Quantitative Immunohistochemistry Research Articles

Investigation of Cyclooxygenase-2 Expression in Tissue: Protein and mRNA Levels Compared Between Patients with Oral Squamous Cell Carcinoma and Oral Lichen Planus

Aim: Cyclooxygenase-2 (COX-2) is a crucial enzyme in the prostaglandin cascade. Overexpression of the COX-2 enzyme has been associated with the pathogenesis and progression of certain cancers, including oral cancer. This study aimed to investigate the quantitative expression and immunohistochemistry (IHC) expression of COX-2 in tissue samples from patients with oral squamous cell carcinoma (OSCC) and oral lichen planus (OLP) to gain insight into its potential role in the pathogenesis of these oral diseases. Methods: A total of 76 samples were analyzed, consisting of 25 cases of OSCC, 27 cases of OLP, and 24 cases of normal oral mucosa. COX-2 expression was analyzed using IHC staining and quantitative polymerase chain reaction (qPCR). Statistical analysis was performed using the Chi-squared, Spearman’s, and Fisher’s exact tests, with significance declared at a P value of <.05. Results: The mRNA expression of COX-2 in OSCC tissues (3.17 ± 1.02) was found to be significantly higher than in OLP (1.65 ± 0.72) and normal (1.08 ± 0.79; P < .01). In addition, protein expression of COX-2 was significantly higher in cancer tissues than in normal and OLP ( P < .05). The oral cavity was the most common tumor site in the studied cases, representing 56% of OSCC cases, as determined by qPCR analysis ( P < .05). Conclusion: Understanding the involvement of COX-2 in these oral diseases has implications for the development of targeted therapies and personalized treatment approaches. Also, the identification of COX-2 as a potential biomarker may contribute to improved diagnostic accuracy and prognosis in patients with OSCC and OLP. Moreover, the qPCR method could be as effective a technique as IHC to evaluate COX-2 amplification.

Supraphysiological Dose of Testosterone Impairs the Expression and Distribution of Sex Steroid Receptors during Endometrial Receptivity Development in Female Sprague-Dawley Rats.

This study aims to investigate the effect of a supraphysiological dose of testosterone on the levels of sex steroid hormones and the expression and distribution of sex steroid receptors in the uterus during the endometrial receptivity development period. In this study, adult female Sprague-Dawley rats (n = 24) were subcutaneously administered 1 mg/kg/day of testosterone alone or in combination with the inhibitors (finasteride or anastrozole or both) from day 1 to day 3 post-coitus, while a group of six untreated rats served as a control group. The rats were sacrificed on the evening of post-coital day 4 of to measure sex steroid hormone levels by ELISA. Meanwhile, gene expression and protein distribution of sex steroid receptors were analysed by quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC), respectively. In this study, treatment with a supraphysiological dose of testosterone led to a significant reduction in oestrogen and progesterone levels compared to the control. The mRNA expression of the androgen receptor increased significantly in all treatment groups, while the mRNA expression of both the progesterone receptor and the oestrogen receptor-α decreased significantly in all treatment groups. The IHC findings of all sex steroid receptors were coherent with all mRNAs involved. This study shows that a supraphysiological dose of testosterone was able to interrupt the short period of the implantation window. This finding could serve as a basis for understanding the role of testosterone in endometrial receptivity in order to develop further therapeutic approaches targeting androgen-mediated disorders of endometrial receptivity.

Role of HPV E7/miR-143-3p/SH2D3A pathway in regulating the occurrence and development of cervical cancer.

This study aims to explore the role of SH2D3A in cervical cancer, as well as its potential interaction with human papillomavirus (HPV) E7 and microRNA (miRNA). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to compare the expressions of SH2D3A in tissues. To assess the effects of SH2D3A on cervical cancer cell phenotypes, SH2D3A was knocked down in SiHa and HeLa cells, followed by cell proliferation (Cell Counting Kit-8 assay), apoptosis (flow cytometry), and invasion (Transwell assay) analyses. A transplantation tumor model was established to compare the tumorigenic ability of cervical cancer cells before and after SH2D3A silencing. Bioinformatics analysis predicted and dual-luciferase reporter assays verified the sponge adsorption effect of SH2D3A on miRNA. Western blot and qRT-PCR analyses were conducted to examine the impact on target genes following the downregulation of HPV E7 and SH2D3A. SH2D3A expression was significantly elevated in cervical cancer tissues. SH2D3A silencing inhibited cell proliferation and invasion, induced apoptosis, and reduced tumorigenesis in nude mice. Bioinformatics tools identified a binding relationship between SH2D3A and miR-143-3p, confirmed by the luciferase reporter assays. Western blot analysis revealed that SH2D3A knockdown led to decreased levels of Janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) proteins. Additionally, qRT-PCR showed that SH2D3A mRNA levels decreased after HPV E7 silencing, whereas miR-143-3p levels significantly increased. HPV E7 influences SH2D3A expression through miR-143-3p, thereby regulating the JAK1/STAT3 pathway. This mechanism promotes the occurrence and development of cervical cancer.

