Bin Xie,Feng Peng,Fengping He,Yixing Cheng,Jiangtao Cheng,Zhibing Zhou,Wei Mao
Ras-association domain family 1A (RASSF1A) is one of the most methylated genes in lung cancer (LC). We investigate whether the high DNA methylation level of RASSF1A can relieve the resistance of RASSF1A to LC by inhibiting RASSF1A's transcription factor binding to RASSF1A. RASSF1A expression in tissues and cells was tested utilizing quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. RASSF1A expression and RASSF1A methylation level in LC cells exposed to 5-Aza-dc were assessed by qRT-PCR and quantitative methylation-specific PCR. The association between CTCF and RASSF1A was assessed using hTFtarget, ChIP, and luciferase reporter gene analysis. The effects of 5-Aza-dc, CTCF, and RASSF1A on cell biological behaviors and epithelial-mesenchymal transition (EMT)-related markers were assessed by cell function experiments and Western blot. Moreover, we constructed the xenograft tumor and pulmonary nodule metastasis models, and assessed tumor volume and weight. RASSF1A expression and pulmonary nodule metastasis were tested utilizing qRT-PCR, Western blot, and H&E staining. RASSF1A was under-expressed in LC tissues and cells. 5-Aza-dc enhanced RASSF1A level and weakened RASSF1A methylation level in LC cells. RASSF1A silencing neutralized 5-Aza-dc-mediated repressing effects on LC cell biological function and EMT. The loss of CTCF binding to RASSF1A in LC cells was associated with DNA methylation. The effect of 5-Aza-dc on RASSF1A level, LC cell malignant behaviors, and EMT-related factors were strengthened by CTCF upregulation. RASSF1A overexpression suppressed LC tumor growth and pulmonary nodule metastasis in vivo. DNA methylation blocked the modulation of RASSF1A expression by CTCF and relieved the resistance of RASSF1A to LC.
Yu Liu,Shuyi Liu,Deshuai Zhen,Ji Huang,Fengjiao He
The detection of DNA methylation at cytosine/guanine dinucleotide (CpG) islands in promoter regions of tumor suppressor genes has great potential for early cancer screening, diagnosis, and prognosis monitoring. Nevertheless, achieving accurate, sensitive, cost-effective, and quantitative detection of target methylated DNA remains challenging. Herein, we propose a novel piezoelectric sensor (series piezoelectric quartz crystal (SPQC)) based on transcription activator-like effectors (TALEs) for detecting DNA methylation of Ras association domain family 1 isoform A (RASSF1A) tumor suppressor genes (R-5mC). The sensor employs TALEs-Ni magnetic beads to specifically recognize and separate the R-5mC, thereby improving the detection selectivity. The TALEs-Ni magnetic beads-R-5mC complex is sheared by a nucleic acid enzyme (DNAzyme) to release the single-stranded DNA (ST). ST initiates a catalyzed hairpin assembly (CHA) reaction on the surface of the electrode, which in turn triggers the hybridization chain reaction (HCR) and silver staining for enhanced detection sensitivity. The strategy exhibits a linear response in the detection of R-5mC in the range of 1 fM to 1 nM with a detection limit of 0.79 fM. R-5mC as low as 0.01% can be detected, even in the presence of large numbers of unmethylated DNA. The detection of R-5mC in circulating cell-free DNA (cfDNA) derived from clinical plasma specimens of lung cancer patients yielded satisfactory results.
Dechan Lu,Yanping Chen,Longfeng Ke,Zufang Huang,Yudong Lu,Jing Wang
DNA methylation is a promising biomarker for early cancer detection; however, traditional detection methods require bisulfite treatment and nucleic acid amplification, which are costly and impractical for point-of-care use. We propose a facile method that leverages immunoassays and surface-enhanced Raman scattering (SERS) readouts for the multiplex, sensitive, and portable detection of lung cancer-related DNA methylation biomarkers. This approach employs 5-methylcytosine antibody-modified magnetic probes to enrich methylated DNA sequences and uses core–shell nanoparticles with Probe DNA as SERS nanotags for multiplex detection, eliminating the need for bisulfite treatment and nucleic acid amplification. The multiplex SERS immunoassay detects synthetic methylated SHOX2 and RASSF1A with detection limits of 0.52 pM and 0.66 pM, respectively. It shows a strong correlation with quantitative PCR results in analyzing methylated SHOX2 and RASSF1A from cell lines and formalin-fixed paraffin-embedded tissue samples (n = 35). Additionally, the random forest analysis based on methylated SHOX2 and RASSF1A expression distinguishes lung cancer from benign lung diseases with a clinical sensitivity of 96 %, specificity of 90 %, and an AUC value of 0.972. This method offers a significant advancement for DNA methylation research and clinical applications.
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