Gene Oncology Research Articles

P4HA3 promotes head and neck squamous cell carcinoma progression via the WNT/β-catenin signaling pathway

Here, we explored the role of Prolyl 4-Hydroxylase Subunit Alpha 3 (P4HA3), the most recently identified member of the prolyl-4-hydroxylase (P4H) family, in head and neck squamous cell carcinoma (HNSCC) progression. P4HA3 is upregulated during cancer progression; however, its specific role in HNSCC progression remains elusive. Thus, this study aimed to elucidate the regulatory function of P4HA3 in HNSCC development and progression and to describe the underlying mechanisms. Initially, we analyzed the correlation between the expression of P4HA3 and the WNT pathway genes and clinicopathologic features in HNSCC based on microarray data from The Cancer Genome Atlas (TCGA). Next, we used Gene Oncology (GO) functional data to describe several potentially associated pathways in HNSCC. Then, we knocked down P4HA3 in SCC15 and SCC25 cells, two classic HNSCC cell lines, and assessed the resulting changes using RT-qPCR. Furthermore, we used Western blot to evaluate the regulatory role of P4HA3 in the epithelial-to-mesenchymal transition (EMT) and the WNT/β-catenin signaling pathway. To explore the effect of P4HA3 knockdown on tumor progression, in vivo experiments were conducted using a murine model. Immunohistochemistry assays were then employed to identify proteins associated with EMT and the WNT/β-catenin signaling pathway in tumor tissues. Upregulated P4HA3 in HNSCC patient tumor tissues was positively correlated with poor prognosis. Notably, P4HA3 knockdown significantly inhibited the proliferative and invasive abilities of HNSCC. The levels of genes and proteins associated with EMT and the WNT/β-catenin signaling pathway were also markedly reduced by P4HA3 knockdown. Importantly, the in vivo experiments demonstrated that P4HA3 can promote subcutaneous tumorigenesis in nude mice and knockdown of P4HA3 induce a significant ihibitation of EMT and WNT/β-catenin pathway detected by immunohistochemistry assay in tumor tissues. In summary, we demonstrated that P4HA3 is a promising diagnostic and therapeutic biomarker for HNSCC. As an oncogene, P4HA3 increases HNSCC proliferation by inducing the EMT and activating the WNT/β-catenin signaling pathway.

The Immune System-A Double-Edged Sword for Adenovirus-Based Therapies.

Pathogenic adenovirus (Ad) infections are widespread but typically mild and transient, except in the immunocompromised. As vectors for gene therapy, vaccine, and oncology applications, Ad-based platforms offer advantages, including ease of genetic manipulation, scale of production, and well-established safety profiles, making them attractive tools for therapeutic development. However, the immune system often poses a significant challenge that must be overcome for adenovirus-based therapies to be truly efficacious. Both pre-existing anti-Ad immunity in the population as well as the rapid development of an immune response against engineered adenoviral vectors can have detrimental effects on the downstream impact of an adenovirus-based therapeutic. This review focuses on the different challenges posed, including pre-existing natural immunity and anti-vector immunity induced by a therapeutic, in the context of innate and adaptive immune responses. We summarise different approaches developed with the aim of tackling these problems, as well as their outcomes and potential future applications.

Proteomic profiling points to the significance of adipocyte-derived extracellular vesicles in diet-induced obesity

Extracellular vesicles (EVs) are lipid-bilayer membrane-enclosed vesicles well known to mediate cell-cell communication to maintain normal physiological functions. The diverse cargo of EVs encompassing proteins, lipids and miRNAs represent potential biomarkers and therapeutic targets. However, the role of adipocyte-derived EVs (AdEVs) in obesity is still unknown. To address this, we performed proteomic profiling of AdEVs isolated from the C57BL/6 male mice that were fed either a normal chow diet (NCD) or a high-fat high-sucrose diet (HFHSD) for 12 weeks starting at 4 weeks of age. AdEVs were obtained, through ultracentrifugation, from the conditioned media of cultured adipocytes derived from gonadal adipose tissue. Nanoparticle tracking analysis (NTA) of AdEVs showed that adipocytes from mice fed HFHSD tended to release more AdEVs despite the comparable size of AdEVs relative to those of mice fed NCD. Subsequently, proteins were isolated, digested and labeled with TMTpro16-plex for proteomic analysis. Raw data were processed in Scaffold, and a general bioinformatic and functional analysis conducted in Ingenuity Pathway Analysis Qiagen (IPA) ( https://digitalinsights.qiagen.com/explore/ga-ipa?utm_source=QDI_GA_SA_IPA_1122_PF&cmpid=QDI_GA_IPA ) and Srplot identified over 1100 proteins in AdEVs (protein threshold: 99%, minim peptide:2, peptide threshold: 95%, 0.00% FDR). Of these, 58 proteins were identified as upregulated while 41 proteins were downregulated in HFHSD-fed mice (Fold Change > 2, adjusted p < 0.05). With IPA analysis, the top 3 influenced pathways including mitochondrial dysfunction (27%), Sirtuin signaling pathway (22%), and Granzyme A signaling (38%) are predicted to be activated while the oxidative phosphorylation (47%), estrogen receptor signaling (15%) and neutrophil extracellular trap signaling pathway (14%) are predicted to be inhibited. The Gene Oncology (GO) functional enrichment analysis in Srplot revealed the involvement of identified differentially expressed proteins in various biological processes such as lipid metabolism, catabolic process processes, and cellular respiration. Experiments are underway to validate specific proteins involved in these biological processes. Furthermore, analysis of cellular components highlighted three notable terms including myelin sheath, collagen-containing extracellular matrix and organelle inner membrane. In conclusion, the present study elucidates the significance of AdEVs in the context of diet-induced obesity and underscores the potential role of mitochondrial dysfunction in the initiation and progression of obesity and its associated diseases. NIH Veterans Affairs. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1346: Combined role of interleukin-15 stimulated natural killer cell-derived extracellular vesicles and carboplatin in Osimertinib resistant H1975 lung cancer with EGFR mutations

