BACKGROUND:Formin-related protein-1(FRL1) has reportedly been overexpressed in a variety of malignancies, such as clear cell renal cell carcinoma (ccRCC). However, the clinical value and molecular mechanisms underlying ccRCC tumorigenesis and progression in association with FRL1 remain poorly understood.METHODS:Immunohistochemical analysis was performed on 119 paraffin-embedded RCC tissue samples to detect FRL1 expression and analyze its prognostic value. Colony formation, the CCK-8 assay, flow cytometry, and in vivo nude mice subcutaneous experiments were used to identify the effects of FRL1 on growth and proliferation. In vitro tests for wound healing, migration, and invasion were used to assess the involvement of FRL1 in invasion and metastatic potential. The process of epithelial-mesenchymal transition process (EMT) and the MMP2 expression were detected in stably transfected RCC cells via western blotting, as well as in tumor tissue paraffin sections from xenograft model.RESULTS:Both FRL1 mRNA and protein levels were noticeably elevated in ccRCC cell lines and samples. Aberrant overexpression of FRL1 was associated with unfavorable clinicopathological features of ccRCC and indicated poor prognosis. Ectopic overexpression of FRL1 increased the growth-promoting traits of ccRCC cells as well as the migratory and invasive capacity of RCC cells, whereas FRL1-silencing caused the opposite results. In addition, FRL1 promoted epithelial-mesenchymal transition (EMT) and upregulated the expression of matrix metalloproteinase 2 (MMP2). Finally, overexpression of FRL1 upregulated phosphorylation level of ERK1/2 with no effect on total level of ERK1/2 in the RCC cells. MAPK/ERK inhibitor reversed the promotional effects of FRL1.CONCLUSION:FRL1 was overexpressed in ccRCC tissues and predicted poor prognosis. FRL1 contributes to invasion and aggressive phenotype of ccRCC by facilitating EMT through MAPK/MMP2 axis.

Background: Clear cell renal cell carcinoma (ccRCC) is a worldwide malignancy with high morbidity and mor-tality. Translation initiation factor 4A1 (eIF4A1), which is an ATP-dependent RNA helicase as a part of eIF4F complex, has been linked to malignant transformation and progression, and a variety of cancers display dysre-gulation of this enzyme. However, its role in ccRCC remains unclear. In our study, we examined its potential effects in ccRCC. Methods: Based on Proteomic data, TCGA and ONCOMINE database, RCC cell lines and tissues, the expression of eIF4A1 between ccRCC and normal tissues were investigated. A correlation was evaluated between the prog-nostic model for OS and ccRCC progression. Analysis of functional enrichment and PPI network were performed. After examining differentially expressed genes between the eIF4A1 high and low-expression groups, we per-formed GSEA analysis. Furthermore, we investigated immune cell infiltration of eIF4A1. Then we determined eIF4A1 functions in the establishment and maintenance of cell viability, migration and invasion of cell lines. Flow cytometry was utilized to detect cell cycle. Results: The eIF4A1 was up-regulated in ccRCC tissues and cell lines. An increased level of eIF4A1 was linked to lower survival rates and impaired immunity. Depletion of eIF4A1 could arrest tumor cells in G1 phase, so as to seriously limit cell proliferation and weaken the capacity of cell migration. Conclusion: ccRCC patients with high eIF4A1 expression are at increased risk of poor prognosis, furthermore eIF4A1 plays a prominent role in facilitating tumor cell proliferation and migration which may further be a potential prognostic biomarker and therapeutic target.

BACKGROUND:Cancer stem cells (CSCs) have an key role in the beginning, progression and treatment of bladder cancer. In the current study, our target was to identify CSCS-related genes in bladder cancer.METHODS:Bladder cancer (BLCA) transcriptome data were acquired from The Cancer Genome Atlas (TCGA) database. WGCNA was used to screen genes connected with the mRNA expression-based stemness index (mRNAsi).Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to analyze the biological function of mRNAsi-related genes. Univariate Cox regression and LASSO Cox regression algorithms were applied to build a risk score model. Additionally, a ceRNA regulatory netwok based on key mRNAsi-related genes was established via TargetScan, miRDB, miRTarBas and miRcode database,and lncRNA SNHG12 was selected for further in vitro and invivo functional assays.RESULTS:Between BLCA and normal samples were identified 1560 differentially expressed genes (DEGs).845 DEGs were most significantly associated with mRNAsi according to WGCNA analysis, which were mainly enriched in GO terms and KEGG pathways related to cell proliferation. Univariate Cox regression and LASSO Cox regression algorithms screened 25 mRNAsi-related genes to construct the risk score model with the significant ability to estimate prognosis of BLCA patients. A ceRNA network, including 8 lncRNA, 11 miRNA and 9 mRNAsi-related mRNA, was constructed.We found that lncRNAs ADAMTS9-AS1 and SNHG12 were observably related to the survival of BLCA patients. To verify this finding, we selected SNHG12 for further study. RT-PCR experiments revealed that SNHG12 was high expression in both bladder cancer tissues and cells.SNHG12 promoted proliferation, invasion, migration, apoptosis and stemness of bladder cancer cells in vitro and tumour proliferation in vivo.CONCLUSION:Our study identified 25 biomarkers associated with stemness indices in BLCA and established a ceRNA network based on key mRNAsi-related genes.SNHG12 promoted BLCA proliferation, invasion, migration, apoptosis and stemness in vitro. It was also showed that SNHG12 promoted tumour growth.

Purpose: In this study, we have undertaken the whole proteomic analysis and got a better understanding of biological processes involved in the development and progression of ccRCC. We hope promising biomarkers can be uncovered to facilitate early diagnosis, predict the prognosis and progression, more importantly, to be applied as potential therapeutic targets. Experimental design: Fresh frozen tissue samples were surgically resected from patients with local or locally advanced ccRCC. Trypsin digested proteins were analyzed using TMT-based LC-MS/MS proteomic approach, followed by bioinformatic analysis. A potential prognostic marker FMNL1 was chosen to be validated in TCGA_KIRC datasets (n=525 and 72), further validation sets (n=10 and 10) and expanded validation sets (n=81 and 16). The effects of FMNL1 on proliferation, migration and invasion were determined by colony formation, wound healing, and transwell assays in 786-O and Caki-1 cells in vitro study. Results: A total of 657 differentially expressed proteins were identified and quantified between ccRCC and adjacent normal tissues (p-value<0.05, FC>2 or<1/2), of which 186 proteins were up-regulated and 471 proteins were down-regulated. Bioinformatic analysis showed enriched metabolic biological processes and pathways. Univariate and multivariate analysis defined FMNL1 as an independent negative prognostic marker in the TCGA datasets. High expression of FMNL1 correlated significantly with tumor stage and distant metastasis (P<0.05) both in the TCGA-KIRC datasets and expanded validation sets. Kaplan-Meier survival curve illustrated that the patients with high FMNL1 protein level had shorter OS time in the expanded validation sets (p=0.0273). In vitro experiments presented the functional effects of FMNL1 knockdown on the inhibition of proliferation, migration and invasion in cancer cell lines. Conclusion and clinical relevance: The proteomic results uncovered sophisticated metabolic reprogramming of ccRCC and indicated that the upregulation of rate-limiting enzymes in glycolysis and mitochondrial impairment may be the cause of metabolic reprogramming in ccRCC. Moreover, FMNL1 has been identified as a promising prognostic marker, and knockdown of FMNL1 could inhibit ccRCC cell proliferation, migration and invasion, which might be used as a new effective therapeutic strategy to inhibit the progression of ccRCC.