Proteoglycan 4 (Lubricin) and Regulation of Xanthine Oxidase in Synovial Macrophage as A Mechanism of Controlling Synovitis.

Synovial macrophages (SMs) are important effectors of joint health and disease. A novel Cx3CR1 + TREM2 + SM population expressing the tight junction protein claudin-5, was recently discovered in synovial lining. Ablation of these SMs was associated with onset of arthritis. Proteoglycan 4 (PRG4) is a mucinous glycoprotein that fulfills lubricating and homeostatic roles in the joint. The aim of this work is to study the role of PRG4 in modulating synovitis in the context of SM homeostasis and assess the contribution of xanthine oxidase (XO)-hypoxia inducible factor alpha (HIF-1α) axis to this regulation. We used Prg4 FrlioxP/FrtloxP ;R26 FlPoER/+ , a novel transgenic mouse, where the Prg4 Frt allele normally expresses the PRG4 protein and was designed to flank the first two exons of Prg4 with a flippase recognition target and "LOXP" sites. Inducing flippase activity with tamoxifen (TAM) inactivates the Frt allele and thus creates a conditional knockout state. We studied anti-inflammatory SMs and XO by quantitative immunohistochemistry, isolated RNA and studied immune pathway activations by multiplexed assays and isolated SMs and studied PRG4 signaling dysfunction in relation to glycolytic switching due to pro-inflammatory activation. Prg4 inactivated mice were treated with oral febuxostat, a specific XO inhibitor, and quantification of Cx3CR1 + TREM2 + SMs, XO immunostaining and synovitis assessment were conducted. Prg4 inactivation induced Cx3CR1 + TREM2 + SM loss (p < 0.001) and upregulated glycolysis and innate immune pathways in the synovium. In isolated SMs, Xdh (p < 0.01) and Hif1a (p < 0.05) were upregulated. Pro-inflammatory activation of SMs was evident by enhanced glycolytic flux and XO-generated reactive oxygen species (ROS). Febuxostat reduced glycolytic flux (p < 0.001) and HIF-1α levels (p < 0.0001) in SMs. Febuxostat also reduced systemic inflammation (p < 0.001), synovial hyperplasia (p < 0.001) and preserved Cx3CR1 + TREM2 + SMs (p < 0.0001) in synovia of Prg4 inactivated mice. PRG4 is a biologically significant modulator of synovial homeostasis via inhibition of XO expression and downstream HIF-1a activation. PRG4 signaling is anti-inflammatory and promotes synovial homeostasis in chronic synovitis, where direct XO inhibition is potentially therapeutic in chronic synovitis.

JARID2 activation by NFYA promotes stemness of triple-negative breast cancer cells through the PI3K/AKT pathway

ABSTRACT Background This study aimed to investigate the role of Jumonji AT Rich Interacting Domain 2 (JARID2) in regulating triple-negative breast cancer (TNBC) stemness and its mechanism. Research design and methods Bioinformatics analysis examined JARID2 expression, prognosis, and transcription factors. Quantitative polymerase chain reaction, western blot, and immunohistochemistry detected expression. Dual luciferase reporter gene and chromatin immunoprecipitation assays verified binding. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay detected viability and proliferation. Sphere formation assay detected the sphere formation efficiency. Flow cytometry detected CD44+/CD24− -marked stem cells. A xenograft tumor model verified the effect of JARID2 in vivo. Results JARID2 and nuclear transcription factor Y subunit α (NFYA) were upregulated in TNBC tissues and positively correlated. Knockdown of JARID2 or NFYA inhibited cell stemness by inhibiting the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway. Enforced JARID2 expression rescued the suppressive effect of NFYA knockdown on the PI3K/AKT signaling pathway and cell stemness. Knockdown of JARID2 inhibited tumor growth and cell stemness in mice but was alleviated by concurrent overexpression of NFYA. Conclusions NFYA promotes TNBC cell stemness by upregulating JARID2 expression and regulating the PI3K/AKT signaling pathway, suggesting JARID2 as a potential target for innovating drugs that target TNBC stem cells.

Study on the Effect of the Vaginal Administration of Conjugated Estrogens Cream Combined with Progesterone on the Endometrium of Rats and Its Mechanism of Action.

