Mechanism Of DAPK Gene Methylation And MAPK/ERK Signaling Pathway In Papillary Thyroid Carcinoma

Objective: To explore the mechanism of the tumor suppressor gene DAPK promoter methylation and MAPK / ERK signaling pathway in the occurrence and development of papillary thyroid carcinoma,and the therapeutic effect of the demethylation drug5-Aza-CdR on it,provide theoretical basis for targeted drug treatment of clinical papillary thyroid carcinoma.Methods The 10-mmol / L demethylated preparation 5-Aza-CdR was applied to human thyroid papillary carcinoma TPC-1 cell line and human normal thyroid Nthy-ori-3 cell line,using methylation-specific PCR technology(MSP)to observe the changes of methylation of DAPK gene before and after treatment,and to detect the mRNA and protein expression of DAPK,DNMTs,ERK1/2,p-ERK1/2 before and after treatment by using Real-Time PCR and Western-Blot technology.Results(1)Methylated primer amplified fragments did not appear in human normal thyroid cells,but methylated primer amplified fragments appeared in human thyroid papillary cancer cells.5-Aza-CdR was applied to human thyroid papillary cancer cells,DAPK methylation primer amplification fragments decreased,methylation expression decreased;(2)Real-time results showed that,compared with human normal thyroid Nthy-ori-3 cells,the mRNA expression levels of DNMT1,DNMT3 A,DNMT3B,ERK1/2 and p-ERK1/2 are higher;After treatment with 5-Aza-CdR for human thyroid papillary carcinoma cell line TPC-1,the mRNA expression of DAPK increased and the difference was statistically significant(P <0.05),while the mRNA expression of DNMT1,DNMT3 A,DNMT3B,ERK1/2,and p-ERK1/2 decreased,and the difference was statistically significant(P <0.05));(3)Western blot results showed that,compared with the human normal thyroid cell line Nthy-ori-3,the expression of DAPK gene in human thyroid papillary carcinoma cell line TPC-1 was very low,DNMT1,DNMT3 A,DNMT3B,ERK1/2,p-ERK1/2 have higher protein expression levels;after 5-Aza-CdR treatment,the protein expression of DAPK increased and the difference was statistically significant(P <0.05),while the expression of DNMT1,DNMT3 A,DNMT3B,ERK1/2,and p-ERK1/2 proteins decreased,the difference was statistically significant(P <0.05);DAPK was negatively correlated with mRNA and protein expression of ERK1/2 and p-ERK1/2.Conclusions(1)There is methylation of DAPK in human thyroid papillary carcinoma.5-Aza-CdR may restore the silent DAPK gene to normal function by regulating the mRNA and protein expression of DNMT1,DNMT3 A,DNMT3B;(2)the lack of gene methylation expression may lead to the excessive activation of MAPK/ERK signaling pathway,which may play a key role in the occurrence and development of papillary thyroid carcinoma;(3)5-Aza--CdR can reverse the DAPK methylation status may become a new drug for demethylation treatment of papillary thyroid carcinoma and provide a new treatment for papillary thyroid carcinoma.