Abstract Extracellular vesicles (EVs) derived from immune cells (e.g., NK cells, dendritic cells) may be able to treat malignancies that are resistant to other treatments (e.g., chemotherapy) relying on the hypothesis that EVs carry almost the same cargo of the parent cells. In this study, we evaluated IL-15 stimulated natural killer cell-derived EVs (NK-EVs) as therapeutic agents in vitro and in vivo in Osimertinib resistant lung cancer (H1975R) with EGFR mutations(L858R). NK-EVs were isolated by ultracentrifugation and nanoparticle tracking analysis revealed a size distribution of 89.5 ± 3.4 nm, zeta potential of -31.38 ± 0.25 mV. Atomic force microscopy imaging revealed vesicles with a spherical form and comparable sizes, meeting the criteria of exosomal EVs. Further, western blot studies demonstrated the presence of regular EV markers along with specific NK markers (perforin and granzyme). EVs were also characterized by using proteomic analysis which demonstrated that EVs had proteins for natural killer cell mediated cytotoxicity (Granzyme B) and T cell activation (Perforin and Plastin-2). Gene oncology (GO) analysis also showed that these are differentially expressed proteins (DEPs) that are involved in programmed cell death and positive regulation of cell death. Further, isolated NK-EVs (1*1011 particles/mL) were cytotoxic to H1975R cells in vitro with 55% and 68% cell viability in 2D and 3D cell cultures respectively against control. Carboplatin’s IC50 was reduced by approximately 1.9 and 1.7-fold (p less than 0.001) in 2D and 3D cell culture respectively when combined with NK-EVs. The EVs were then combined with carboplatin (25 mg/kg) and administered by i.p. route to H1975R tumor xenografts and significant reduction in tumor volume in vivo (≈1000 mm3 compared to ≈2000 mm3 of control group, p less than 0.001) was observed after 10 days. Our findings show for the first time that NK-EVs target the PD-L1/PD-1 immunological checkpoint and induce apoptosis and anti-inflammatory response by downregulation of SOD2, PARP, BCL2, SET, NF-κB and TGF-ß. MicroRNAs regulating cytotoxic proteins like perforin, granzyme and plastin were also identified using sequencing and the miRNAs: hsa-miR-5193/149-5P/3133/193-5p are picked for further studies. These miRNAs have been encapsulated in NK-EVs by electroporation and will be evaluated against Lung PDX xenografts along with carboplatin loaded NK-EVs. These miRNAs are directly involved in T cell or NK cell mediated cytotoxicity and are being currently investigated. The ability to isolate functional NK-EVs on a large scale and using them with platinum-based drugs may lead to new clinical applications. The results of the present study suggest the possibility of the combination of NK-cell-derived EVs and carboplatin as a viable immunochemotherapeutic strategy for resistant cancers. Citation Format: Aakash Nathani, Li Sun, Islauddin Khan, Mounika Aare, Arvind Bagde, Yan Li, Mandip Sachdeva. Combined role of interleukin-15 stimulated natural killer cell-derived extracellular vesicles and carboplatin in Osimertinib resistant H1975 lung cancer with EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1346.

Characteristics of innate and adaptive immune disorder in IgA nephropathy based on integrated bioinformatics.

Our study aims to investigate the immunological pathogenesis underlying immunoglobulin A nephropathy (IgAN) and explore potential biomarkers for IgAN diagnosis. Differentially expressed genes (DEGs) of formalin-fixed and paraffin-embedded (FFPE) samples were screened between IgAN patients and healthy people based on GSE115857. Gene oncology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) enrichment was performed to identify related biological processes and pathways. CIBERSORT was utilized to seek the relationship of immune cell infiltration with IgAN. Finally, the expression of paraoxonase 2 (PON2) related to innate immune response was verified in FFPE samples of minimal change disease and IgAN patients by immunohistochemistry and PAS staining. 25 down-regulated genes and 12 up-regulated genes were identified in IgAN patients, which mainly responded to endothelial cell proliferation, inflammatory response, and angiogenesis. Toll-like receptor signaling pathway and Epstein-Barr virus (EBV) infection might be involved in IgAN pathogenesis. In addition, the infiltration of macrophages M0, naïve B cells, and follicular helper T (Tfh) cells was positively correlated in IgAN patients. Macrophages M1 and M2 infiltration were up-regulated in IgAN patients, which indicated that innate immune response was closely associated with IgAN. Besides, the results of immunohistochemistry showed that PON2 was obviously positively expressed in acute and chronic lesions of IgAN patients. In addition to abnormalities in the adaptive immune response, macrophages M1/M2 and innate immune disorder may participate in IgAN pathogenesis. PON2 may become the feasible targets for further investigation of IgAN.

