Identification of a dysregulated CircRNA-associated gene signature for predicting prognosis, immune landscape, and drug candidates in bladder cancer.

Increasing evidences have demonstrated that circular RNA (circRNAs) plays a an essential regulatory role in initiation, progression and immunotherapy resistance of various cancers. However, circRNAs have rarely been studied in bladder cancer (BCa). The purpose of this research is to explore new circRNAs and their potential mechanisms in BCa. A novel ceRNA-regulated network, including 87 differentially expressed circRNAs (DE-circRNAs), 126 DE-miRNAs, and 217 DE-mRNAs was constructed to better understanding the biological processes using Cytoscape 3.7.1 based on our previously high-throughput circRNA sequencing and five GEO datasets. Subsequently, five randomly selected circRNAs (upregulated circ_0001681; downregulated circ_0000643, circ_0001798, circ_0006117 and circ_0067900) in 20 pairs of BCa and paracancerous tissues were confirmed using qRT-PCR. Functional analysis results determined that 772 GO functions and 32 KEGG pathways were enriched in the ceRNA network. Ten genes (PFKFB4, EDNRA, GSN, GAS1, PAPPA, DTL, TGFBI, PRSS8, RGS1 and TCF4) were selected for signature construction among the ceRNA network. The Human Protein Atlas (HPA) expression of these genes were consistent with the above sequencing data. Notably, the model was validated in multiple external datasets (GSE13507, GSE31684, GSE48075, IMvigor210 and GSE32894). The immune-infiltration was evaluated by 7 published algorithms (i.e., TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, MCPCOUNTER, XCELL and EPIC). Next, Correlations between riskscore or risk groups and clinicopathological data, overall survival, recognized immunoregulatory cells or common chemotherapeutic agents of BCa patients were performed using wilcox rank test, chi-square test, cox regression and spearman's correlation analysis; and, these results are significant. According to R package "GSVA" and "clusterProfiler", the most significantly enriched HALLMARK and KEGG pathway was separately the 'Epithelial Mesenchymal Transition' and 'Ecm Receptor Interaction' in the high- vs. low-risk group. Additionally, the functional experiments in vitro also revealed that the overexpression of has_circ_0067900 significantly impaired the proliferation, migration, and invasion capacities of BCa cells. Collectively, the results of the current study provide a novel landscape of circRNA-associated ceRNA-regulated network in BCa. The ceRNA-associated gene model which was constructed presented a high predictive performance for the prognosis, immunotherapeutic responsiveness, and chemotherapeutic sensitivity of BCa. And, has_circ_0067900 was originally proposed as tumor suppressor for patients with BCa.

Shen C ，Li Z ，Zhang Y ，Zhang Z ，Wu Z ，Da L ，Yang S ，Wang Z ，Zhang Y ，Qie Y ，Zhao G ，Lin Y ，Huang S ，Zhou M ，Hu H ... -  《Frontiers in Oncology》

Identification of Enzalutamide Resistance-Related circRNA-miRNA-mRNA Regulatory Networks in Patients with Prostate Cancer.

This study aimed to identify enzalutamide resistant-related (EnzR-related) circRNAs and to characterize and validate circRNA-miRNA-mRNA ceRNA regulatory network and corresponding prognostic signature of prostate cancer patients. We obtained circRNA expression microarray from the Gene Expression Omnibus (GEO) database and performed differential expression analysis to identify EnzR-related circRNAs using the limma package. The miRNA and mRNA expression profiling were downloaded and performed differential expression analysis, then overlapped with predicted candidates. Next, we established circRNA-miRNA-mRNA ceRNA network and PPI network utilized Cytoscape software and STRING database, respectively. In addition, univariate and Lasso Cox regression analyses were applied to generate a prognostic signature. Receiver operating characteristic (ROC) curves and Kaplan-Meier analysis were used to evaluate the reliability and sensitivity of the signature. Ultimately, we chose hsa_circ_0047641 to validate the feasibility of the EnzR-related ceRNA regulatory pathway using qRT-PCR, CCK8 and Transwell assays. We identified 13 EnzR-related circRNAs and constructed a ceRNA regulatory network that contained two downregulated circRNAs (has-circ-00000919 and has-circ-0000036) and two upregulated circRNAs (has-circ-0047641 and has-circ-0068697), and their sponged 6 miRNAs and 167 targeted mRNAs. Subsequently, these targeted mRNAs were performed to implement PPI analysis and to identify 10 Hub genes. Functional enrichment analysis provided new ways to seek potential biological functions. Besides, we established a prognostic signature of PCa patients based on 8 prognostic-associated mRNAs. We confirmed that the survival rates of PCa patients with high-risk subgroup were slightly lower than those with low-risk subgroup in the TCGA dataset (p<0.001), and ROC curves revealed that the AUC value for prognostic signature was 0.816. Finally, the functional analysis suggested that knockdown of hsa_circ_0047641 could inhibit the progression of PCa and could reverse Enz-resistance in vitro. We identified 13 EnzR-related circRNAs, and constructed and confirmed that EnzR-related circRNA-miRNA-mRNA ceRNA network and corresponding prognostic signature could be a useful prognostic biomarker and therapeutic target.

