DNA methylation influences the CTCF‐modulated transcription of RASSF1A in lung cancer cells

Abstract

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.

Insulin-like Growth Factor-2 (IGF-2) in Fibrosis

“…The non-coding H19 gene, which is 128 kb downstream of the IGF-2 gene, is widely regarded as the IGF-2/H19 locus with the IGF-2 gene, because their genetic imprints are jointly determined by differentially methylated regions (DMRs) within the imprinting control region (ICR) [ 10 ]. The transcription regulator CCCTC-binding factor (CTCF), as a highly conserved zinc finger nuclear protein, inhibits gene expression by binding to different sites of gene promoters, enhancers, and insulators [ 11 ]. In the human genome, more than 15,000 CTCF binding sites have been identified [ 12 ], including the IGF-2/H19 locus.…”