%0 Journal Article
%T The transcription activity of OTX2 on p16 expression is significantly blocked by methylation of CpG shore in non-promoter of lung cancer cell lines.
%A Peng H
%A Fu W
%A Chang C
%A Gao H
%A He Q
%A Liu Z
%A Cui M
%A Wang H
%A Yu Y
%A Wu Y
%A Zhang X
%A Jiang S
%A Xu C
%A Shen X
%A Zhang Z
%A Zhou Y
%A Li D
%A Wang Q
%J Transl Cancer Res
%V 12
%N 10
%D 2023 Oct 31
%M 37969391
%F 0.496
%R 10.21037/tcr-23-909
%X <B>UNASSIGNED: </B>The aberrant expression of the classical tumor suppressor gene p16 is a frequent event in lung cancer mainly due to the hypermethylation of its 5'-cytosine-phosphate-guanine-3' island (Cgi). However, whether methylation happens in other regions and how p16 expression and function are affected are largely unknown.<BR><B>UNASSIGNED: </B>Clustered Regularly Interspaced Short Palindromic Repeats/dCas9 (CRISPR/dCas9) technology was used for methylation editing at specific site of p16. The effects of methylation editing were detected by 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt (MTS), transwell migration and wound healing tests. Chromatin immnoprecipitation-quantitative polymerase chain reaction (CHIP-qPCR) was performed to explore the impact of Cgi shore methylation on the binding abilities of transcription factors (TFs) including YY1, SP1, ZNF148 and OTX2 to p16 gene. A rescue experiment was performed to verify the regulatory effect of OTX2 on p16. The negative relationship between p16 expression and the methylation level of Cgi shore in non-promoter region was further verified with datasets from The Cancer Genome Atlas (TCGA) program and lung adenocarcinoma (LUAD) patients' samples.<BR><B>UNASSIGNED: </B>The suppressive effect of p16 Cgi shore methylation on its expression was demonstrated in both HEK293 and A549 cells using CRISPR/dCas9-mediated specific site methylation editing. Methylation of the Cgi shore in the p16 non-promoter region significantly decreased its expression and promoted cell growth and migration. The ability of OTX2 bound to p16 was significantly reduced by 19.35% after methylation modification. Over-expression of OTX2 in A549 cells partly reversed the inhibitory effect of methylation on p16 expression by 19.04%. The verification results with TCGA and LUAD patients' samples supported that the p16 Cgi shore is a key methylation regulatory region.<BR><B>UNASSIGNED: </B>Our findings suggested that methylation of the Cgi shore in the p16 non-promoter region can hamper the transcriptional activity of OTX2, leading to a reduction in the expression of p16, which might contribute to the development of lung cancer.