The purpose of this study was to investigate the impact of conjugated estrogen cream, in conjunction with progesterone, on the endometrium, following vaginal administration, and assess the combined dose-effect relationship with progesterone. Initially, bilateral ovaries from mature, female, Sprague Dawley rats were excised to establish a hypoestrogenic (perimenopausal) model. A conjugated estrogen-progesterone combination cream was administered vaginally for a duration of 12 days. Subsequently, this study used pathological sections, Enzyme-Linked Immunosorbent Assay (ELISA) for pharmacodynamic studies, network pharmacology to explore possible signalling pathways associated with the drug and menopausal syndrome, and partial validation using a real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (ICH). The results demonstrate that, relative to the model group, the conjugated estrogen monotherapy significantly increased the uterine weight coefficients (p < 0.0001) and endometrial thickness (p < 0.001) and upregulated the expression of Cyclin D1 and VEGF. Moreover, this treatment downregulated PTEN expression. The co-administration of progesterone reversed these effects in a dose-dependent manner. Overall, the vaginal administration of a combination of progesterone and conjugated estrogen cream demonstrated the ability to mitigate endometrial hyperplasia induced by conjugated estrogen vaginal cream monotherapy. Furthermore, the effect of progesterone exhibited a dose-dependent response.

IDH2 regulates macrophage polarization and tumorigenesis by modulating mitochondrial metabolism in macrophages

BackgroundTargeting the tumor microenvironment represents an emerging therapeutic strategy for cancer. Macrophages are an essential part of the tumor microenvironment. Macrophage polarization is modulated by mitochondrial metabolism, including oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and reactive oxygen species content. Isocitrate dehydrogenase 2 (IDH2), an enzyme involved in the TCA cycle, reportedly promotes cancer progression. However, the mechanisms through which IDH2 influences macrophage polarization and modulates tumor growth remain unknown.MethodsIn this study, IDH2-deficient knockout (KO) mice and primary cultured bone marrow-derived macrophages (BMDMs) were used. Both in vivo subcutaneous tumor experiments and in vitro co-culture experiments were performed, and samples were collected for analysis. Western blotting, RNA quantitative analysis, immunohistochemistry, and flow cytometry were employed to confirm changes in mitochondrial function and the resulting polarization of macrophages exposed to the tumor microenvironment. To analyze the effect on tumor cells, subcutaneous tumor size was measured, and growth and metastasis markers were identified.ResultsIDH2-deficient macrophages co-cultured with cancer cells were found to possess increased mitochondrial dysfunction and fission than wild-type BMDM. Additionally, the levels of M2-associated markers decreased, whereas M1-associated factor levels increased in IDH2-deficient macrophages. IDH2-deficient macrophages were predominantly M1. Tumor sizes in the IDH2-deficient mouse group were significantly smaller than in the wild-type mouse group. IDH2 deficiency in macrophages was associated with inhibited tumor growth and epithelial–mesenchymal transition.ConclusionsOur findings suggest that IDH2 deficiency inhibits M2 macrophage polarization and suppresses tumorigenesis. This study underlines the potential contribution of IDH2 expression in macrophages and tumor microenvironment remodeling, which could be useful in clinical cancer research.

Oncostatin M promotes epithelial barrier dysfunction in patients with eosinophilic chronic rhinosinusitis with nasal polyps

ABSTRACT Background Oncostatin M (OSM) may be involved in the promotion of mucosal epithelial barrier dysfunction in patients with eosinophilic chronic rhinosinusitis with nasal polyps (Eos CRSwNP) by inducing matrix metalloproteinase (MMP) −1 and −7. The aim was to evaluate the roles and mechanisms of action of OSM on MMP-1 and −7 synthesis from nasal epithelial cells (NECs). Methods OSM, OSM receptor (OSMR), MMP-1 and −7 expression was evaluated in nasal mucosa or primary NECs from scrapings by quantitative polymerase chain reaction (qPCR), immunofluorescence and immunohistochemistry. OSM and other cytokines were used to stimulate air-liquid interface (ALI) cultured NECs. qPCR, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were used to evaluate the expression of OSMR, MMP-1, −7 and occludin in NECs. Results Elevated levels of OSMRβ, MMP-1 and −7 were found in the tissues and scraped NECs of Eos CRSwNP in comparison to them obtained from the inferior turbinate (IT) and control subjects. The levels of OSM and OSMRβ mRNA in tissues were positively correlated with the levels of MMP-1 and −7. OSM stimulation of NECs increased the expression of MMP-1 and −7, and the responses were suppressed by a STAT3 inhibitor, and a PI3K inhibitor respectively. In parallel studies, we found that stimulation with OSM disrupted the localization of occludin, a tight junction protein in NECs. The response was suppressed by a pan-MMP inhibitor. Conclusion OSM induces the synthesis and release of MMP-1 and −7 in NECs. Furthermore, MMP-1 and −7 promote mucosal epithelial barrier dysfunction in patients with Eos CRSwNP.

