UPF1甲基化和miR-744-5p/CCND1在甲状腺乳头状癌中的作用机制研究

摘要 目的：探讨UPF1甲基化和miR-744-5p/CCND1在甲状腺乳头状癌中的作用机制研究。方法：将人甲状腺乳头状癌细胞株TCP-1和正常甲状腺上皮细胞Nthy-ori-3分别用去甲基化试剂5-Aza-CdR进行干预，分别在干预前后采用甲基化特异性PCR技术检测UPF1基因甲基化变化，采用Western-Blotting 检测干预UPF1、DNMT1、miR-744-5p、CCND1蛋白相对表达，采用transwell细胞侵袭实验检测细胞侵袭情况。结果：PCR扩增显示，UPF1基因在Nthy-ori-3组仅出现非甲基化引物扩增条带（U条带），在TCP-1组仅出现甲基化引物扩增条带（M条带）。经5-Aza-Cdr作用后，UPF1基因甲基化扩增条带减少，甲基化表达降低。各组UPF1、DNMT1、miR-744-5p、CCND1蛋白相对表达差异具有统计学意义（P＜0.05）。与Nthy-ori-3组比较，TCP-1组DNMT1、UPF1蛋白相对表达明显提高，miR-744-5p、CCND1蛋白相对表达明显降低（P＜0.05）；与TCP-1组比较，TCP-1干预组DNMT1、UPF1蛋白相对表达明显降低，miR-744-5p、CCND1蛋白相对表达明显提高（P＜0.05）。与TCP-1组比较，TCP-1干预组细胞侵袭、迁移数量明显减少（P＜0.05）。结论：UPF1甲基化存在于甲状腺乳头状癌中，UPF1基因甲基化的表达缺失可能抑制miR-744-5p/CCND1轴，在甲状腺乳头状癌发生发展中发挥关键作用。

Mechanism of UPF1 Methylation and miR-744-5p/CCND1 in Papillary Thyroid Carcinoma

ABSTRACT Objective: To investigate the mechanism of UPF1 methylation and miR-744-5p/CCND1 in papillary thyroid cancer. Methods: The human papillary thyroid cancer cell line TCP-1 and normal thyroid epithelial cell Nthy ori-3 were intervened with demethylation reagent 5-Aza CdR, respectively. Methylation specific PCR technology was used to detect the methylation changes of UPF1 gene before and after the intervention, and Western blotting was used to detect the relative expression of intervention UPF1, DNMT1, miR-744-5p, and CCND1 proteins. Results: PCR amplification showed that the UPF1 gene only showed non methylated primer amplification bands (U bands) in the Nthy ori-3 group, and only methylated primer amplification bands (M bands) in the TCP-1 group. After the action of 5-Aza-Cdr, the methylation amplification bands of UPF1 gene decreased, and the methylation expression decreased. There was a statistically significant difference in the relative expression of UPF1, DNMT1, miR-744-5p, and CCND1 proteins among each group (P<0.05). Compared with the Nthy ori-3 group, the relative expression of DNMT1 and UPF1 proteins in the TCP-1 group was significantly increased, while the relative expression of miR-744-5p and CCND1 proteins was significantly reduced (P<0.05); Compared with the TCP-1 group, the relative expression of DNMT1 and UPF1 proteins in the TCP-1 intervention group was significantly reduced, while the relative expression of miR-744-5p and CCND1 proteins was significantly increased (P<0.05). Compared with the TCP-1 group, the number of cell invasion and migration in the TCP-1 intervention group was significantly reduced (P<0.05). Conclusion: UPF1 methylation is present in papillary thyroid cancer, and the expression loss of UPF1 gene methylation may inhibit the miR-744-5p/CCND1 axis, playing a key role in the occurrence and development of papillary thyroid cancer.