential expression pattern of inflammation-related genes in the mRNA microarray data and results of bioinformatics analysis. Results The average blood pressure was significantly higher in the EH group than those in the control group (P < 0.01), while there was no statistical significance for the levels of serum lipids and blood glucose between the 2 groups. Compared with healthy Kazakh controls, a total of 522 mRNA were significantly differentially expressed from PBMCs of hypertensive Kazakh patients with fold change of ≥1.5 and P value <5% in expression. Among them, 411 were upregulated and 111 were downregulated. GO enrichment analysis of upregulated mRNA identified numerous inflammation-related biological processes and molecular functions. Many of these processes and molecular function were mainly involved in immune response-regulating signaling pathway, immune response-activating signal transduction, antigen binding, immunoglobulin receptor binding and peptide antigen binding, etc. GSEA and KEGG pathway analysis for the upregulated mRNA indicated several positively enriched gene sets and pathways related to inflammatory response, such as focal adhesion, cell adhesion molecules, chemokine signaling pathway, leukocyte transendothelial migration, cytokine–cytokine receptor interaction, and B-cell receptor signaling pathway. qRT-PCR validation results of 14 mRNA related to inflammatory response were consistent with the microarray data. Conclusions This is the first study to identify gene expression profile involved in hypertensive inflammation in Kazakh Chinese population in Xinjiang with EH, and not only provides specific molecular biomarkers for predicting and diagnosing progression of EH, but also will provide targeted-specific treatments of this disorder in Kazakh Chinese population in Xinjiang.

Caspase-3 in glioma indicates an unfavorable prognosis by involving surrounding angiogenesis and tumor cell repopulation.

Effective biomarkers for estimating glioma prognosis are deficient. Canonically, caspase-3 acts as an "apoptosis executioner". However, its prognostic role in glioma and mechanistic effects on prognosis remain unclear. With glioma tissue microarrays, the prognostic roles of cleaved caspase-3 and its association with angiogenesis were explored. Next, by analyzing the mRNA microarray data from the CGGA, the prognostic role of CASP3 expression and correlations between CASP3 and markers of glioma angiogenesis and proliferation were investigated. To biologically interpret the prognostic role of caspase-3 in glioma, the influence of caspase-3 on surrounding angiogenesis and glioma cell repopulation was investigated with an in vitro cell co-culture model, which comprises irradiated U87 cells and un-irradiated firefly luciferase (Fluc)-labeled HUVEC (HUVEC-Fluc) or U87 (U87-Fluc) cells. The over-expressed dominant-negative caspase-3 was used to suppress normal caspase-3 activity. High levels of cleaved caspase-3 expression were associated with poor survival outcomes in glioma patients. Higher microvessel density was observed in patients with high levels of cleaved caspase-3 expression. By mining the microarray data in CGGA, it was revealed that higher CASP3 expression was found in glioma patients with lower Karnofsky Performance score, higher WHO grade, malignant histological subtype, wild-type IDH. Higher CASP3 expression indicated a worse survival rate in glioma patients. Patients with high CASP3 expression and negative IDH mutation showed the worst survival rate. Positive correlations were found between CASP3 and markers of tumor angiogenesis and proliferation. Subsequent data based on an in vitro cell co-culture model revealed that caspase-3 in irradiated glioma cells mediated pro-angiogenic and repopulation-promoting effects via regulating COX-2 signaling. With glioma tissue microarrays, high levels of COX-2 expression showed inferior survival outcomes in glioma patients. Glioma patients with high levels of cleaved caspase-3 and COX-2 expression showed the worst survival outcomes. This study innovatively identified an unfavorable prognostic role of caspase-3 in glioma. The pro-angiogenic and repopulation-prompting effects of caspase-3/COX-2 signaling may explain its unfavorable prognostic role and offer novel insights into therapy sensitization and curative effect prediction of glioma.

Microphthalmia-Associated Transcription Factor: A Differentiation Marker in Uveal Melanoma.

