Comprehensive analysis of scRNA-Seq and bulk RNA-Seq reveals dynamic changes in the tumor immune microenvironment of bladder cancer and establishes a prognostic model.

BACKGROUND: The prognostic management of bladder cancer (BLCA) remains a great challenge for clinicians. Recently, bulk RNA-seq sequencing data have been used as a prognostic marker for many cancers but do not accurately detect core cellular and molecular functions in tumor cells. In the current study, bulk RNA-seq and single-cell RNA sequencing (scRNA-seq) data were combined to construct a prognostic model of BLCA. METHODS: BLCA scRNA-seq data were downloaded from Gene Expression Omnibus (GEO) database. Bulk RNA-seq data were obtained from the UCSC Xena. The R package "Seurat" was used for scRNA-seq data processing, and the uniform manifold approximation and projection (UMAP) were utilized for downscaling and cluster identification. The FindAllMarkers function was used to identify marker genes for each cluster. The limma package was used to obtain differentially expressed genes (DEGs) affecting overall survival (OS) in BLCA patients. Weighted gene correlation network analysis (WGCNA) was used to identify BLCA key modules. The intersection of marker genes of core cells and genes of BLCA key modules and DEGs was used to construct a prognostic model by univariate Cox and Least Absolute Shrinkage and Selection Operator (LASSO) analyses. Differences in clinicopathological characteristics, immune microenvironment, immune checkpoints, and chemotherapeutic drug sensitivity between the high and low-risk groups were also investigated. RESULTS: scRNA-seq data were analyzed to identify 19 cell subpopulations and 7 core cell types. The ssGSEA showed that all 7 core cell types were significantly downregulated in tumor samples of BLCA. We identified 474 marker genes from the scRNA-seq dataset, 1556 DEGs from the Bulk RNA-seq dataset, and 2334 genes associated with a key module identified by WGCNA. After performing intersection, univariate Cox, and LASSO analysis, we obtained a prognostic model based on the expression levels of 3 signature genes, namely MAP1B, PCOLCE2, and ELN. The feasibility of the model was validated by an internal training set and two external validation sets. Moreover, patients with high-risk scores are predisposed to experience poor OS, a larger prevalence of stage III-IV, a greater TMB, a higher infiltration of immune cells, and a lesser likelihood of responding favorably to immunotherapy. CONCLUSION: By integrating scRNA-seq and bulk RNA-seq data, we constructed a novel prognostic model to predict the survival of BLCA patients. The risk score is a promising independent prognostic factor that is closely correlated with the immune microenvironment and clinicopathological characteristics.

Bayesian-based analysis of the causality between 731 immune cells and erectile dysfunction: a two-sample, bidirectional, and multivariable Mendelian randomization study.

Background: The causal relationship between certain immune cells and erectile dysfunction (ED) is still uncertain. Aim: The study sought to investigate the causal effect of 731 types of immune cells on ED through Mendelian randomization (MR) using genome-wide association studies (GWAS). Methods: Genetic instruments for 731 immune cells were identified through GWAS, and ED data were obtained from the FinnGen database. Univariable and multivariable bidirectional MR studies were conducted to explore potential causal relationships between these immune cells and ED. The inverse-variance weighted method was primarily used, with Cochran's Q test and MR-Egger intercept test assessing pleiotropy and heterogeneity. Bayesian weighted Mendelian randomization (BWMR) was also employed. Outcomes: Six immune cells were identified as related to ED. CD45 on Natural Killer (NK) cells, CD33dim HLA DR+ CD11b + Absolute Count, CD19 on IgD- CD38dim B cells, and CD3 on CD39+ resting CD4 regulatory T cells were identified as risk factors, whereas CD20 on IgD+ CD38dim B cells and Activated & resting CD4 regulatory T cell %CD4+ T cells were protective factors. Further multivariable MR analysis confirmed that 5 of these immune cells independently impacted ED, except for CD45 on NK cells. Reverse MR analysis indicated that ED occurrence decreases certain immune cell counts, but BWMR found no causal relationship for CD20 on IgD+ CD38dim B cells. Results: Our MR analysis confirmed a potential bidirectional causal relationship between immune cells and ED, providing new insights into potential mechanisms and therapeutic strategies. Clinical Translation: This study provides evidence for the impact of certain immune cells on the development of ED and suggests potential therapeutic targets. Strengths and Limitations: We performed both univariable and multivariable MR to strengthen the causal relationship between exposures and outcomes. However, the population in this study was limited to European ancestry. Conclusion: Our MR analysis confirmed a potential bidirectional causal relationship between immune cells and ED. This provides new insights into potential mechanisms of pathogenesis and subsequent therapeutic strategies.

Integrating Bulk and Single-Cell RNA Sequencing Reveals Heterogeneity, Tumor Microenvironment, and Immunotherapeutic Efficacy Based on Sialylation-Related Genes in Bladder Cancer.