Yu J ，Sun S ，Mao W ，Xu B ，Chen M ... -  《OncoTargets and Therapy》

Construction of circRNA-miRNA-mRNA network and identification of novel potential biomarkers for non-small cell lung cancer.

The underlying circular RNAs (circRNAs)-related competitive endogenous RNA (ceRNA) mechanisms of pathogenesis and prognosis in non-small cell lung cancer (NSCLC) remain unclear. Differentially expressed circRNAs (DECs) in two Gene Expression Omnibus datasets (GSE101684 and GSE112214) were identified by utilizing R package (Limma). Circinteractome and StarBase databases were used to predict circRNA associated-miRNAs and mRNAs, respectively. Then, protein-protein interaction (PPI) network of hub genes and ceRNA network were constructed by STRING and Cytoscape. Also, analyses of functional enrichment, genomic mutation and diagnostic ROC were performed. TIMER database was used to analyze the association between immune infiltration and target genes. Kaplan-Meier analysis, cox regression and the nomogram prediction model were used to evaluate the prognostic value of target genes. Finally, the expression of potential circRNAs and target genes was validated in cell lines and tissues by quantitative real-time PCR (qRT-PCR) and Human Protein Atlas (HPA) database. In this study, 15 DECs were identified between NSCLC tissues and adjacent-normal tissues in two GEO datasets. Following the qRT-PCR corroboration, 7 DECs (hsa_circ_0002017, hsa_circ_0069244, hsa_circ_026337, hsa_circ_0002346, hsa_circ_0007386, hsa_circ_0008234, hsa_circ_0006857) were dramatically downregulated in A549 and SK-MES-1 compared with HFL-1 cells. Then, 12 circRNA-sponged miRNAs were screened by Circinteractome and StarBase, especially, hsa-miR-767-3p and hsa-miR-767-5p were significantly up-regulated and relevant to the prognosis. Utilizing the miRDB and Cytoscape, 12 miRNA-target genes were found. Functional enrichment, genomic mutation and diagnostic analyses were also performed. Among them, FNBP1, AKT3, HERC1, COL4A1, TOLLIP, ARRB1, FZD4 and PIK3R1 were related to the immune infiltration via TIMER database. The expression of ARRB1, FNBP1, FZD4, and HERC1 was correlated with poor overall survival (OS) in NSCLC patients by cox regression and nomogram. Furthermore, the hub-mRNAs were validated in cell lines and tissues. We constructed the circRNA-miRNA-mRNA network that might provide novel insights into the pathogenesis of NSCLC and reveal promising immune infiltration and prognostic biomarkers.

Yang J ，Hao R ，Zhang Y ，Deng H ，Teng W ，Wang Z ... -  《Cancer Cell International》

Construction and Validation of a Prognostic Model Based on Pyroptosis-related Genes in Bladder Cancer.