Microphthalmia-associated transcription factor (MITF) is an important regulator of melanogenesis and melanocyte development. In cutaneous melanoma, MITF loss has been linked to an increased expression of stem cell markers, a shift in epithelial-to-mesenchymal transition (EMT)-related factors, and increased inflammation. We explored the role of MITF in Uveal Melanoma (UM) using a cohort of 64 patients enucleated at the Leiden University Medical Center. We analysed the relation between MITF expression and clinical, histopathological and genetic features of UM, as well as survival. We performed differential gene expression and gene set enrichment analysis using mRNA microarray data, comparing MITF-low with MITF-high UM. MITF expression was lower in heavily pigmented UM than in lightly pigmented UM (p = 0.003), which we confirmed by immunohistochemistry. Furthermore, MITF was significantly lower in UM with monosomy 3/BAP1 loss than in those with disomy 3/no BAP1 loss (p < 0.001) and with 8q gain/amplification 8q (p = 0.02). Spearman correlation analysis showed that a low MITF expression was associated with an increase in inflammatory markers, hallmark pathways involved in inflammation, and epithelial-mesenchymal transition. Similar to the situation in cutaneous melanoma, we propose that MITF loss in UM is related to de-differentiation to a less favourable EMT profile and inflammation.

Astragalus polysaccharide ameliorates steroid-induced osteonecrosis of the femoral head by regulating miR-200b-3p-mediated Wnt/β-catenin signaling pathway via inhibiting SP1 expression

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is the necrosis of the femur bone caused by prolonged and massive use of corticosteroids. The present study probed into the significance of Astragalus polysaccharide (APS) in SONFH progression.MethodsSONFH cell model was constructed using murine long bone osteocyte Y4 (MLO-Y4) cells and then treated with APS. mRNA microarray analysis selected differentially expressed genes between control group and SONFH group. RT-qPCR determined SP1 and miR-200b-3p expression. Levels of SP1, β-catenin, autophagy-related proteins (LC3II/LC3I, Beclin1, p62) and apoptosis-related proteins (Bax, C-caspase3, C-caspase9, Bcl-2) were tested by Western blot. ChIP and luciferase reporter assays confirmed relationship between SP1 and miR-200b-3p. Fluorescence intensity of LC3 in cells was detected by immunofluorescence. Flow cytometry assessed cell apoptosis. Osteonecrosis tissues from SONFH mice were examined by HE and TRAP staining.ResultsAPS induced autophagy and suppressed apoptosis in SONFH cell model. APS inhibited SP1 expression and SP1 overexpression reversed effects of APS on SONFH cell model. Mechanistically, SP1 targeted miR-200b-3p to inhibit Wnt/β-catenin pathway. MiR-200b-3p depletion rescued the promoting effect of SP1 on SONFH cell model by activating Wnt/β-catenin pathway. HE staining showed that APS treatment reduced the empty lacunae and alleviated inflammation in trabecular bone of SONFH mice. TRAP staining revealed decreased osteoclasts number in SONFH mice after APS treatment.ConclusionAPS regulated osteocyte autophagy and apoptosis via SP1/miR-200b-3p axis and activated Wnt/β-catenin signaling, thereby alleviating SONFH, shedding new insights for therapy of SONFH.

The role and mechanism of FTO in pulmonary vessels

ABSTRACT Pulmonary vascular remodeling (PVR) is the main factor of pulmonary hypertension (PH). The pathological characteristics of PVR are vascular smooth muscle hyperplasia, hypertrophy, and extensive damage. In vivo experiments, the expression of FTO in PH rat lung tissues of different rat models of hypoxia PH was observed by immunohistochemical method. mRNA microarray analysis was used to analyze the differential expressed genes in rat lung tissues. In vitro experiments, we developed models of overexpression and knockdown of FTO to study the effect of FTO protein expression on cell apoptotic, cell cycle, and the abundance of m6A. The expression of FTO was increased in PH rats. FTO knockdown can inhibit the proliferation of PASMCs, thereby regulating the cell cycle and reducing the expression of Cyclin D1 and the abundance of m6A, while overexpression of FTO leads to increased expression of Cyclin D1 and the abundance of m6A. FTO destroys the stability of Cyclin D1 by regulating the abundance of Cyclin D1 m6A, causing cell cycle arrest and inducing cell proliferation, thus inducing the occurrence and development of PVR in PH.

Identification of POLQ as a key gene in cervical cancer progression using integrated bioinformatics analysis and experimental validation.

