Journal: Chinese Journal of Cancer Research
Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis
doi: 10.21147/j.issn.1000-9604.2019.06.09
Figure Lengend Snippet: During transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT), stomatin was downregulated through methylation-dependent transcription mechanism. (A) Real-time polymerase chain reaction (PCR) was performed to validate the transcriptional change of stomatin identified in the microarray. **, P<0.001; (B) Two CpG islands spanning from −768 to −223 bp and from −143 to +122 bp upstream of STOM gene were identified by EMBOSS cpgplot program (upper). Schematic representation of −768/−223 and −143/+122 (marked with the grey square) CpG islands in the upstream of stomatin gene (lower). Red box marked the exon. The translation start site was position +1; (C) Measuring stomatin promoter methylation levels by Sequenom MassARRAY assay; (D) Sequenom MassARRAY analysis of methylation percentages of CpG sites at the stomatin promoter. The top panel showed the methylation percentages of 30 sites in human bronchial epithelial (HBE) and A549 cells. Dots with different color depths represent different methylation status. Dark dots represent high degrees of methylation. Light dots represent low degrees of methylation. The methylation percentages of 30 CpG sites were compared by two-tailed paired Student’s t test. In line graph, each dot represents an individual CpG dinucleotide. Green dots represent CpG dinucleotides in HBE cells. Red dots represent CpG sites in A549 cells. The corresponding detection sites were connected in a straight line. Real-time PCR was performed to evaluate the expression of stomatin in HBE and A549 cells (P<0.01); (E) The same sequenom MassArray and statistical analyses were also implemented to assess the methylation status of the indicated promoter region of stomatin in A549 and A549-5Aza cells (P<0.01); (F) A549 cells were exposed to TGFβ1 (5 ng/mL) for 2 d or 5 d with or without 5-Aza. Expression of DNMT3A and stomatin was analyzed by western blotting. β-actin was used as a loading control; (G) Sequenom MassArray analysis was implemented to assess the stomatin promoter methylation levels in A549, A549-TGFβ1, and A549-TGFβ1/5-Aza cells (P=0.005, P=0.004, respectively).
Article Snippet: The methylation status of Stomatin promoter was detected using Sequenom MassARRAY quantitative methylation analysis platform (BGI tech, Shenzhen, China).
Techniques: Methylation, Real-time Polymerase Chain Reaction, Microarray, Sequenom Massarray Assay, Two Tailed Test, Expressing, Western Blot, Control
