A SNP of miR-146a is involved in bladder cancer relapse by affecting the function of bladder cancer stem cells via the miR-146a signallings.

MiR-146a-5p in urine samples was recently reported to be possibly used as a prognostic marker for bladder cancer (BC). Interestingly, YAP1 and COX2 were both demonstrated to function as stem cell regulators in BC. Therefore, in this study, we aimed to establish the molecular mechanism underlying the role of miR-146a, YAP1 and COX2 in BC relapse. We also studied the possibility of using the C > G genotype of miR-146a rs2910164 SNP as an indicator of BC relapse. A total of 170 BC patients were assigned into different groups based on their genotypes of rs2910164 SNP. Kaplan-Meier survival curves were plotted to compare the recurrence-free rate among these groups. Real-time PCR, Western Blot, bioinformatic analysis, luciferase assay and IHC assay were conducted to study the role of rs2910164 SNP in the progression of BC. Accordingly, GC/CC-genotyped patients presented a higher risk of recurrence when compared with GG-genotyped patients, while the expression of BC regulators was influenced by the presence of rs2910164. COX2 mRNA and YAP1 mRNA were, respectively, validated as direct target genes of miR-146a, and the expression of YAP1 and COX2 mRNA/protein was both suppressed by miR-146a precursors. The expression of ALDH1A1 mRNA/protein was inhibited upon the down-regulation of YAP1, while the expression of let7 and SOX2 mRNA/protein was inhibited upon the down-regulation of COX2. In conclusion, two signalling pathways, miR-146a/YAP1/ALDH1A1 and miR-146a/COX2/PGE2/let7/SOX2, were modulated by miR-146a. As an SNP regulating the expression of miR-146a, the rs2910164 G > C SNP could be utilized as a biomarker for BC relapse.

Development of Oxidation Damage Base-Based Fluorescent Probe for Direct Detection of DNA Methylation.

DNA methylation has become a promising epigenetic biomarker for cancer diagnosis, prognosis, and therapy monitoring. Herein, we demonstrate for the first time the development of a new oxidation damage base (8-oxo-7,8-dihydroguanine (8-oxoG))-modified fluorescent probe for direct detection of DNA methylation. This fluorescent probe is labeled with carboxy-X-rhodamine (ROX) and black hole quencher 2 (BHQ2) at the 5' and 3' termini, respectively, with one 8-oxoG base modification in the middle position, and it can discriminate the methylated cytosine from the unmethylated cytosine. The presence of target methylated DNA may induce the recycle cleavage of fluorescent probes with the assistance of human 8-oxoG DNA glycosylase 1 (hOGG1) enzyme, resulting in an enhanced fluorescence signal. In comparison with the reported bisulfite treatment-based indirect approaches, this fluorescent probe can be used for direct detection of DNA methylation under isothermal conditions without the requirement of a stringent primer/template design, any thermal cycling, and ligation procedures, greatly simplifying the experimental processes. Moreover, this fluorescent probe exhibits good specificity and high sensitivity, and it can distinguish a 0.01% methylation level even in the presence of excess unmethylated DNA. Furthermore, this fluorescent probe can be used to detect DNA methylation in genomic DNA extracted from human colon cancer cells, holding great potential in epigenetic study and early clinical diagnosis.

Up-regulation of microRNA-200c-3p inhibits invasion and migration of renal cell carcinoma cells via the SOX2-dependent Wnt/ß-catenin signaling pathway.

BACKGROUND: MicroRNA-200c-3p (miR-200c-3p) has been revealed to be related to renal cell carcinoma (RCC) progression, while the inner mechanisms remain unknown. In our study, we intend to unearth the capability of miR-200c-3p in RCC development via the Wnt/ß-catenin signaling pathway through binding to SOX2. METHODS: miR-200c-3p, SOX2, ß-catenin and GSK3ß expression in both tissues and cells of RCC were detected by RT-qPCR or western blot analysis. miR-200c-3p was restored or silenced to determine their biological functions of RCC cells. Expression of SOX2 and related proteins in the Wnt/ß-catenin signaling pathway were evaluated by RT-qPCR and western blot analysis. The effect of the combination of downregulated miR-200c-3p and downregulated SOX2 on cell biological behavior change was also determined. RESULTS: Initially, we found that miR-200c-3p was declined while SOX2, ß-catenin and GSK3ß was elevated in RCC tissues and cells. A498 cells with the largest difference in miR-200c-3p expression and OS-RC-2 cells with the smallest difference were selected for subsequent experiments. Additionally, upregulated miR-200c-3p and downregulated SOX2 was determined to suppress proliferation, migration, invasion and induce apoptosis of RCC cells. Furthermore, miR-200c-3p inhibited SOX2 to inactivate the Wnt/ß-catenin signaling pathway. CONCLUSION: Collectively, this study highlights that upregulated miR-200c-3p inhibits expression of SOX2, thereby inhibiting development of RCC cells via modulating the Wnt/ß-catenin signaling pathway activation.

Label-free and amplified detection of apoptosis-associated caspase activity using branched rolling circle amplification.

We develop a label-free fluorescence method for ultrasensitive detection of apoptosis-associated caspase activity based on branched rolling circle amplification. This work demonstrates for the first time the use of a nucleic acid amplification strategy for the amplified detection of caspase activity, which allows for highly sensitive detection of endogenous caspase even at the single-cell level. This method can be further applied for kinetic analysis and inhibitor screening.