As the most common gynecologic malignancy worldwide, cervical cancer (CC) is a serious hazard to health. Therefore, the present study aimed to identify the key genes in CC progression using integrated bioinformatics analysis and experimental validation. The mRNA microarray GSE63514 and microRNA (miRNA) microarray GSE86100 were obtained from the Gene Expression Omnibus database, and the differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) in the progression of CC were identified. Thereafter, GO and KEGG functional enrichment analysis, protein‑protein interaction (PPI) network and significant subnetworks construction, and miRNA‑target regulatory network construction were performed. Based on the results of integrated bioinformatics analysis, the DEGs structural maintenance of chromosomes 4 (SMC4), ATPase family, AAA domain‑containing 2 (ATAD2) and DNA polymerase θ (POLQ) were identified as hub genes in the PPI network and were involved in the first significant subnetwork. In addition, these DEGs were predicted to be regulated by miR‑106B, miR‑17‑5P, miR‑20A and miR‑20B, which were identified as DEMs. Of note, SMC4 and ATAD2 are tumor‑promotors in CC. In the present study, small interfering (si)RNAs were used to knock down POLQ expression. Cell Counting Kit‑8, Transwell, cell cycle and apoptosis analyses revealed that the downregulation of POLQ restrained cell proliferation, migration and invasion, and promoted apoptosis and the arrest of the cell cycle in the G2 phase. In conclusion, POLQ, which may have a close interaction with SMC4 and ATAD2, may serve a vital role in the progression of CC.

Expression Profile of Pyroptosis-Related Genes and the Associated Regulatory Axis in Primary Gout Patients.

Analyzed the expression characteristics of pyroptosis-related genes (PRGs) in peripheral blood mononuclear cells (PBMCs) of gout patients by microarray, and constructed ceRNA network to explore the molecular mechanism of RNA-mediated pyroptosis regulation. Human mRNA, lncRNA, circRNA microarray data were used to identify differentially expressed in PBMCs from patients with primary gout and healthy controls. Differential PRGs in PBMCs of gout patients identified by Genecard database and mRNA microarray data. GO and KEGG enrichment analyses of these genes were then conducted. Protein-protein interaction networks and cytoHubba were used to identify hub genes. Combining the lncRNA and circRNA microarray data, a ceRNA network was constructed by Cytoscape to screen out key non-coding RNA molecules that can regulate target PRGs. Finally, the relative expression levels of target miRNA and circRNA in 60 gout patients and 40 healthy subjects were detected by qRT-PCR. The results revealed 30 differentially expressed PRGs. GO and KEGG analysis of these genes were mainly concentrated in the production and regulation of cytokines, NOD-like receptor signaling pathway and so on. Nine hub genes were screened by PPI network, including IL1B, DDX3X, NLRP3, NLRP9, AIM2, CASP8, P2XR7, CARD8 and IFI16. The has_circRNA_102906\hsa_circRNA_102910\hsa_circRNA_102911-hsa-miR-129-5p-DDX3X\NLRP3\NLRP9 regulatory network was constructed. The expression of has_circRNA_102906, hsa_circRNA_102910, hsa_circRNA_102911 were up-regulated and hsa-miR-129-5p down-regulated in PBMCs of gout patients. The relative expression of hsa_circRNA_102911 was positively correlated with clinical inflammatory indicators associated with gout, and the area under the curve of hsa_circRNA_102911 for gout diagnosis was 0.85 (95% CI: 0.775-0.925; p < 0.001). There are several differentially expressed PRGs in PBMCs of gout patients, which are involved in the regulation of gout inflammation through multiple pathways. hsa_circRNA_102911-hsa-miR-129-5p-DDX3X\NLRP3\NLRP9 may be the key regulatory pathway for pyroptosis to regulate gout inflammation, and hsa_circRNA_102911 may be a potential biomarker for the diagnosis of primary gout.