Study on molecular mechanism of intervertebral disc degeneration by single cell hdWGCNA combined with transcriptome sequencing

BackgroundIntervertebral disc degeneration (IVDD) is one of the important causes of lower back pain, seriously affecting people's health and quality of life. This research employs single-cell analysis to identify the specific cellular subtypes and key regulatory genes associated with IVDD. MethodsWe analyzed the single-cell data and screened cells that closely associated with the development of IVDD. The differential expression of feature genes between IVDD and control groups was analyzed. Additionally, drugs and regulatory transcription factors that interact with feature genes were predicted and clinically validated by reverse transcription quantitative real-time PCR (RT-qPCR), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). ResultsOur study identified the Chond2 cell subtype associated with IVDD and selected four feature genes influencing the development of IVDD, namely IGFBP3, ACAN, VAPA and TMEM45A, through the high-dimensional weighted gene co-expression network analysis (hdWGCNA) analysis, least absolute shrinkage and selection operator (LASSO), and random forest (RF). Besides, compared to the MDD group, IGFBP3 and TMEM45A were significantly upregulated in the SDD group, while ACAN and VAPA showed no significant difference between the two groups. ELISA testing revealed a positive correlation between IGFBP3 concentration and the grading of IVDD. Furthermore, Celecoxib may be used to treat IVDD by inhibiting IGFBP3. ConclusionOur study identified the Chond2 cell subtype associated with IVDD and selected four feature genes influencing the development of IVDD, namely IGFBP3, ACAN, VAPA and TMEM45A. Our findings establish a robust theoretical foundation for the clinical diagnosis and treatment of IVDD patients.

A novel m6A reader RBFOX2 expression is increased in oral squamous cell carcinoma and promotes tumorigenesis

Background and ObjectiveOral squamous cell carcinoma (OSCC) is a significant global health concern due to its aggressive nature and poor prognosis. Recent research has highlighted the important role of RNA modifications in cancer biology, especially N6-methyladenosine (m6A) modifications, which are controlled by a complex interplay of m6A regulators. This study specifically investigates RBFOX2, a new m6A reader, and its involvement in OSCC. MethodsOur study primarily utilized OSCC tissue, adjacent normal tissue samples, OSCC cell lines, and normal healthy oral keratinocytes to validate RBFOX2 mRNA expression. Additionally, we used the TCGA-HNSCC dataset for large cohort analysis and clinicopathological characterization. Furthermore, we visualized the RBFOX2 network to identify the primary functions of the protein. ResultsOur research shows a noticeable increase in RBFOX2 expression in OSCC tissues compared to adjacent non-tumorous tissues, as determined by quantitative PCR and immunohistochemistry analyses. Functional pathway enrichment analysis revealed that RBFOX2 is involved in the receptor tyrosine kinase signaling pathway and the Hippo signaling pathway, which plays a critical role in oral cancer development and progression. Clinically, elevated RBFOX2 expression correlated with advanced tumor stages and poorer patient outcomes, demonstrating its prognostic value. These findings indicate that RBFOX2 acts as an oncogenic driver in OSCC as an m6A reader, facilitating the expression of m6A-modified oncogenes. ConclusionOur study identifies RBFOX2 as a critical player in OSCC pathogenesis and opens avenues for novel therapeutic strategies targeting the m6A regulatory machinery in this malignancy.

Increased CCT5 expression is a potential unfavourable factor promoting the growth of nasopharyngeal carcinoma.

