Zand Hamid, Hosseini Seyed Ahmad, Cheraghpour Makan, Alipour Meysam, Sedaghat Fatemeh
Department of Cellular and Molecular Nutrition, Faculty of Nutrition Science and Food Technology, National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Adv Biomed Res. 2024 Nov 30;13:114. doi: 10.4103/abr.abr_75_24. eCollection 2024.
Acquisition of stem-like properties requires overcoming the epigenetic barrier of differentiation and re-expression of several genes involved in stemness and the cell cycle. DNA methylation is the classic epigenetic mechanism for de/differentiation. The writers and erasers of DNA methylation are not site-specific enzymes for altering specific gene methylation. Thus, the aim of the present study is investigation of the interaction of ten eleven translocations (TETs) with nuclear factor kappa B (NF-κB) in hypomethylation of stemness genes.
This experimental study was performed on HT-29 cells as human colorectal cancer cell lines. The interaction between TETs and DNA-methyltransferases 3 beta (DNMT3s) with p65 was achieved by coimmunoprecipitation. TETs were knocked down using siRNA, and the efficacy was analyzed by reverse-transcriptase polymerase chain reaction. The promoter methylation status of the target genes (NANOG, MYC) was determined by the methylation-sensitive high-resolution melting method.
TET3 and DNMT3b functionally interacted with p65 in samples through 25 ng/ml TNF-α treatment for 48 h in HT-29 cells. Transfection with siRNA significantly decreased the expression of TET enzymes after 72 h. Interestingly, treatment with TET siRNAs enhanced methylation of MYC and NANOG genes in samples with 25 ng/ml TNF-α treatment for 72 h in HT-29 cells. Moreover, methylation effects of TET3 were stronger than those of TET1 and TET2.
These results suggest that inflammation may alter the methylation status of genes required for stemness and predispose the cells to neoplastic alterations.
获得干细胞样特性需要克服分化的表观遗传障碍，并重新表达一些参与干性和细胞周期的基因。DNA甲基化是去分化/分化的经典表观遗传机制。DNA甲基化的写入酶和擦除酶不是用于改变特定基因甲基化的位点特异性酶。因此，本研究的目的是探讨十 - 十一易位（TETs）与核因子κB（NF-κB）在干性基因低甲基化中的相互作用。
本实验研究以人结肠癌细胞系HT-29细胞为研究对象。通过免疫共沉淀实现TETs与DNA甲基转移酶3β（DNMT3s）与p65之间的相互作用。使用小干扰RNA（siRNA）敲低TETs，并通过逆转录聚合酶链反应分析其效果。通过甲基化敏感的高分辨率熔解方法测定靶基因（NANOG、MYC）的启动子甲基化状态。
在HT-29细胞中，通过25 ng/ml肿瘤坏死因子-α（TNF-α）处理48小时，TET3和DNMT3b在样品中与p65发生功能相互作用。转染siRNA 72小时后显著降低了TET酶的表达。有趣的是，在HT-29细胞中，用TET siRNAs处理增强了在25 ng/ml TNF-α处理72小时的样品中MYC和NANOG基因的甲基化。此外，TET3的甲基化作用比TET1和TET2更强。
这些结果表明，炎症可能会改变干性所需基因的甲基化状态，并使细胞易发生肿瘤性改变。