MP56-06 PATHOLOGIC DOWNSTAGING RATE AND PREDICTIVE BIOMARKER EXPLORATION IN PATIENTS WITH BIOPSY-PROVEN CLINICALLY NODE POSITIVE BLADDER CANCER UNDERGOING CHEMOTHERAPY FOLLOWED BY RADICAL CYSTECTOMY

You have accessJournal of UrologyCME1 Apr 2023MP56-06 PATHOLOGIC DOWNSTAGING RATE AND PREDICTIVE BIOMARKER EXPLORATION IN PATIENTS WITH BIOPSY-PROVEN CLINICALLY NODE POSITIVE BLADDER CANCER UNDERGOING CHEMOTHERAPY FOLLOWED BY RADICAL CYSTECTOMY Amy Lim, Akshay Sood, Vikram Narayan, Mohammed Seif, Justin Matulay, Kelly Bree, Matt Campbell, Ashish Kamat, Colin Dinney, and Neema Navai Amy LimAmy Lim More articles by this author , Akshay SoodAkshay Sood More articles by this author , Vikram NarayanVikram Narayan More articles by this author , Mohammed SeifMohammed Seif More articles by this author , Justin MatulayJustin Matulay More articles by this author , Kelly BreeKelly Bree More articles by this author , Matt CampbellMatt Campbell More articles by this author , Ashish KamatAshish Kamat More articles by this author , Colin DinneyColin Dinney More articles by this author , and Neema NavaiNeema Navai More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003309.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Studies assessing the efficacy of preoperative chemotherapy in bladder cancer rarely include clinically node positive (cN+) patients. Further, the cN+ patients that are included are frequently categorized as cN+ based on imaging only. Therefore, true nodal response rates and oncological outcomes in this select patient group are generally unknown. Additionally, studies examining responder characteristics are lacking. METHODS: Data were collected retrospectively on patients with cTanyN1-3M0 bladder cancer who underwent radical cystectomy at the University of Texas MD Anderson Cancer Center (2006-2017). Complete nodal response (pN0) and bladder tumor response (pT0), recurrence free survival (RFS) and overall survival (OS) for patients with a positive lymph-node (LN) biopsy were recorded. Tissue mRNA microarray expression profiling was performed on LN and primary tumor tissue (pre-therapy) and compared among responders (pN0) and non-responders. RESULTS: Among the 120 patients identified with cTanyN1-3M0 disease, 26 (21.7%) patients had a positive LN biopsy. Induction chemotherapy was administered in all patients. Nine (34.6%) were pN0 and 9 (34.6%) were pT0 after primary chemotherapy. Seven were pT0N0. Lower stage disease on final pathology was associated with improved RFS and OS (p<0.001). Fourteen differentially expressed genes in the LNs and 68 differentially expressed genes in the bladder were predictive of pN0 response to primary chemotherapy in the LNs (FDR<0.1; Figure). CONCLUSIONS: Among cN+ patients, the true response rate to primary chemotherapy appears to be∼35%. Pathologic bladder response correlates well with nodal response. Finally, our study suggests that a panel of genes may be useful in distinguishing cN+ chemo-responders vs not, and thus could help reduce over- and undertreatment. These exploratory findings need prospective validation. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e775 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Amy Lim More articles by this author Akshay Sood More articles by this author Vikram Narayan More articles by this author Mohammed Seif More articles by this author Justin Matulay More articles by this author Kelly Bree More articles by this author Matt Campbell More articles by this author Ashish Kamat More articles by this author Colin Dinney More articles by this author Neema Navai More articles by this author Expand All Advertisement PDF downloadLoading ...

MTHFD1L confers a poor prognosis and malignant phenotype in esophageal squamous cell carcinoma by activating the ERK5 signaling pathway

MTHFD1L, a key enzyme of folate metabolism, is seldom reported in cancer. In this study, we investigate the role of MTHFD1L in the tumorigenicity of esophageal squamous cell carcinoma (ESCC). ESCC tissue microarrays (TMAs) containing 177 samples from 109 patients were utilized to evaluate whether MTHFD1L expression, determined using immunohistochemical analysis, is a prognostic indicator for ESCC patients. The function of MTHFD1L in the migration and invasion of ESCC cells was studied with wound healing, Transwell, and three-dimensional spheroid invasion assays in vitro and a lung metastasis mouse model in vivo. The mRNA microarrays and Ingenuity pathway analysis (IPA) were used to explore the downstream of MTHFD1L. Elevated expression of MTHFD1L in ESCC tissues was significantly associated with poor differentiation and prognosis. These phenotypic assays revealed that MTHFD1L significantly promote the viability and metastasis of ESCC cell in vivo and in vitro. Further detailed analyses of the molecular mechanism demonstrated that the ESCC progression driven by MTHFD1L was through up-regulation ERK5 signaling pathways. These findings reveal that MTHFD1L is positively associated with the aggressive phenotype of ESCC by activating ERK5 signaling pathways, suggesting that MTHFD1L is a new biomarker and a potential molecular therapeutic target for ESCC.