Chaperonin containing TCP1 subunit 5 (CCT5) encodes the CCT5 protein subunit of chaperonin-containing TCP-1 (CCT/TRiC) complex, and is shown to be upregulated in tumour pathogenesis. The study aim was to investigate the differential expression of CCT5 between nasopharyngeal carcinoma (NPC) and noncancerous nasopharyngeal tissues, and the correlation between CCT5 expression and clinicopathological parameters/prognosis in patients with NPC. Microarray assay data were evaluated for differential expression between NPC and noncancerous nasopharyngeal tissues. CCT5 expression in NPC and noncancerous nasopharyngeal tissues was determined at mRNA and protein levels by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry. Relationships between CCT5 expression in NPC, clinical parameters, and prognosis were statistically analysed. CCT5-mediated cell proliferation was assessed using EdU and cell counting kit-8. Western blot and co-immunoprecipitation were utilized to explore E3 ubiquitin-protein ligase parkin (PARK2)-induced degradation of CCT5. Microarray data showed CCT5 levels to be significantly increased in NPC versus noncancerous nasopharyngeal tissues, which was confirmed by qRT-PCR and immunohistochemical assays. Increased CCT5 protein levels positively correlated with tumour size, tumour recurrence, and clinical stage, and inversely correlated with patient's overall survival. Multivariate Cox regression analysis showed that enhanced CCT5 protein expression is an independent prognostic factor for patients with NPC. Overexpression of CCT5 markedly induced NPC cell proliferation. Finally, PARK2, as a suppressive E3 ubiquitin-ligase enzyme, was shown to bind CCT5 and induce degradation in NPC. Increased CCT5 may be an unfavourable factor promoting NPC growth. Binding of PARK2 to CCT5 was associated with CCT5 degradation, suggesting that PARK2 is an upstream negative modulator in NPC.

Ferroptosis-related gene signature predicts prognosis and immune microenvironment in prostate cancer.

Ferroptosis, an iron-dependent form of programmed cell death, significantly impacts cancer, yet its link to prostate cancer (PCa) prognosis remains underexplored. This study aims to develop and validate a ferroptosis-related gene signature to predict PCa prognosis and immune microenvironment differences, potentially identifying therapeutic targets. RNA-sequencing data of 478 PCa patients and corresponding clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. We investigated the disease-free survival (DFS) rates of the high- and low-risk groups using the Kaplan-Meier method. Functional differences between the high- and low-risk groups were investigated by a gene set enrichment analysis (GSEA), and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The link between ferroptosis risk score and immune status was examined using CIBERSORT. The expression levels of core prognostic genes in benign prostatic hyperplasia (BPH) and PCa were verified using quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemistry (IHC). A novel ferroptosis-related prognostic gene signature was established and tested in the Gene Expression Omnibus (GEO) database based on univariate and multivariate Cox regression analyses. Patients with PCa were classified into high- and low-risk groups based on this ferroptosis signature. Patients in the high-risk group had worse outcomes than those in the low-risk group. The predictive accuracy of the model was demonstrated by a receiver operating characteristic (ROC) analysis. An additional enrichment analysis of TCGA cohort revealed the immune-related pathways were significantly upregulated in the high-risk group, with areas under the curve (AUCs) of 0.85 at 1 year, 0.82 at 3 years, and 0.76 at 5 years. In the GEO cohort, the AUCs reached 0.69 at 1 year, 0.74 at 3 years, and 0.75 at 5 years. An additional enrichment analysis indicated a significant upregulation of cytokine-related pathways, immune receptor activity, and other immune-related pathways in the high-risk group. Furthermore, the analysis revealed that the proportions of mast cells and plasma cells were significantly lower in the high-risk group compared to the low-risk group of PCa patients. Conversely, the proportion of regulatory T cells (Tregs) was significantly higher in the high-risk group than in the low-risk group. According to the qRT-PCR, Western blot, and IHC results, DRD4, SRC, AKR1C2, and AIFM2 expression was significantly higher in PCa than BPH. We also showed that the ferrostatin 1-treated LNCaP cells had higher expression levels of DRD4, SRC, and AKR1C2. A prognostic signature of eight ferroptosis-related genes (FRGs) that may accurately predict PCa patient outcomes was constructed and validated. FRGs may contribute to anti-tumor immunity and serve as therapeutic targets in PCa.

Zoledronic acid-treated rats show altered jaw histology and gene expression in non-exposed MRONJ