Background: As known abnormal sialylation exerts crucial roles in the growth, metastasis, and immune evasion of cancers, but the molecular characteristics and roles in bladder cancer (BLCA) remain unclear. This study intends to establish BLCA risk stratification based on sialylation-related genes and elucidate its role in prognosis, tumor microenvironment, and immunotherapy of BLCA. Methods: Bulk RNA-seq and scRNA-seq data were downloaded from open-access databases. The scRNA-seq data were processed using the R package "Seurat" to identify the core cell types. The tumor sub-typing of BLCA samples was performed by the R package "ConsensusClusterPlus" in the bulk RNA-seq data. Signature genes were identified by the R package "limma" and univariate regression analysis to calculate risk scores using the R package "GSVA" and establish risk stratification of BLCA patients. Finally, the differences in clinicopathological characteristics, tumor microenvironment, and immunotherapy efficacy between the different groups were investigated. Results: 5 core cell types were identified in the scRNA-seq dataset, with monocytes and macrophages presenting the greatest percentage, sialylation-related gene expression, and sialylation scores. The bulk RNA-seq samples were classified into 3 tumor subtypes based on 19 prognosis-related sialylation genes. The 10 differential expressed genes (DEGs) with the smallest p-values were collected as signature genes, and the risk score was calculated, with the samples divided into high and low-risk score groups. The results showed that patients in the high-risk score group exhibited worse survival outcomes, higher tumor grade, more advanced stage, more frequency of gene mutations, higher expression levels of immune checkpoints, and lower immunotherapy response. Conclusion: We established a novel risk stratification of BLCA from a glycomics perspective, which demonstrated good accuracy in determining the prognostic outcome, clinicopathological characteristics, immune microenvironment, and immunotherapy efficacy of patients, and we are proposing to apply it to direct the choice of clinical treatment options for patients.

Prognosis analysis and validation of lipid metabolism-associated lncRNAs and tumor immune microenvironment in bladder cancer.

BACKGROUND: Numerous types of research revealed that long noncoding RNAs (lncRNAs) played a significant role in immune response and the tumor microenvironment of bladder cancer (BLCA). Dysregulated lipid metabolism is considered to be one of the major risk factors for BLCA, the study aimed to detect the lipid metabolism-related lncRNAs (LMRLs) along with their potential prognostic values and immune correlations in BLCA. METHODS: We collected lipid metabolism-related genes, expression profiles, and clinical information on BLCA from the Molecular Signature Database (MSigDB) and the TCGA database, respectively. Differentially expressed lipid metabolism genes (DE-LMRGs) and differentially expressed long non-coding RNAs (DE-lncRNAs) were selected using the limma package. Spearman correlation analysis was employed to explore the correlations between DE-lncRNAs and DE-LMRGs and to further develop protein-protein interaction (PPI) networks and perform mutational analysis. The least absolute shrinkage and selection operator (LASSO) and univariate Cox analysis were then employed to construct a prognostic risk model. The performance of the model was evaluated using Kaplan-Meier survival analysis, receiver operating characteristic (ROC) curves, and consistency indices. In addition, we downloaded the GSE31684 dataset for external validation of the prognostic signature. Moreover, we explored the association of the risk model with immune cell infiltration and chemotherapy response analysis to reveal the tumor immune microenvironment of BLCA. Finally, RT-qPCR was utilized to validate the expression of prognostic genes. RESULTS: A total of 48 DE-LncRNAs and 33 DE-LMRGs were found to be robustly correlated, and were used to construct a lncRNA-mRNA co-expression network, in which ACACB, ACOX2, and BCHE showed high mutation rates. Then, a risk model based on three LMRLs (RP11-465B22.8, MIR100HG, and LINC00865) was constructed. The risk model effectively distinguished between the clinical outcomes of BLCA patients, with high-risk scores indicating a worse prognosis and with substantial prognostic prediction accuracy. The model's results were consistent in the GSE31684 dataset. In addition, a nomogram was constructed based on the risk score, age, pathological T-stage, and pathological N-stage, which showed robust predictive power. Immune landscape analysis indicated that the risk model was significantly associated with T-cell CD4 memory activation, M1 macrophage, M2 macrophage, dendritic cell activation, and T-cell regulatory. We predicted that 49 drugs would perform satisfactorily in the high-risk group. Additionally, we found five m6A regulators associated with the high- and low-risk groups, suggesting that upstream regulation of LncRNA could be a novel target for BLCA treatment. Finally, RT-qPCR showed that RP11-465B22.8 was highly expressed in BLCA, while MIR100HG and LINC00865 were downregulated in BLCA. CONCLUSION: Our findings suggest that the three LMRLs may serve as potential prognostic and immunotherapeutic biomarkers in BLCA. In addition, our study provides new ideas for understanding the pathogenic mechanisms and developing therapeutic strategies for BLCA patients.