Characterization of extrachromosomal circular DNAs in plasma of patients with clear cell renal cell carcinoma.

BACKGROUND AND PURPOSE: Extrachromosomal circular DNAs (eccDNAs) have been recognized for their significant involvement in numerous biological processes. Nonetheless, the existence and molecular characteristics of eccDNA in the peripheral blood of patients diagnosed with clear cell renal cell carcinoma (ccRCC) have not yet been reported. Our aim was to identify potentially marked plasma eccDNAs in ccRCC patients. METHODS AND MATERIALS: The detection of plasma eccDNA in ccRCC patients and healthy controls was performed using the Tn5-tagmentation and next-generation sequencing (NGS) method. Comparisons were made between ccRCC patients and healthy controls regarding the distribution of length, gene annotation, pattern of junctional nucleotide motif, and expression pattern of plasma eccDNA. RESULTS: We found 8,568 and 8,150 plasma eccDNAs in ccRCC patients and healthy controls, respectively. There were no statistical differences in the length distribution, gene annotation, and motif signature of plasma eccDNAs between the two groups. A total of 701 differentially expressed plasma eccDNAs were identified, and 25 plasma eccDNAs with potential diagnostic value for ccRCC have been successfully screened. These up-regulated plasma eccDNAs also be indicated to originate from the genomic region of the tumor-associated genes. CONCLUSION: This work demonstrates the characterization of plasma eccDNAs in ccRCC and suggests that the up-regulated plasma eccDNAs could be considered as a promising non-invasive biomarker in ccRCC.

Overexpression of Spondin-2 Is Associated with Recurrence-Free Survival in Patients with Localized Clear Cell Renal Cell Carcinoma.

BACKGROUND: The spondin-2 (SPON2) gene is overexpressed in multiple malignant tumors and may promote tumor aggressiveness. However, its expression profile and functional roles in clear cell renal cell carcinoma (ccRCC) are still unclear. METHODS: SPON2 expression in ccRCC was evaluated using expression data from TCGA and GEO databases, then confirmed by local patient population (94 patients). The clinical significance of SPON2 expression was evaluated. Downregulation of SPON2 was performed using small-interfering RNA (siRNA). The effects of SPON2 silencing on cell proliferation, apoptosis, invasion, and migration in vitro were investigated. RESULTS: SPON2 was overexpressed in the majority of the ccRCC at both mRNA and protein levels. SPON2 expression was significantly correlated with stage, grade, and recurrence (all P < 0.05) in patients with localized ccRCC. The receiver operating characteristic (ROC) curve showed that SPON2 expression could serve as a predictor of recurrence. SPON2 expression was significantly associated with recurrence-free survival (RFS) in patients with localized ccRCC. Knocking down SPON2 resulted in suppressed cell invasion and migration in vitro. CONCLUSION: SPON2 expression might function as a prognostic biomarker in patients with localized ccRCC.

Overexpression of COL5A1 promotes tumor progression and metastasis and correlates with poor survival of patients with clear cell renal cell carcinoma.

BACKGROUND AND AIMS: COL5A1 has been identified to be involved in metastasis of clear cell renal cell carcinoma (ccRCC) by bioinformatic analysis. This study aimed to investigate COL5A1 expression and its clinical significance in ccRCC. The function of COL5A1 in ccRCC was further investigated. METHODS: COL5A1 expression was examined in 256 ccRCC tissues and paired adjacent normal renal tissues by immunohistochemistry and real-time quantitative PCR. The clinical significance of COL5A1 expression was evaluated. Downregulation of COL5A1 was achieved using siRNA. The effects of COL5A1 silencing on cell proliferation, apoptosis, migration, invasion in vitro, and tumor growth in vivo were investigated. RESULTS: COL5A1 expression was upregulated in the majority of the ccRCC tissues at both protein and mRNA levels. COL5A1 expression was significantly correlated with tumor diameter, tumor stage, tumor grade, distant metastasis, recurrence, necrosis, and sarcomatoid (all P<0.05). COL5A1 expression was also significantly associated with overall survival of ccRCC patients (HR 1.876; P=0.027) and recurrence-free survival of localized ccRCC patients (HR 4.751; P<0.001). The accuracy of TNM, University of California Los Angeles Integrated Staging System, and Mayo clinic stage, size, grade, and necrosis prognostic models was improved when COL5A1 expression was added. CONCLUSION: COL5A1 knockdown significantly inhibited cell proliferation, induced cell apoptosis, inhibited cell migration and invasion in vitro, and inhibited tumor growth in vivo. Therefore, COL5A1 may be a novel prognostic biomarker and a promising therapeutic target for ccRCC.

H2O2 inhibits proliferation and mediates suppression of migration via DLC1/RhoA signaling in cancer cells.

BACKGROUND: RhoGTPase-activating proteins (RhoGAPs) regulate RhoGTPases in cells, but whether individual reactive oxygen species (ROS) regulate RhoGAPs is unknown. Our previous published papers have shown that deleted in liver cancer 1 (DLC1) inhibits cancer cell migration by its RhoGAP activity. The present study was designed to explore the role of H2O2 in regulation of DLC1. MATERIALS AND METHODS: We treated cells with H2O2 for 24h and phenotypic changes were analyzed by MTT, RT-PCR, Western blotting, immunofluorescence staining and wound healing assays. RESULTS: H2O2 downregulated cyclin D1 and cyclin E to inhibit proliferation, and upregulated BAX to induce apoptosis in MCF-7 cells. Compared with non-tumorigenic cells, H2O2 increased expression of DLC1 and reduced activity of RhoA in cancer cells. Stress fiber production and migration were also suppressed by H2O2 in MDA-MB-231 cells. CONCLUSIONS: Our study suggests that H2O2 inhibits proliferation through modulation of cell cycle and apoptosis-related genes, and inhibits migration by decreasing stress fibers via DLC1/RhoA signaling.