Bladder cancer (BCa) is a highly prevalent disease with a poor prognosis. There is no better forecasting method for it yet. Current studies demonstrate that pyroptosis is involved in the development and progression of various cancers. This study employed bioinformatics techniques to analyze the data of BCa patients obtained from the TCGA and GEO databases in order to construct a prognostic risk model. The TCGA dataset was used for the training set, and the multiple external datasets (including GSE13507, GSE31684, GSE48075, IMvigor210, and GSE32894) were applied as the validation sets. Prognostic-associated pyroptosis genes screened by univariate Cox regression analysis were utilized to construct the lasso Cox regression model. GO and KEGG analysis results identified the selected genes that are primarily involved in the inflammation and cell death processes. The related patients were grouped into low- and high-risk groups. Kaplan-Meier survival analysis was performed to compare survival differences between the risk groups. The accuracy of this risk prediction model was assessed by ROC. We also applied the Human Protein Atlas (HPA) to detect the protein expression of these genes. Subsequently, qRT-PCR was performed to verify the expression of these model genes. There are 29 pyroptosis-related genes with significant expression differences between BCa and corresponding adjacent tissues, and 11 genes (SH2D2A, CHMP4C, MRFAP1L1, GBP2, EHBP1, RAD9A, ANXA1, TMEM109, HEYL, APOL2, ORMDL1) were picked by univariate and LASSO Cox regression analysis. Immunological cell infiltration and ssGSEA results further indicated that the low and high-risk groups were substantially correlated with the immune status of BCa patients. According to TCGA and multiple external datasets, Kaplan-Meier survival curves showed the overall survival rate of the high-risk group to be decreased. ROC curves showed the model established to be accurate and reliable. Moreover, the HPA database also demonstrated the verification of the modeled genes' expression in BCa and normal bladder tissue using the HPA database. qRT-PCR results also suggested the up-regulated EHBP1 and down-regulated RAD9A mRNA expression levels to be confirmed in 15 pairs of BCa and corresponding adjacent tissues. This study presents the development and validation of a novel gene signature associated with pyroptosis, which holds the potential for predicting patient outcomes in BCa and providing insights into the immune microenvironment of BCa.

Shen C ，Han C ，Li Z ，Yan Y ，Li C ，Chen H ，Fan Z ，Hu H ... -  《-》

Identification of Serum Exosome-Derived circRNA-miRNA-TF-mRNA Regulatory Network in Postmenopausal Osteoporosis Using Bioinformatics Analysis and Validation in Peripheral Blood-Derived Mononuclear Cells.

Osteoporosis is one of the most common systemic metabolic bone diseases, especially in postmenopausal women. Circular RNA (circRNA) has been implicated in various human diseases. However, the potential role of circRNAs in postmenopausal osteoporosis (PMOP) remains largely unknown. The study aims to identify potential biomarkers and further understand the mechanism of PMOP by constructing a circRNA-associated ceRNA network. The PMOP-related datasets GSE161361, GSE64433, and GSE56116 were downloaded from the Gene Expression Omnibus (GEO) database and were used to obtain differentially expressed genes (DEGs). Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were applied to determine possible relevant functions of differentially expressed messenger RNAs (mRNAs). The TRRUST database was used to predict differential transcription factor (TF)-mRNA regulatory pairs. Afterwards, combined CircBank and miRTarBase, circRNA-miRNA as well as miRNA-TF pairs were constructed. Then, a circRNA-miRNA-TF-mRNA network was established. Next, the correlation of mRNAs, TFs, and PMOP was verified by the Comparative Toxicogenomics Database. And expression levels of key genes, including circRNAs, miRNAs, TFs, and mRNAs in the ceRNA network were further validated by quantitative real-time PCR (qRT-PCR). Furthermore, to screen out signaling pathways related to key mRNAs of the ceRNA network, Gene Set Enrichment Analysis (GSEA) was performed. A total of 1201 DE mRNAs, 44 DE miRNAs, and 1613 DE circRNAs associated with PMOP were obtained. GO function annotation showed DE mRNAs were mainly related to inflammatory responses. KEGG analysis revealed DE mRNAs were mainly enriched in osteoclast differentiation, rheumatoid arthritis, hematopoietic cell lineage, and cytokine-cytokine receptor interaction pathways. We first identified 26 TFs and their target mRNAs. Combining DE miRNAs, miRNA-TF/mRNA pairs were obtained. Combining DE circRNAs, we constructed the ceRNA network contained 6 circRNAs, 4 miRNAs, 4 TFs, and 12 mRNAs. The expression levels of most genes detected by qRT-PCR were generally consistent with the microarray results. Combined with the qRT-PCR validation results, we eventually identified the ceRNA network that contained 4 circRNAs, 3 miRNAs, 3 TFs, and 9 mRNAs. The GSEA revealed that 9 mRNAs participate in many important signaling pathways, such as "olfactory transduction", "T cell receptor signaling pathway", and "neuroactive ligand-receptor interaction". These pathways have been reported to the occurrence and development of PMOP. To sum up, key mRNAs in the ceRNA network may participate in the development of osteoporosis by regulating related signal pathways. A circRNA-associated ceRNA network containing TFs was established for PMOP. The study may help further explore the molecular mechanisms and may serve as potential biomarkers or therapeutic targets for PMOP.

Dong Q ，Han Z ，Tian L 《Frontiers in Endocrinology》