BackgroundDiagnosis and management of medication-related osteonecrosis of the jaws (MRONJ) prior to clinical exposure induced by trauma may lead to improved patient management. Currently, few studies have examined early histologic and molecular MRONJ-related changes in the jaws. PurposeThis study aimed to identify histological and gene expression changes in the maxilla and mandible of Sprague-Dawley (SD) rats after zoledronic acid (ZA) treatment. Study Design, Setting, and SampleThis was an in-vivo animal study. The experiments were conducted in the laboratory at the Stomatology Hospital of Tianjin Medical University. A total of 12 SD rats were included. Predictor variableThe predictor variable was ZA exposure. Twelve SD rats were divided into two groups: experimental (n = 6) and control (n = 6), and they were intraperitoneally injected with ZA and saline, respectively. Main outcome variableThe outcome variables were histological and molecular changes. The maxilla, mandible, and ilium bone tissue samples were examined using Masson's trichrome and hematoxylin-eosin staining. Gene expression changes were identified using transcriptome sequencing, Kyoto encyclopedia of genes and genomes (KEGG), and gene interactome network analysis. The key changes were validated using the quantitative real-time polymerase chain reaction and immunohistochemistry. CovariatesNone. AnalysesThe t-test, Chi-square test, and Fisher's exact probability method were used for statistical analyses using the Statistical package for the social sciences software (version 26.0). ResultsAll animals remained healthy during the experiments. Histological staining revealed that the percentage of empty bone lacunae in the maxilla and mandible was significantly higher than that in the ilium (P < 0.01). In total, 552 genes were screened using transcriptome sequencing. The sonic hedgehog (Shh) signaling pathway was highly enriched. The key gene for the Shh interaction was distal-less homeobox 5 (Dlx-5). The Shh, Dlx-5, and bone morphogenetic protein 2 genes and protein expression levels in the maxilla and mandible were higher in the experimental group than in the control group (P < 0.05). Conclusions and RelevanceMRONJ-induced osteonecrosis and gene expression changes precede trauma-induced clinical changes in the SD rat model. These findings may provide additional support for timely and clinically early diagnosis and intervention.

Revealing the key role of cuproptosis in osteoporosis via the bioinformatic analysis and experimental validation of cuproptosis-related genes.

The incidence of osteoporosis has rapidly increased owing to the ageing population. Cuproptosis, a novel mechanism that regulates cell death, may be a new therapeutic approach. However, the relevance of cuproptosis in the immune microenvironment and osteoporosis immunotherapy is still unknown. We intersected the differentially expressed genes from osteoporotic samples with 75 cuproptosis-related genes to identify 16 significantly expressed cuproptosis genes. We further explored the connection between the cuproptosis pattern, immune microenvironment, and immunotherapy. The weighted gene co-expression network analysis algorithm was used to identify cuproptosis phenotype-associated genes, and we used quantitative real-time PCR and immunohistochemistry in mouse femur tissues to verify hub gene (MAP2K2, FDX1, COX19, VEGFA, CDKN2A, and NFE2L2) expression. Six hub genes and 59 cuproptosis phenotype-associated genes involved in immunisation were identified among the osteoporosis and control groups, and the majority of these 59 genes were enriched in the inflammatory response, as well as in signal transducers, Janus kinase, and transcription pathway activators. In addition, two different clusters of cuproptosis were found, and immune infiltration analysis showed that gene Cluster 1 had a greater immune score and immune infiltration level. Further analysis revealed that three key genes (COX19, MAP2K2, and FDX1) were highly correlated with immune cell infiltration, and external experiments validated the association of these three genes with the prognosis of osteoporosis. We used the three key mRNAs COX19, MAP2K2, and FDX1 as a classification model that may systematically elucidate the complex connection between cuproptosis and the immune microenvironment of osteoporosis. New insights into osteoporosis pathogenesis and immunotherapy prospects may be gained from this study.

TIPE2 inhibits the migration and invasion of epithelial ovarian cancer cells by targeting Smad2 to reverse TGF-β1-induced EMT.

Epithelial ovarian cancer is the deadliest gynecologic malignancy, characterized by high metastasis. Transforming growth factor-β1 (TGF-β1) drives epithelial- mesenchymal transformation (EMT), a key process in tumor metastasis. Tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TIPE2) acts as a negative regulator of innate and adaptive immunity and involves in various cancers. However, its relationship with TGF-β1 in ovarian cancer and its role in reversing TGF-β1-induced EMT remain unclear. This study examined TIPE2 mRNA and protein expression using quantitative RT-PCR (qRT-PCR), western blot and immunohistochemistry. The effects of TIPE2 overexpression and knockdown on the proliferation, migration and invasion of epithelial ovarian cancer cells were assessed through 5-ethynyl-2-deoxyuridine, colony-forming, transwell migration and invasion assays. The relationship between TIPE2 and TGF-β1 was investigated using qRT-PCR and enzyme-linked immunosorbent assay, while the interaction between TIPE2 and Smad2 was identified via co-immunoprecipitation. The results revealed that TIPE2 protein was significantly down-regulated in epithelial ovarian cancer tissues and correlated with the pathological type of tumor, patients' age, tumor differentiation degree and FIGO stage. TIPE2 and TGF-β1 appeared to play an opposite role to each other during the progression of human ovarian cancer cells. Furthermore, TIPE2 inhibited the metastasis and EMT of ovarian cancer cells by combining with Smad2 invitro or in an intraperitoneal metastasis model. Consequently, these findings suggest that TIPE2 plays a crucial inhibitory role in ovarian cancer metastasis by modulating the TGF-β1/Smad2/EMT signaling pathway and may serve as a potential target for ovarian cancer, providing important direction for future diagnostic and therapeutic strategies.