Combined Role of Interleukin-15 Stimulated Natural Killer Cell-Derived Extracellular Vesicles and Carboplatin in Osimertinib-Resistant H1975 Lung Cancer Cells with EGFR Mutations.

In this study, we evaluated IL-15 stimulated natural killer cell-derived EVs (NK-EVs) as therapeutic agents in vitro and in vivo in Osimertinib-resistant lung cancer (H1975R) with EGFR mutations (L858R) in combination with carboplatin (CBP). NK-EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, and atomic force microscopy imaging revealed vesicles with a spherical form and sizes meeting the criteria of exosomal EVs. Further, Western blot studies demonstrated the presence of regular EV markers along with specific NK markers (perforin and granzyme). EVs were also characterized by proteomic analysis, which demonstrated that EVs had proteins for natural killer cell-mediated cytotoxicity (Granzyme B) and T cell activation (perforin and plastin-2). Gene oncology analysis showed that these differentially expressed proteins are involved in programmed cell death and positive regulation of cell death. Further, isolated NK-EVs were cytotoxic to H1975R cells in vitro in 2D and 3D cell cultures. CBP's IC50 was reduced by approximately in 2D and 3D cell cultures when combined with NK-EVs. The EVs were then combined with CBP and administered by i.p. route to H1975R tumor xenografts, and a significant reduction in tumor volume in vivo was observed. Our findings show for the first time that NK-EVs target the PD-L1/PD-1 immunological checkpoint to induce apoptosis and anti-inflammatory response by downregulation of SOD2, PARP, BCL2, SET, NF-κB, and TGF-ß. The ability to isolate functional NK-EVs on a large scale and use them with platinum-based drugs may lead to new clinical applications. The results of the present study suggest the possibility of the combination of NK-cell-derived EVs and CBP as a viable immunochemotherapeutic strategy for resistant cancers.

Comprehensive analysis of ETS1 expression and its prognostic value in clear cell renal cell carcinoma.

ETS1, a member of the large ETS domain family of transcription factors, plays a role in the progression of many types of carcinoma. ETS1 expression has been linked to a more favorable prognosis in renal cell carcinoma. The objective of this study was to assess the predictive significance of ETS1 in individuals suffering from clear cell renal cell carcinoma (ccRCC). The correlation between ETS1 expression and ccRCC was analyzed. Data on ETS1 and clinical information for ccRCC patients were obtained from the Cancer Genome Atlas database and analyzed using R software. Then, we presented validation results using RT-qPCR (quantitative reverse transcription PCR). The receiver operator characteristic (ROC) curves were generated using the pROC software package to determine the cutoff values for ETS1. Additionally, the ImmuneScore, StromalScore, and ESTIMATEScore were calculated using the ESTIMATES algorithm. The connection between ccRCC and ETS1 was investigated using enrichment analysis based on Gene Oncology and the Kyoto Encyclopedia of Genes and Genomes. The tumor immunity estimation resource (TIMER) and the integrated repository portal for tumor-immune system interactions (TISIDB) databases were utilized to analyze the association between ETS1 expression and immune cell infiltration in ccRCC. The impact of ETS1 on the survival of ccRCC patients was evaluated using the PrognoScan database. We evaluated the Tumor Mutation Burden (TMB) value between the two sets of samples with high and low ETS1 expression, as well as the differences in gene mutations between the two groups. The mRNA expression of ETS1 in ccRCC was higher compared to normal tissues. Results showed a significant positive correlation between elevated ETS1 expression levels and improved overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFS), with a P < 0.05. Furthermore, high ETS1 expression levels were closely linked to early tumor stage and prolonged survival time. TMB in the ETS1-high expression group was significantly less than that in the ETS1-low expression group. Downregulation of ETS1 expression correlated with poor prognosis and immune infiltration in ccRCC, further suggesting that ETS1 may be a biomarker for better prognosis in ccRCC patients.

Involvement of ICAM5 in Carcinostasis Effects on LUAD Based on the ROS1-Related Prognostic Model.

Lung cancer is the most common type of cancer in the world. In lung adenocarcinoma (LUAD), studies on receptor tyrosine kinase ROS proto-oncogene 1 (ROS1) have mainly focused on the oncogenic effects of its fusion mutations, whereas ROS1 has been reported to be aberrantly expressed in a variety of cancers and can extensively regulate the growth, survival, and proliferation of tumor cells through multiple signaling pathways. The comprehensive analysis of ROS1 expression has not been fully investigated regarding its predictive value for LUAD patients. Gene expression profiles collected from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases were used to build and validate prognostic risk models. The association of ROS1 with overall survival and the immune landscape was obtained from the Tumor Immune Estimation Resource (TIMER) database. The following analyses were performed using the R package to determine the model's validity: pathway dysregulation analysis, gene set enrichment analysis, Gene Oncology analysis, immune invasion analysis, chemotherapy, radiotherapy, and immunotherapy sensitivity analysis. Finally, we conducted a pan-cancer analysis and performed in vitro experiments to explore the regulatory role of intercellular adhesion molecule 5 (ICAM5) in the progression of LUAD. We constructed a 17-gene model that categorized patients into two risk groups. The model had predictive accuracy for tumor prognosis and was specific for patients with high ROS1 expression. Comprehensive analysis showed that patients in the high-risk group were characterized by marked dysregulation of multiple pathways (eg, unfolded protein response), immune suppression of the tumor microenvironment, and poor benefit from immunotherapy and radiotherapy compared with patients in the low-risk group. PLX4720 may be a suitable treatment for the high-risk patient population. The ICAM5 gene has been demonstrated to inhibit the proliferation, cell cycle, invasion, and migration of LUAD cells. We constructed a 17-gene prognostic risk model and found differences in immune-related cells, biological processes, and prognosis among patients in different risk groups based on the correlation between ROS1 and immunity. Personalized therapy may play an essential role in treatment. We further investigated the role of ICAM5 in inhibiting the malignant bioactivity of LUAD cells.

