Objective To investigate the expression characteristics of the transcription factor E2F2 in clear cell renal cell carcinoma (ccRCC), its impact on patient prognosis, and its potential role in the tumor immune microenvironment, with validation using clinical samples and functional assays. Methods RNA sequencing data and clinical information of ccRCC patients were obtained from the TCGA database; GSE53757 and GSE66272 datasets were downloaded from the GEO database as external validation cohorts. The expression difference of E2F2 between ccRCC tissue and normal tissue was compared, and the relationship between E2F2 expression and clinical pathological characteristics was analyzed. Kaplan-Meier (KM) survival analysis with log-rank test was performed to evaluate the effects of E2F2 on overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS). The prognostic efficacy of E2F2 in predicting 1-, 3- and 5-year survival was evaluated using receiver operating characteristic (ROC) curve. GSEA was performed to identify E2F2-related signaling pathways, and the ESTIMATE and CIBERSORT algorithms were used to assess the relationship between E2F2 expression level and tumor immune cell infiltration and immune microenvironment. Twenty pairs of surgically resected and pathologically confirmed ccRCC tissues and matched adjacent non-tumor tissues were collected from our hospital, and immunohistochemistry (IHC) was performed to detect the protein expression of E2F2. Human ccRCC cell line 786-O was used for functional assays;shRNA was used to knock down E2F2 expression (constructing stable shE2F2#1 and shE2F2#2 cell lines), and qRT-PCR and Western blot were performed to verify knockdown efficiency, followed by MTT, colony formation, and Transwell migration assays to evaluate the effects of E2F2 on the biological behaviors of ccRCC cells. Results E2F2 was significantly upregulated in ccRCC tissues (the same results in the verification set). Higher E2F2 expression was associated with advanced histological grade, clinical stage and higher TNM classification (P＜0.05). KM analysis showed that patients in the E2F2 high-expression group had worse OS, PFS, and DSS (P=0.019, 0.036, ＜0.001); the ROC curves showed area under the curve (AUC) of E2F2 of predicting 1-, 3- and 5- year survival of ccRCC patients were 0.844, 0.851 and 0.815, respectively. GSEA revealed that the E2F2 low-expression group was enriched in multiple metabolism-related pathways, whereas the E2F2 high-expression group was associated with immune-related pathways. Immune analysis demonstrated that high E2F2 expression was associated with increased immune scores, increased proportion of immune cells, higher tumor mutation burden, and potentially stronger immunotherapy response. IHC results showed that the E2F2 immunoreactivity score in ccRCC tissues was significantly higher than that in adjacent non-tumor tissues (P＜0.05). Functional assays indicated that E2F2 knockdown significantly inhibited the proliferation, colony formation, and migration of ccRCC cells. Conclusion E2F2 is highly expressed in ccRCC and may be involved in tumorigenesis and progression through modulation of the immune microenvironment. Its expression level is closely associated with patient prognosis, and E2F2 has the potential to serve as a prognostic biomarker and immunotherapeutic target in ccRCC.

Objective:Bioinformatics was used to analyze the gene expression profile of renal chromophobe cell carcinoma (RCCC) to find out the key genes of RCCC.Methods:Chromophobe renal cell carcinoma gene chip data GSE15641 and GSE11151 were downloaded from the GEO database. Using R software packages such as " Affy" and " limma" in R software to screen differentially expressed genes, combining with David and STRING online bioinformatics tools to analyze the regulatory network of differentially expressed genes and construct protein-protein interaction (PPI) network, the Hub gene was screened through the Cytohubba plug-in of Cytoscape software.Results:A total of 261 differentially expressed genes were screened, including 194 down-regulated genes and 67 up-regulated genes. Gene enrichment (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to explore their biological functions. In GO enrichment analysis, biological processes were mainly enriched in cell secretion, gluconeogenesis and cell proliferation regulation; in cell composition, they were mainly enriched in exosomes, plasma membranes and their components; in molecular function, they were mainly enriched in heparin binding; in KEGG pathway analysis, they were mainly enriched in metabolic pathway, antibody biosynthesis pathway and renin angiotensin system pathway. PPI network was constructed by using online bioinformatics tools. The top 10 Hub genes were screened by using cytohubba plug-in in Cytoscape software, which were pipecolic acid and sarcosine oxidase (PIPOX), hydroxyacid oxidase 2 (HAO2), kynurenine 3-monooxygenase (KMO), solute carrier family 2 member 2 (SLC2A2), formimidoyltransferase cyclodeaminase (FTCD), angiogenin (ANG), APOBEC1 complementation factor (A1CF), aldehyde dehydrogenase 8 family member A1 (ALDH8A1), vitamin D binding protein (GC), histidine rich glycoprotein (HRG).Conclusions:Bioinformatics analysis of differentially expressed genes in renal chromophobe cell carcinoma can effectively explore the interaction information of these differentially expressed genes, and provide new ideas for the treatment of renal chromophobe cell carcinoma.

AIM:To investigate the role of Rho-associated kinase ( ROCK) inhibitor fasudil in the formation of rabbit urethral stricture after injury and to observe the cell activity , migration and extracellular matrix synthesis in the rabbit urethra fibroblasts.METHODS:The rabbit model of urethral stricture was established by microsurgical techniques .The rabbits were divided into sham operation group , operation group and fasudil (3 mg/kg, 10 mg/kg, 30 mg/kg) groups.The diameter of the stenosis was measured by retrograde urethrography 3 months after surgery .The fibroblasts were isolated from urethral scar, and then incubated with fasudil (12.5 μmol/L, 25 μmol/L, 50 μmol/L) in the presence of transforming growth factor-β1 (TGF-β1, 10 μg/L).The untreated cells were used for control .The cell activity was measured by MTT assay.The cell migration ability was tested by the method of Transwell chambers .The protein expression of ROCK , α-smooth muscle actin (α-SMA) , collagen I and collagen III was determined by Western blot analysis .RESULTS:Fasudil significantly reduced formation of urethral stricture after injury (P<0.05).Cultured rabbit fibroblasts with different con-centrations of fasudil inhibited the cell activity and cell migration ability (P<0.05).The protein expression of ROCK,α-SMA, collagen I and collagen III was also inhibited by treatment with fasudil in a dose -dependent manner ( P<0.05 ) . CONCLUSION:Fasudil inhibits the formation of extracellular matrix and reduces the incidence of urethral stricture after injury by down-regulating TGF-β1-induced Rho/ROCK pathway activation in the rabbit urethra fibroblasts .