Exploration of Key Genes Combining with Immune Infiltration Level andTumor Mutational Burden in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a lethal malignancy due to its heterogeneity and aggressive behavior. Recently, somatic mutations and tumor cell interactions with the surrounding tumor immune microenvironment (TIME) have been reported to participate in HCC carcinogenesis and predict HCC progression. In this study, we aimed to investigate the association between tumor mutational burden (TMB) and TIME in HCC. Additionally, we sought to identify differentially expressed genes (DEGs) associated with HCC prognosis and progression. The expression, clinical, and mutational data were downloaded from the cancer genome atlas (TCGA) database. The immune infiltration levels and TMB levels of the HCC samples were estimated and the samples were divided into immune cluster (ICR)-1 and 2 based on immune infiltration score and high and low TMB groups based on TMB score. Thereafter, differential gene expression analysis was conducted to identify the DEGs in the ICR1/2 and high/low TMB groups, and the intersecting DEGs were selected. Thereafter, Cox regression analysis was performed on 89 significant DEGs, among which 19 were associated with prognosis. These 19 DEGs were then used to construct a prognostic model based on their expression levels and regression coefficients. Thereafter, we analyzed the DEGs in mutant and wildtype TP53 HCC samples and identified high BCL10 and TRAF3 expression in the mutant TP53 samples. BCL10 and TRAF3 expression was detected by real-time quantitative reverse transcription PCR and immunohistochemistry, and their clinical correlation, biological function, and immune infiltration levels were analyzed by chi-square analyses, Gene Set Enrichment Analysis (GSEA), and "ssGSEA", respectively. The results of our study revealed that immune infiltration level was correlated with TMB and that they synergistically predicted poor prognosis of HCC patients. DEGs enriched in immune-related pathways could serve as indicators of immunotherapy response in HCC. Among these DEGs, BCL10 and TRAF3 were highly expressed in HCC tissues, especially in the mutant TP53 group, and they co-operatively exhibited immunological function, thereby affecting HCC progression and prognosis. In this study, we identified BCL10 and TRAF3 as potential prognostic indicators in HCC patients. Additionally, we found that BCL10 and TRAF3 influence TMB and TIME in HCC patients and can be used for the development of immune-based therapies for improving the long-term survival of HCC patients.

The consistently up-regulated expression of NLRP3 in severe asthma patients from mRNA microarray and ovalbumin-induced mouse model of asthma.

Severe asthma (SA) is a chronic lung disease, resistant to current treatments, symbolized by repeated symptoms of reversible airflow obstruction, airway hyper-responsiveness, and inflammation. The aim of this study was to identify genes exhibiting differential expression in individuals without asthma and SA patients. We aimed to pinpoint hub differentially expressed genes (DEGs) by utilizing a mouse model of asthma sensitized to ovalbumin (OVA). Microarray data for SA were acquired from the Gene Expression Omnibus (GEO) databases. DEGs were identified, and functional enrichment analyses were carried out. STRING and Cytoscape were utilized to design a protein-protein interaction (PPI) network and conduct module analysis. An OVA-induced asthma mice model was established. Lung tissue from the mice was collected for quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemistry (IHC) to assess the expression of DEGs. A total of 545 DEGs were identified, among which 172 genes were upregulated in SA patients compared to healthy controls. The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) was significantly up-regulated in SA patients [adjusted P value (Padj) =0.001]. Analysis of lung tissue using qRT-PCR, western blot, and IHC revealed higher expression of NLRP3 in OVA-induced asthma mice compared to the control group. Enrichment analysis suggests the involvement of NLRP3 in pathways related to pyroptosis, c-type lectin receptor signaling, and NOD-like receptor signaling. Through bioinformatics analysis, we identified a multitude of DEGs that could potentially contribute significantly to the development of SA. Notably, our findings highlight NLRP3 as a potential pivotal player in asthma pathogenesis, underscoring its prospective utility as a biomarker for SA.

Exploration of the ubiquitination-related molecular classification and signature to predict the survival and immune microenvironment in colon cancer.