GRP94 in cerebrospinal fluid may contribute to a potential biomarker of depression: Based on proteomics

The present study was designed to investigate potential biomarkers of depression and targets of antidepressants from the perspective of hippocampal endoplasmic reticulum stress (ERS) based on cerebrospinal fluid (CSF) proteomics. Firstly, a six-week depression model was established and treated with fluoxetine (FLX). We found antidepressant-FLX could ameliorate depression-like behaviors and cognition in depressed rats caused by chronic unpredictable mild stress (CUMS). FLX significantly increased neuronal numbers in dentate gyrus (DG) and CA3 regions of hippocampus. CSF proteome data revealed thirty-seven differentially expressed proteins (DEPs) co-regulated by CUMS and FLX, including GRP94 and EIF2α. Results of Gene Oncology (GO) annotation and KEGG pathway enrichment for DEPs mainly included PERK-mediated unfolded protein response, endoplasmic reticulum, and translational initiation. The expression levels of GRP94, p-PERK, p-EIF2α, CHOP and Caspase-12 were increased in hippocampus of CUMS rats, and FLX worked the opposite way. FLX had strong affinity and binding activity with GRP94 protein, and four key proteins on the PERK pathway (PERK, EIF2α, p-EIF2α, CHOP). We proposed that FLX may exert antidepressant effects and neuroprotective action by alleviating excessive activation of the hippocampal PERK pathway and reducing neuronal deficits in depressed rats. PERK, EIF2α, p-EIF2α, and CHOP may be potential targets for antidepressant-FLX. GRP94 in CSF may be a potential biomarker of depression and the therapeutic effects of antidepressants.

NDUFA4L2 is a novel biomarker for colorectal cancer through bioinformatics analysis.

Colorectal cancer (CRC) is a major cause of cancer-related deaths worldwide. NDUFAL42 is an important mitochondrial respiratory chain subunit that plays a critical role in cellular energy metabolism. However, the role of NDUFA4L2 in CRC remains unclear. Therefore, we used the data obtained from The Cancer Genome Atlas (TCGA) database to prove the relationship between NDUFA4L2 and CRC. The expression levels of NDUFA4L2 in CRC tissues were analyzed by immunohistochemical staining of NDUFA4L2 from the HPA database. Wilcoxon rank sum test, Chi-square test, Fisher exact test and logistic regression were used to evaluate relationships between clinical-pathologic features and NDUFA4L2 expression. Receiver operating characteristic (ROC) curves were used to describe binary classifier value of NDUFA4L2 using area under curve (AUC) score. Kaplan-Meier method and Cox regression analysis were used to evaluate factors contributing to prognosis. Gene oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used to predict the function of differentially expressed genes associated with NDUFA4L2. Gene set enrichment analysis (GSEA) was used to predict canonical pathways associated with NDUFA4L2.Immune infiltration analysis was performed to identify the significantly involved functions of NDUFA4L2. Protein-protein interaction (PPI) networks were established and 20 hub genes identified with Cytoscape software. Increased NDUFA4L2 expression in CRC was associated with T stage (P = .019), N stage (P < .001), Pathologic stage (P = .020), Residual tumor (P = .023), Perineural invasion (P = .039), Lymphatic invasion (P = .007), Histological type(P < .001), PFI event (P = .007) and DSS event (P = .004).ROC curve suggested the significant diagnostic and prognostic ability of NDUFA4L2 (AUC = 0.878). High NDUFA4L2 expression predicted a poorer Overall-survival (P = .021), poorer progression-free interval (P = .001), and poorer Disease Specific Survival (P = .002). GO, KEGG, GSEA and immune infiltration analysis showed that NDUFA4L2 expression was correlated with regulating the function of DNA and some types of immune infiltrating cells. NDUFA4L2 expression was significantly correlated with poor survival and immune infiltrations in CRC, and it may be a promising prognostic biomarker in CRC.

Differential expression of LLGL2 in prostate ductal adenocarcinoma and acinar adenocarcinoma and its significance

Objective: To investigate the expression differences of LLGL2 between prostatic ductal adenocarcinoma (PDA) and prostatic acinar adenocarcinoma, and its potential clinical significance. Methods: Eighteen patients diagnosed of PDA or prostatic acinar adenocarcinoma with PDA component by histopathology during January 2015 and December 2019 in the Beijing Hospital, China were retrospectively studied. The transcriptome analysis was conducted using the tissue of PDA and prostatic acinar adenocarcinoma. Differentially expressed genes and the differences in expression profiles were identified. Further, differentially expressed proteins were verified by immunohistochemistry. Results: The tissue from 8 of the 18 patients were used for transcriptome analysis, the results of which were compared with data from public databases. 129 differentially expressed genes were identified. 45 of them were upregulated while 84 were downregulated. The results of gene enrichment analysis and gene oncology (GO) analysis revealed that the differentially expressed genes were mostly enriched in the hypertrophic cardiomyopathy and interleukin-17 related pathways. GPAT2, LLGL2, MAMDC4, PCSK9 and SMIM6 were differentially expressed between PDA and prostatic acinar adenocarcinoma. Moreover, LLGL2 was more likely expressed in the cytoplasm (P=0.04) than the nucleus (P<0.01) in PDA, compared with prostatic acinar adenocarcinoma. Conclusions: The gene expression profiling indicates that PDA are very similar to prostatic acinar adenocarcinoma. Among the differentially expressed proteins screened and verified in this study, the expression of GPAT2, LLGL2, MAMDC4 and PCSK9 is increased in PDA, while that of SMIM6 is reduced in PDA. The expression of LLGL2 shows significantly different patterns between PDA and prostatic acinar carcinoma, and thus may help differentiate PDA from prostatic acinar adenocarcinoma in clinical practice.

