| ObjectiveTo explore the relationship between the methylation of MAGE-D4 gene promoter and its mRNA expression in non-small cell lung cancer(NSCLC).To analyze the effect of MAGE-D4 up-regulated by demethylation on the biological behavior of NSCLC cells in vitro and clarify the role and significance of MAGED4 in the occurrence and development of NSCLC.Methods1.The promoter region and CpG islands of MAGE-D4 gene were predicted by bioinformatics,and methylation specific PCR(MSP)primers were designed by methprimer software.2.The mRNA expression of MAGE-D4 in 52 NSCLC tissues was analyzed by qRT-PCR.3.The methylation status of MAGE-D4 promoter in 52 NSCLC tissues was detected by MSP.The relationship between promoter methylation of MAGE-D4 and its mRNA expression was analyzed.The correlation between promoter methylation of MAGE-D4 and clinicopathological parameters of NSCLC patients was discussed.4.qRT-PCR and BSP were used to detect the effect of MAGE-D4 promoter demethylation on its mRNA expression in NSCLC cells(A549,H460 and H1299)after treatment with methyltransferase inhibitor(5-AZA-CdR).The key methylation sites that regulate gene expression in the MAGE-D4 promoter region were further clarified.5.The effects of MAGE-D4 on the proliferation,apoptosis,cell cycle,migration and invasion of A549 and H460 cells were analyzed by MTT assay,flow cytometry,transwell experiment and cell scratch experiment,in order to analyze the effect of MAGE-D4 up-regulated by demethylation on the occurrence and development of NSCLC.Results1.The promoter region of MAGE-D4 is located at-173bp～+212bp(X chromosome 52184650～52185035),and the length is 385bp.The CpG island in the MAGE-D4 promoter region is located at-220bp～+242bp(X chromosome 52181823～52182285)which contains 37 CpG sites,and the length is 462bp.Four pairs of primers were designed by Methprimer for MSP amplification(MSP-1 and MSP-2)and BSP amplification(BSP-1 and BSP-2),respectively.2.The expression of MAGE-D4 mRNA in NSCLC tissues was 6.33±4.80,which was significantly higher than that of adjacent tissues(2.08±0.98),P=0.000.3.In MSP-S primer group,the methylation frequencies of MAGE-D4 promoter were 34.61%in cancer tissues and 67.31%in adjacent tissues(P=0.000).The expression level of MAGE-D4 mRNA in methylation samples of cancer tissues was significantly lower than that in unmethylation samples(3.03±1.55vs9.86±3.95,r=-0.832,P=0.000);In MSP-L primer group,the methylation frequencies of MAGE-D4 promoter were 34.61%and 73.08%in NSCLC tissues and adjacent tissues,respectively(P=0.000).The expression level of MAGE-D4 mRNA in methylation samples of cancer tissues was significantly lower than that in unmethylation samples(2.86±1.52 vs 9.76±3.99,r=-0.892,P=0.000).Correlation analysis showed that the methylation of MAGE-D4 was negatively correlated with its mRNA expression.Hypomethylation of MAGE-D4 promoter was significantly associated with tumor diameter(P<0.01),but not significantly correlated with other clinicopathological parameters.4.After treatment with 5-Aza-CdR,the expression levels of MAGE-D4 mRNA were significantly increased,while the total methylation frequencies were decreased in three NSCLC cell lines.The expression level of MAGE-D4 mRNA in A549 cell increased from 0.94±0.40 to 5.86±0.35,and the methylation frequency of its promoter region decreased from 29.3%to 21.8%(P=0.000,r=0.607).The expression level of MAGE-D4 mRNA in H460 cell increased from 10.05±0.11 to 28.55±0.86,and the methylation frequency decreased from 27.9%to 7.7%(P=0.000,r=-0.893).The expression level of MAGE-D4 mRNA in H1299 cell increased from 5.42±1.27 to 10.97±0.28,and the methylation frequency decreased from 4.3%to 3.2%(P=0.000,r=-0.103).The results showed that demethylation of MAGE-D4 promoter could up-regulate the gene expression of transcription level.In H460 cell,the effect of demethylation regulation in BSP-2 region was more significant than that in BSP-1 region(P<0.05),and the key site of MAGE-D4 methylation regulation may be 152 site in BSP-1 region.5.The expression of MAGE-D4 was up-regulated by 5-AZA-CdR demethylation,which promoted the proliferation,migration and invasion of A549 and H460 cells.The up-regulation of MAGE-D4 expression inhibited the apoptosis(P<0.005)and promoted cell cycle from G1/G0 to G2/M of H460 cell(P=0.000).The results showed that promoter demethylation could up-regulate the expression of MAGE-D4,which promoted the malignant biological behavior of NSCLC cells in vitro.ConclusionMAGE-D4 mRNA is highly expressed in NSCLC tissues and cell lines,and its expression may be regulated by the methylation of promoter region.The key regulatory region of MAGE-D4 promoter region is BSP-2,and the key methylation site is 152 site of BSP-1.The expression of MAGE-D4 was upregulated by 5-AZA-CdR demethylation,which promoted the proliferation,migration and invasion of NSCLC cells.Apoptosis of NSCLC cells was inhibited and cell cycle was accelerated,which promoted the development and metastasis of NSCLC. |
PDF Full Text Request