Ubiquitination, a major post-translational modification, significantly impacts tumorigenesis, progression, and prognosis. This study aims to classify colon cancer at the molecular level and create a reliable signature using ubiquitination-related genes (URGs) to assess the immune microenvironment and prognosis. We employed non-negative matrix factorization to subtype colon cancer based on ubiquitination-related gene (URG) expression patterns. Quantitative scores for 28 immune cell infiltrates and the tumor microenvironment were computed using single-sample gene set enrichment analysis (ssGSEA) and the Estimate algorithm. Subtype feature genes were selected through Lasso logistic regression and SVM-RFE algorithm. The ubiquitination-related signature was constructed using univariate Cox, Lasso, and stepwise regression methods to categorize patients into high and low-risk groups. Validation included log-rank tests, receiver operating characteristic (ROC) analysis, decision curve analysis (DCA), and external dataset validation. Immune therapy response was compared using Tumor Immune Dysfunction and Exclusion (TIDE), Immunophenoscore (IPS), and submap analyses. Clinical variables and risk scores were integrated into an enhanced nomogram. The early diagnostic value of four URGs was confirmed via quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. The cell proliferation was assessed through colony formation, EdU staining, and xenograft tumorigenesis assays. Prognostic ubiquitination-related genes (URGs) stratified patients into subtypes, revealing differences in survival, immune cell infiltration, and pathological staging. A signature of 6 URGs (ARHGAP4, MID2, SIAH2, TRIM45, UBE2D2, WDR72) was identified from 57 subtype-related genes. The high-risk group exhibited characteristics indicative of enhanced epithelial-mesenchymal transition, immune escape, immunosuppressive myeloid-derived suppressor cells, regulatory T cell infiltration, and lower immunogenicity. In contrast, the low-risk group demonstrated the opposite trend but showed a better response to CTLA4 checkpoint inhibitors. The predictive performance of the nomogram significantly improved with the integration of risk score, stage, and age. ARHGAP4 and SIAH2 exhibit promising early diagnostic capabilities. Additionally, WDR72 knockdown significantly inhibited CRC cell proliferation both in vitro and in vivo. Our developed ubiquitination-related signature and genes serve as promising biomarkers for colon cancer prognosis, immune microenvironment, and diagnosis.

MDia2 is an important mediator of MRTF-A-dependent regulation of breast cancer cell migration.

Dysregulated actin cytoskeleton gives rise to aberrant cell motility and metastatic spread of tumor cells. This study evaluates the effect of overexpression of wild-type versus functional mutants of MRTF-A on migration and invasion of breast cancer (BC) cells. Our studies indicate that SRF's interaction is critical for MRTF-A-induced promotion of both two-dimensional and three-dimensional cell migration, while the SAP-domain function is important selectively for three-dimensional cell migration. Increased MRTF-A activity is associated with more effective membrane protrusion, a phenotype that is attributed predominantly to SRF's interaction with MRTF. We demonstrate formin-family protein mDia2 as an important mediator of MRTF-stimulated actin polymerization at the leading edge and cell migration. Multiplexed quantitative immunohistochemistry and transcriptome analyses of clinical BC specimens further demonstrate a positive correlation between nuclear localization of MRTF with malignant traits of cancer cells and enrichment of MRTF-SRF gene signature in pair-matched distant metastases versus primary tumors. In conclusion, this study establishes a novel mechanism of MRTF-dependent regulation of cell migration and provides evidence for the association between MRTF activity and increased malignancy in human BC, justifying future development of specific small molecule inhibitors of the MRTF-SRF transcriptional complex as potential therapeutic agents in BC.

The novel circFKBP8/miR-432-5p/E2F7 cascade functions as a regulatory network in breast cancer

BackgroundCircular RNAs (circRNAs) are capable of affecting breast cancer (BC) development. However, the role and underneath mechanism of circFKBP8 (also known as hsa_circ_0000915) in BC remain largely unknown.MethodsExpression analyses were performed using quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry (IHC) assays. Effects on cell functional phenotypes were determined by assessing cell proliferation, migratory capacity, invasion, and stemness in vitro. The relationship between microRNA (miR)-432-5p and circFKBP8 or E2F transcription factor 7 (E2F7) was examined by RNA pull-down, dual-luciferase reporter, and RNA immunoprecipitation (RIP) assays. Xenograft assays were used to identify the function of circFKBP8 in vivo.ResultsCircFKBP8 was presented at high levels in BC tissues and cells. High circFKBP8 expression was associated with worse overall survival in BC patients. CircFKBP8 suppression inhibited BC cell proliferation, migratory capacity, invasion and stemness in vitro. CircFKBP8 suppression blocked xenograft tumor growth in vivo. Mechanistically, circFKBP8 functioned as a miR-432-5p sponge to modulate E2F7 expression. CircFKBP8 modulated BC cell malignant behaviors by miR-432-5p, and miR-432-5p affected these cell phenotypes through E2F7.ConclusionOur observations prove that circFKBP8 promotes BC malignant phenotypes through the miR-432-5p/E2F7 cascade, offering a promising therapeutic and prognostic target for BC.