Hypoxia-induced lncRNA MRVI1-AS1 accelerates hepatocellular carcinoma progression by recruiting RNA-binding protein CELF2 to stabilize SKA1 mRNA

BackgroundLong non-coding RNAs (lncRNAs) perform a vital role during the progression of hepatocellular carcinoma (HCC). Here, we aimed to identify a novel lncRNA involved in HCC development and elucidate the underlying molecular mechanism.MethodsThe RT-qPCR and TCGA dataset analysis were applied to explore the expressions of MRVI1-AS1 in HCC tissues and cell lines. Statistical analysis was applied to analyze the clinical significance of MRVI1-AS1 in HCC. The functions of MRVI1-AS1 in HCC cells metastasis and growth were explored by transwell assays, wound healing assay, MTT assay, EdU assay, the intravenous transplantation tumor model, and the subcutaneous xenograft tumor model. Microarray mRNA expression analysis, dual luciferase assays, and actinomycin D treatment were used to explore the downstream target of MRVI1-AS1 in HCC cells. RIP assay was applied to assess the direct interactions between CELF2 and MRVI1-AS1 or SKA1 mRNA. Rescue experiments were employed to validate the functional effects of MRVI1-AS1, CELF2, and SKA1 on HCC cells.ResultsMRVI1-AS1 was found to be dramatically upregulated in HCC and the expression was strongly linked to tumor size, venous infiltration, TNM stage, as well as HCC patients’ outcome. Cytological experiments and animal experiments showed that MRVI1-AS1 promoted HCC cells metastasis and growth. Furthermore, SKA1 was identified as the downstream targeted mRNA of MRVI1-AS1 in HCC cells, and MRVI1-AS1 increased SKA1 expression by recruiting CELF2 protein to stabilize SKA1 mRNA. In addition, we found that MRVI1-AS1 expression was stimulated by hypoxia through a HIF-1-dependent manner, which meant that MRVI1-AS was a direct downstream target gene of HIF-1 in HCC.ConclusionIn a word, our findings elucidated that hypoxia-induced MRVI1-AS1 promotes metastasis and growth of HCC cells via recruiting CELF2 protein to stabilize SKA1 mRNA, pointing to MRVI1-AS1 as a promising clinical application target for HCC therapy.

SOX9 Expression Is Increased in Alzheimer's Disease (AD) and Is Associated With Disease Progression and APOE4 Genotype: A Computational Approach.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by depositions of amyloid-β protein leading to neuronal loss. Despite our understanding of the disease several gaps remain, including the role of astrocytes andastrocytic genes in the disease development and progression. Recently, some reports have suggested that SOX9 transcription factor (TF), an important mediator of astrocyte differentiation and maturation, might be linked to AD. Using human AD publicly available dataset, we aimed to analyze SOX9 expression and its relation to disease. The AD gene expression data set was obtained from National Center for Bioinformatics-Gene Expression Omnibus (NCBI-GEO). The GSE48350 consisted of mRNA microarray data from 55 normal controls (173 samples) and 26 AD cases (81 samples) obtained, from four brain regions.The SOX9 expression profile and correlations were analyzed using the R2 Genomics Analysis and Visualization platform. The SOX9 was significantly upregulated (p<0.001) in AD tissue compared to control cases. The increased expression appeared to be more in the entorhinal cortex (EC) and hippocampus (HC) regions. The SOX9 expression positively correlated with BRAAK stages (p<0.05). Interestingly in AD patients the SOX9 expression was significantly less in APOE3/3 genotypes compared with genotypes containing APOE4 allele. The SOX9 expression negatively correlated with oxidative phosphorylation genes which could suggest a metabolic role for the TF. From these data we hypothesize that SOX9 acts as a metabolic regulator responding to lipid metabolism disruption associated with APOE4 genotypes. In turn, SOX9 expression could be associated with astrocyte maturation and survival in the disease contributing thus to disease burden and disease progression.