Exploring the mechanism of Astragali radix for promoting osteogenic differentiation based on network pharmacology, molecular docking, and experimental validation.

The present study used network pharmacology and molecular docking to predict the active ingredients and mechanisms of action of Astragalus radix (AR) to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs), and cell experiments were conducted for verification. First, network pharmacology was used to predict the effective components, targets, and mechanisms of action of AR to promote osteogenic differentiation. The effective components and corresponding target proteins of AR, and the target proteins of osteogenic differentiation were collected through the database. The intersection targets of the two were used for the construction and analysis of a protein-protein interaction (PPI) network. Gene Oncology (GO) and Kyoto Encyclopedia of Genes, and Genomes (KEGG) enrichment analyses were conducted. Next, molecular docking technology was carried out to verify the interaction between the active ingredient and the target protein, and to select the appropriate effective active ingredient. Finally, the results of network pharmacology analysis were verified by in vitro experiments. A total of 95 potential targets were retrieved by searching the intersection of AR and osteogenic differentiation targets. PPI network analysis indicated that RAC-α-serine-threonine-protein kinase (Akt1) was considered to be the most reliable target for AR to regulate osteogenic differentiation. GO enrichment analysis included 21 biological processes, 21 cellular components and 100 molecular functions. KEGG enrichment analysis indicated that the class I phosphatidylinositol-3 kinase (PI3K)-serine-threonine kinase (Akt) signaling pathway may play an important role in promoting osteogenic differentiation. The results of molecular docking showed that quercetin's performance was improved compared with that of kaempferol. In vitro experiments showed that quercetin promoted the expression of osteogenic marker proteins (including collagen I, Runt-related transcription factor 2 and osteopontin) in BMSCs and activated the PI3K/Akt signaling pathway. AR acted on Akt1 targets through its main active component quercetin, and promoted the osteogenic differentiation of BM-MSCs by activating the PI3K/Akt signaling pathway.

Novel DNA Promoter Hypermethylation in Nasal Epithelium of Asthma.

Abnormal epigenetic alterations influenced by external factors and affecting DNA expression contribute to the development of asthma. However, the role of the nasal epithelium in airway inflammation remains unknown. The objective of this study is to identify novel DNA promoter hypermethylation, which suppresses mRNA expression in nasal epithelial of asthma. Microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Gene expression and DNA promoter methylation sites in key correlated modules between asthma and normal were identified by weighted gene co-expression network analysis. Gene Oncology and Kyoto Encyclopedia of Genes and Genomes were conducted to analyse the function of genes. Further validation was performed in human BEAS-2B cells challenged by IL-4 or IL-13. Lightcyan, lightgreen, midnightblue, cyan and tan modules in the mRNA expression dataset showed a close relationship with asthma, in which genes were enriched in TNF, IL-17, ErbB, MAPK and Estrogen signalling pathways. Blue and turquoise modules in the methylation profiling dataset were associated with asthma. Forty nine lowly expressed genes were identified to be correlated with aberrant DNA hypermethylation of promoters. Among these genes, the mRNA levels of <i>BCL10, GADD45B</i>, <i>LSR</i> and </i>SQSTM1</i> were downregulated in BEAS-2B cells challenged with IL-4 or IL-13. Four potential genes in the nasal epithelium, by hypermethylating their own DNA promoter, might mediate the inflammatory response in the pathogenesis of asthma. Analyzing epigenomic data by integrated bioinformatics helps to understand the role of DNA methylation in asthma, with the goal of providing new perspectives for diagnosis and therapy.

P-331 Transcriptomic profiling of ovaries with experimental endometrioma in mice reveals the underlying mechanisms of abnormal oocyte quality and functions in endometrioma-associated infertility

