Effect of cytosolic protein 9 gene methylation on malignant progression of oesophageal cancer cells and underlying mechanism
Objective To investigate the effects of cytosolic protein 9(SEPTIN9)gene methylation on oesophageal cancer cell proliferation,apoptosis,migration and invasion.Methods The methylation level of SEPTIN9 gene in normal oesophageal epithelial cells HEEC and oesophageal cancer ECA109 and KYSE150 cells was detected by sulfite sequencing polymerase chain reaction(PCR)method.The methyla-tion inhibitor 5-aza-2'-deoxycytidine(5-Aza-dc)(experimental group)and dimethylsulfoxide(DMSO)(control group)were applied to co-culture with the cells for 72 h,respectively,and the real-time fluores-cence quantitative polymerase chain reaction(RTFQPCR)and protein blotting(Western blotting)were used to detect SEPTIN9 mRNA and its proteins by using the diphenyltetrazolium bromide(MTT)method and fluorescein isothiocyanate labelled phospholipid binding protein(Annexin V-FITC)apoptosis assay to detect proliferation and apoptosis,and Transwell method to detect migration and invasion.Independent samples t-tests was used for comparison between groups.Results The methylation of SEPTIN9 gene in e-sophageal cancer cells ECA109 and KYSE150 was higher than that in normal esophageal epithelial cells HEEC[(83.69±7.15)％vs.(21.36±4.58)％;(84.26±6.97)％vs.(21.36±4.58)％,t=12.714,13.063,P＜0.01].The relative expression of ECA109 and KYSE150 SEPTIN9 mRNA and SEPTIN9 protein in the experimental group was higher than that in the control group(SEPTIN9 mRNA:1.66±0.17 vs.1.23±0.12,1.68±0.20 vs.1.25±0.13;SEPTIN9 protein:1.76±0.18 vs.1.35± 0.15,1.85±0.21 vs.1.38±0.16,t=3.579,3.122,3.031,3.083,P＜0.05).The proliferation a-bility of esophageal cancer cells ECA109 and KYSE150 in the experimental group was lower than that of the control group[(0.38±0.06)vs.(0.91±0.17),(0.40±0.05)vs.(0.97±0.16),t=5.092,5.890,P＜0.01].The apoptosis rate of esophageal cancer cells ECA109 and KYSE150 in the experimental group was higher than that in the control group[(35.69±4.37)％vs.(17.89±2.26)％,(41.18±5.49)％vs.(18.96±3.05)％,t=6.267,6.128,P＜0.01].The number of migrating and invading esophageal cancer cells ECA109 and KYSE150 in the experimental group was less than that in the control group[num-ber of migrating cells:(116.75±12.59)vs.(187.24±19.26),(120.24±13.68)vs.(193.25± 20.34);number of invading cells:(61.34±7.52)vs.(108.35±12.56),(63.58±6.69)vs.(112.24± 15.06),t=5.905,5.159,5.562,5.114,P＜0.01].Conclusion Inhibition of SEPTIN9 gene methyla-tion leads to re-expression of SEPTIN9 gene in oesophageal cancer cells,thereby inhibiting proliferation,migration and invasion of oesophageal cancer cells,and promoting cell apoptosis.
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