Investigation Of The Effect Of P14 Promoter Aberrant Methylation On The Biological Function Of Human Lung Cancer Cells

Objective: In recent years,lung cancer has become the most common respiratory malignancy,and it has the highest mortality rate(26%)among other cancers.According to the pathological types,lung cancer could be divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).P14 ARF gene locus is on the short arm 2region 1 of human chromosome 9(9p21),and it is a tumor suppressor gene.The methylation status of the p14 ARF gene promoter region is significantly different between lung cancer tissues and paracancerous tissues.So,it suggests that the abnormal methylation of p14 ARF gene promoter region is related to the occurrence and development of lung cancer.Our study is to investigate the effect of p14 gene promoter aberrant methylation on the biological function of human lung adenocarcinoma cells.Materials and Methods: We used nested methylation-specific PCR(NMSP)to detect the methylation status of p14 ARF promoter region in human lung cancer cell line SPCA1 and human normal bronchial epithelial cell line BEAS2 B.According to cell type and whether treated with 5-aza-2’-deoxycytidine(5-Aza),cells were divided into 4groups : SPCA1 5-Aza treatment group,SPCA1 control group,BEAS2 B 5-Aza treatment group,BEAS2 B control group.There were 3 duplicate wells in each group.The expression of p14 gene mRNA was detected by Quantitative real-time PCR(qRT-PCR).The expression of ARF protein in each group was detected by Western blot.Apoptosis detected by flow cytometry(FCM),which was used to determine whether the apoptosis of cancer cells was promoted after demethylation,and CCK8 was used for cell viability.Results: NMSP detected abnormal methylation status in the p14 promoter region of SPCA1 cells,while there was no methylation status in BEAS2 B cells.According to qRT-PCR and Western blot analysis,p14 mRNA and ARF protein were not found in SPCA1 cell line which expressed in BEAS2 B cell line.We compared the SPCA1 cell line5-Aza group with the control group and the BEAS2 B cell line 5-Aza group and its control group.After removed the aberrant methylation of the p14 gene promoter region by 5-Aza,we detected the p14 mRNA in SPCA1 cells which was increased 0.63-fold(p<0.05)than control group.Western blot shows that the expression of ARF protein was restored after removed the aberrant methylation of the p14 gene promoter region.The apoptosis rate ofthe experimental group was significantly increased(experimental group:22.36±2.04%,control group: 10.96±0.63%,P<0.05).Also,cell proliferation inhibition rate increased significantly(the apoptosis rate was 39%,40% after 24 h,48h,72 h,96h,respectively).42% and 52%).There was no changes in ARF protein expression,mRNA,apoptosis rate and cell proliferation inhibition rate in BEAS2 B cell line(p>0.05).Conclusions: Abnormal methylation of the p14 ARF promoter region plays an important role in the development of lung cancer cells,which affects p14 gene expression and leads to the inability to synthesize p14 mRNA and ARF protein,which inhibits lung cancer cell apoptosis and accelerates its proliferation.It may become a target for the diagnosis and therapy of lung cancer.It also provides a direction for follow-up research and a possible solution for the study of combined treatment of lung cancer.