Abstract Study question What are the underlying molecular mechanism and significant pathways mediated the endometrioma-associated infertility? Summary answer Significantly altered genes that participated in folliculogenesis, fibrosis, oxidative stress and inflammation are pivotal in the pathogenesis of infertility in endometrioma. What is known already Endometrioma (OMA) is the most common subtype of endometriosis, of which the endometriotic lesions implant in the ovary. Women with OMA are usually coupled with disrupted folliculogenesis, hampered ovulation, impaired oocyte quality, and undesired fertility outcomes. However, the underlying mechanisms of the disrupted folliculogenesis, hampered ovulation and impaired oocyte quality in OMA-associated infertility are still unclear. Next-generation sequencing (NGS) has been widely applied in whole-genome sequencing, RNA sequencing, and epigenomics. It is a promising technology that may provide full picture of the pathogenesis of infertility associated with OMA. Study design, size, duration An experimental mouse model of OMA was established in 4 weeks to study the effects of OMA on fertility outcomes and the underlying mechanism. Eight mice were included in experimental and sham control groups each. Half of the mice were then mated with male compeer and fertility outcomes were assessed after delivery. The other half were euthanized without mating to collect whole ovarian tissues for whole genome bulk RNA sequencing and then immunohistochemistry (IHC) validation. Participants/materials, setting, methods Minced uterine tissues from donor mice were inserted into ovarian bursa in recipient mice. After overnight mating, the pregnant mice were confirmed by positive vaginal plug. Live fetuses, abortion rate, and stillbirth were counted after delivery. Ovaries were either dissected to extract total RNA for sequencing, or paraffin-fixed and embedded for IHC, or superovulated to collect oocytes for counting and staining. Main results and the role of chance Successful establishment of endometriotic lesions in ovarian tissues was confirmed morphologically and histologically in recipient mice. The pregnancy rate was defined as the ratio of the number of pregnant mice to the total number of mated females. Compared to control group, a significantly decreased pregnancy rate was found in the OMA group. The average number and size of pups in the OMA group was significantly reduced. Meanwhile, abortion and stillbirth rates were significant higher in OMA group. Oocytes isolated from OMA ovaries had lower number than controls. Large proportion of the oocytes had significantly higher abnormal spindle rates, indicating mitotic disruption. A large proportion of the oocytes also had impaired cortical granule migration, indicating affected organelle organization. Furthermore, expression of follicle development markers (i.e. Forkhead box O3, anti-müllerian hormone, follicle-stimulating hormone receptor) were significantly decreased in OMA group. RNA sequencing identified several differentially expressed genes. The Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis demonstrated the dysregulation of signaling pathways associated with folliculogenesis, fibrosis, oxidative stress and inflammation, whichmight trigger OMA and lead to abnormal oocyte quality and functions in endometrioma-associated infertility. Limitations, reasons for caution Although ethical issue in human, studies based on this animal model may not reflect the exact situation in human. Moreover, future research is needed to understand the biological functions of differentially expressed pathways in the pathogenesis of OMA-related infertility. Furthermore, the manifestations in individual cells of ovaries are still unclear. Wider implications of the findings The results demonstrated that endometrioma remarkably deteriorated fecundity through several potential pathways. The underlying deteriorating effects and mechanisms might help to understand the biological and molecular mechanism and to develop novel therapeutic targets to improve fertility outcomes in women with endometrioma. Trial registration number not applicable

Proteomic analysis of anti-aging effects of Dendrobium nobile Lindl. alkaloids in aging-accelerated SAMP8 mice

Senescence-accelerated mouse prone 8 (SAMP8) mice exhibit cognitive defects and neuron loss with aging, and were used to study anti-aging effects of Dendrobium nobile alkaloids (DNLA). DNLA (20 and 40 mg/kg) were orally administered to SAMP8 mice from 6 to 10 months of age. At 10-month of age, behavioral tests via Y-maze and Open-field and neuron damage via Nissl staining were evaluated. Protein was extracted and subjected to phosphorylated proteomic analysis followed by bioinformatic analysis. The cognitive deficits and neuron loss in hippocampus and cortex of aged SAMP8 mice were improved by DNLA. Hippocampal proteomic analysis revealed 196 differentially expressed protein/genes in SAMP8 compared to age-matched senescence-accelerated resistant SAMR1 mice. Gene Oncology enriched the tubulin binding, microtubule binding, and other activities. Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed endocytosis, mRNA surveillance, tight junction, protein processing in endoplasmic reticulum, aldosterone synthesis and secretion, and glucagon signaling pathway changes. Upregulated protein/genes in the hippocampus of SAMP8 mice, such as Lmtk3, Usp10, Dzip1, Csnk2b, and Rtn1, were attenuated by DNLA; whereas downregulated protein/genes, such as Kctd16, Psd3, Bsn, Atxn2l, and Kif1a, were rescued by DNLA. The aberrant protein/gene expressions of SAMP8 mice were correlated with transcriptome changes of Alzheimer's disease in the Gene Expression Omnibus (GEO) database, and the scores were attenuated by DNLA. Thus, DNLA improved cognitive dysfunction and ameliorated neuronal injury in aged SAMP8 mice, and attenuated aberrant protein/gene expressions.

Diagnosis of Ovarian Carcinoma Homologous Recombination DNA Repair Deficiency From Targeted Gene Capture Oncology Assays.

Homologous recombination DNA repair deficiency (HRD) is a therapeutic biomarker for sensitivity to platinum and poly(ADP-ribose) polymerase inhibitor therapies in breast and ovarian cancers. Several molecular phenotypes and diagnostic strategies have been developed to assess HRD; however, their clinical implementation remains both technically challenging and methodologically unstandardized. We developed and validated an efficient and cost-effective strategy for HRD determination on the basis of calculation of a genome-wide loss of heterozygosity (LOH) score through targeted, hybridization capture and next-generation DNA sequencing augmented with 3,000 common, polymorphic single-nucleotide polymorphism (SNP) sites distributed genome-wide. This approach requires minimal sequence reads and can be readily integrated into targeted gene capture workflows already in use for molecular oncology. We interrogated 99 ovarian neoplasm-normal pairs using this method and compared results with patient mutational genotypes and orthologous predictors of HRD derived from whole-genome mutational signatures. LOH scores of ≥11% had >86% sensitivity for identifying tumors with HRD-causing mutations in an independent validation set (90.6% sensitivity for all specimens). We found strong agreement of our analytic approach with genome-wide mutational signature assays for determining HRD, yielding an estimated 96.7% sensitivity and 50% specificity. We observed poor concordance with mutational signatures inferred using only mutations detected by the targeted gene capture panel, suggesting inadequacy of the latter approach. LOH score did not significantly correlate with treatment outcomes. Targeted sequencing of genome-wide polymorphic SNP sites can be used to infer LOH events and subsequently diagnose HRD in ovarian tumors. The methods presented here are readily generalizable to other targeted gene oncology assays and could be adapted for HRD diagnosis in other tumor types.

