DNA methylation at specific CpG sites plays a critical role in the diagnosis and treatment of human diseases. In this study, we introduce a site-specific DNA methylation detection biosensor that leverages immune recognition and nonlinear hybridization chain reactions (NHCR). Since anti-methylcytosine antibodies are unable to recognize methylcytosine within a DNA duplex, we specifically target the CpG site by removing the guanine complementary to the target methylcytosine in the capture probe, thereby creating a protruding region accessible for antibody recognition. We utilized NHCR as a signal amplification strategy to enable site-specific DNA methylation detection under isothermal and enzyme-free conditions. The proposed method achieves a detection limit of 0.15 pM and can differentiate methylation levels as low as 0.1 %, even in the presence of a large excess of unmethylated DNA. This approach is capable of detecting DNA methylation at specific CpG sites in genomic DNA extracted from human hepatocellular carcinoma (HCC) tissues, showcasing its considerable potential for biological research and disease diagnosis.
Objectives Transcriptional changes in cartilage can impact function by causing degradation such as that which occurs during the development of osteoarthritis (OA). Epigenetic regulation may be a key factor leading to transcriptional changes in OA. In this study, we performed a combined analysis of DNA methylation and gene expression microarray datasets and identified key transcription factors (TFs) central to the regulation of gene expression in OA. Methods A DNA methylation profile dataset (GSE63106) and a gene expression profiling dataset (GSE114007) were extracted from the Gene Expression Omnibus (GEO). We used ChAMP methylation analysis and the Limma package to identify differentially methylation genes (DMGs) and differentially expressed genes (DEGs) from normal and human knee cartilage samples in OA. Function enrichment analysis of DMGs was conducted using the DAVID database. A combined analysis of DEGs and DMGs was conducted to identify key TFs in OA. We then validated the mRNA expression of selected TFs in normal and OA cartilage by RT-qPCR. Primary chondrocytes were cultured and treated with the DNA methylation inhibitor 5-Aza-2-deoxycytidine (5-Aza) for functional validation. Results We identified 2,170 differential methylation sites (DMS) containing 1005 genes and 1986 DEGs between normal human and OA cartilage. Functional analysis of DMGs revealed that focal adhesion, extracellular matrix (ECM)-receptor interactions, the PI3K-Akt signaling pathway, and the FoxO signaling pathway were involved in OA. Integrated analysis showed a subset of 17 TFs. Four TFs (ELF3, SOX11, RARA, and FOXD2) were validated. RT-qPCR results showed the mRNA expression of SOX11, RARA, and FOXD2 were consistent with the results from the mRNA expression data. However, the expression of ELF3 could not be validated. Upon 5-Aza-2′-deoxycytidine (5-Aza) treatment, the mRNA levels of ELF3 and SOX11 were down-regulated, whilst RARA was up-regulated, and FOXD2 showed no significant change in expression level. Conclusions the effect of DNA methylation on the transcriptional regulation is related to the distribution of methylated sites across the genome. Epigenetic studies on the positions of DMS in transcriptional units can inform a better understanding of the function of DNA methylation and its transcription regulation.
危险品化学品经营许可证（带存储）