A circRNA-miRNA-mRNA network analysis underlying pathogenesis of human heart failure.

The molecular mechanisms of heart failure (HF) are still poorly understood. Circular RNA (circRNA) has been discovered in the heart in increasing numbers of studies. The goal of this research is to learn more about the potential roles of circRNAs in HF. We used RNA sequencing data to identify the characteristics of circRNAs expressed in the heart and discovered that the majority of circRNAs screened were less than 2000 nt. Additionally, chromosomes One and Y had the most and least number of circRNAs, respectively. After excluding duplicate host genes and intergenic circRNAs, a total of 238 differentially expressed circRNAs (DECs) and 203 host genes were discovered. However, only four of the 203 host genes of DECs were examined in HF differentially expressed genes. Another study used Gene Oncology analysis of DECs host genes to elucidate the underlying pathogenesis of HF, and it found that binding and catalytic activity accounted for a large portion of DECs. Immune system, metabolism, and signal transduction pathways were significantly enriched. Furthermore, 1052 potentially regulated miRNAs from the top 40 DECs were collected to build a circRNA-miRNA network, and it was discovered that 470 miRNAs can be regulated by multiple circRNAs, while others are regulated by a single circRNA. In addition, a comparison of the top 10 mRNAs in HF and their targeted miRNAs revealed that DDX3Y and UTY were regulated by the most and least circRNA, respectively. These findings demonstrated circRNAs have species and tissue specific expression patterns; while circRNA expression is independent on host genes, the same types of genes in DECs and DEGs worked in HF. Our findings would contribute to a better understanding of the critical roles of circRNAs and lay the groundwork for future studies of HF molecular functions.

Abstract 3605: Peroxiredoxin IV promotes prostate cancer malignancy through the activation of NF-kB signaling

Abstract Background: The peroxiredoxin (Prx) family of proteins functions as major cellular antioxidants to maintain redox homeostasis as well as mediate oxidative signaling in both physiological and pathological conditions. Our previous studies show that Prx4 is highly expressed in prostate cancer and promotes tumor growth in vitro and in vivo. But how Prx4 contributes to prostate cancer cell invasion and metastasis remains to be elucidated. The purpose of this study is to understand the oncogenic signaling pathways that are mediated by Prx4 in prostate cancer cells. Methods: Bioinformatic analysis of existing database were used to examine the expression of Prx4 in primary and metastatic prostate cancer samples. CRISPR/Cas9 technique was used to establish Prx4 knockout in cultured human prostate cancer cell lines. Matrigel invasion assays were performed to evaluate cell migration and invasion. RNAseq was performed to examine the differences of gene expression between control and Prx4KO cells. Gene Set Enrichment Analysis (GSEA) and Gene Oncology (GO) enrichment analysis were performed using RNAseq results. RT-PCR and immunoblotting were used to validate findings from RNAseq and enrichment analysis. Results: Compared with normal prostate, transcript levels of PRDX4 are found to be upregulated in specimens of patients with prostate cancer, and those with bone metastasis show even higher levels. Knockout of Prx4 in cultured human prostate cancer cells leads to significantly reduced ability of cell migration and invasion. Gene expression profiling reveals that loss of Prx4 leads to the upregulation of epithelial as well as downregulation of mesenchymal markers, and multiple signaling pathway changes including the inhibition of cellular response to inflammatory factors such as tumor necrosis factors and interleukins. These changes are also associated with variety of cellular activities such as wound healing, interferon signaling, and antigen processing &amp; presentation. Among known target genes downstream of NF-κB signaling, E-cadherin, vimentin and matrix metalloproteinase 14 (MMP14) were found to be significantly affected by the loss of Prx4. Therefore, Prx4 plays a critical role in human prostate cancer cell malignancy through regulation of intracellular cell signaling pathways. Conclusions: A combination of bioinformatic, cellular and molecular methods reveals that Prx4 plays a critical role in promoting prostate cancer metastasis. Prx4 is a potential therapeutic target to reduce prostate cancer metastasis. Citation Format: Na Ding, Hong Jiang, Pratik Thapa, Yanning Hao, Aziza Alshahrani, Vivek Rangnekar, Xiaoqi Liu, Qiou Wei. Peroxiredoxin IV promotes prostate cancer malignancy through the activation of NF-kB signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3605.

PD24-11 HOT CLEAR CELL RENAL CELL CARCINOMA IMMUNE STATUS: ILLUSION OR REALITY?

You have accessJournal of UrologyCME1 Apr 2023PD24-11 HOT CLEAR CELL RENAL CELL CARCINOMA IMMUNE STATUS: ILLUSION OR REALITY? Yi Lu and Hongjun Li Yi LuYi Lu More articles by this author and Hongjun LiHongjun Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003302.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The drivers and resistors of the anti-tumor response remain incompletely elucidated. High tumor mutation burden (TMB) and increased infiltration by T cells are associated with the answer to immune checkpoint inhibitors ICIs. We conducted the bioinfomatic study to better understand immunotherapy resistance in clear cell renal cell carcinoma (ccRCC). METHODS: The patients and sequencing data were selected from The Cancer Genome Atlas KIRC (TCGA-KIRC), and a clinical trial dataset. To classify worse-survival ccRCC resistant to immunotherapy, immune prognosis model (IPM) risk score classifier was constructed with prognostic costimulatory molecule genes through survival analysis and validated as well. For function analysis, ESTIMATE, ImmuCellAI, TIDE, and Gene Oncology (GO) were used. RESULTS: The 366 Co-expressed genes (CEGs) correlated with IPM risk score were enriched with p<0.05 and correlation |R-value|>0.5. GO analysis indicated CEGs were enriched within immune-associated gene sets, such as T cell homeostasis. TIDE algorithm predicted all the patients' response to ICIs, and non-responders were enriched in higher IPM risk scores and a real-world ICIs-treated ccRCC cohort validated it with a consistent finding with p=0.028 in chi-square test. ESTIMATE algorithm showed the ESTIMATE, and immune scores of high-risk patients, markedly increased compared with low-risk patients. Nevertheless, tumor purity was thinner than low-risk patients. Besides, TMB and cytolytic activity were higher in high-risk patients. ImmuCellAI algorithm revealed the levels of CD8+ T cells, CD4+ T cells, dendritic cells, and MAIT, together with macrophages, increased in the tumor microenvironment of high-risk patients. The TIDE T cell function estimate was used to test the T cell dysfunction of high-risk patients, which was markedly worse compared with low-risk patients. Meanwhile, immune inhibiting cells, namely exhausted T cells, nTreg cells, iTreg cells, and cancer associated fibroblast (CAF), had an increased fraction in high-risk ccRCC. Remarkably, the microsatellite instability (MSI) score was lower in high-risk ccRCC. Interestingly, IPM risk level was independent from MSI level in prognostic influence. CONCLUSIONS: We found a subtype of refractory ccRCC (IPM high risk), and subsequent function analysis indicated the reason for its insensitivity to immune blockades was T cell dysfunction. In IPM high-risk ccRCC, T cells' high infiltration, hot immune status, is an illusion. The reality is the increased infiltration of dysfunctional CD8+ and CD4+ T cells, immunosuppressive cells (i.e., iT-regs, nT-regs, and CAFs) as well as low MSI. Source of Funding: This work is supported by the grant from National Population Health Science Data Sharing Service Platform Clinical Medical Science Data Center (NCMI-ABD02-201906) to Hongjun Li © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e727 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yi Lu More articles by this author Hongjun Li More articles by this author Expand All Advertisement PDF downloadLoading ...

The first description of the blue swimming crab (Portunus pelagicus) transcriptome and immunological defense mechanism in response to white spot syndrome virus (WSSV)

In the global shellfish farming industry, white spot syndrome virus (WSSV) is a major cause of mortality and a significant factor in economic losses. However, information on molecular immune responses to WSSV in blue swimming crabs (Portunus pelagicus) has never been reported. First, viral loads were measured in the gills, hepatopancreas, intestines, subcuticular epithelium and hemocytes of blue swimming crabs (50 ± 10 g) (n = 4) after WSSV induction at 0, 24, 48 and 96 h post injection (hpi). A significant increase in WSSV particles was observed in gills at 48 and 96 hpi, as supported by histopathology. To further investigate the acute immune response to WSSV, total RNA from the same gill tissues at 0, 24, and 96 hpi was used to construct 16 high-quality RNA-seq cDNA libraries. In summary, 162,740 unigenes were discovered in these transcriptomic libraries analyzed with the GO, KO, KOG, NR, NT, PFAM and SwissProt databases. Intensive sequence analysis against control crabs using three major categories of gene oncology (GO) of DEGs, biological processes (BPs), molecular functions (MFs), and cellular components (CCs), indicated that induction of WSSV in blue swimming crabs strongly affected the immune responses of the target animals significantly during the early stages of infection from 24 to 96 hpi. Furthermore, KEGG identified approximately twenty biological pathways of gene expression that were both downregulated and upregulated. Interestingly, at 24 and 96 hpi, several immune-related genes involved in virus defense in the blue swimming crab, particularly crustin 2, chitinase, anti-lipopolysaccharide, proteinase inhibitor, and lysozyme, were highly expressed during the WSSV early infection stages. At the same time, viral mRNA transcripts, including WSV289, WSV343, WSV306, deoxyuridine 5’ triphosphate nucleohydrolase, RING finger containing E3 ubiquitin-protein ligase WSV403 and WSV404, were recorded in the top twenty upregulated genes. Moreover, some immune-responsive genes related to growth development, such as chitinase, tubulin alpha and beta chains, trypsin, and the cathepsin family, were also differentially expressed during these periods. Expression validation of 20 upregulated and 11 downregulated immune-related genes using qRT‒PCR showed similar patterns with transcriptome information. Overall, the data showed that during WSSV infection, a number of immune-, metabolism-, and growth-related pathways were activated, and several of the pathways involved differed depending on the stage of virus invasion. These findings could effectively help us better understand the impact of WSSV on the physiology of blue swimming crabs and serve as a valuable reference for future research on the immune system and disease control in this target species.