知识与财富的链接
目的 探讨组蛋白去乙酰化酶3（histone deacetylase 3, HDAC3）调节miR-625/组蛋白伴侣抗沉默功能蛋白1B（anti-silencing function 1B, ASF1B）轴对乳腺癌（breast cancer,BC）细胞焦亡的影响及其作用机制。 方法 采用qRT-PCR法检测HDAC3、miR-625和ASF1B在乳腺癌（breast cancer,BC）组织和癌旁正常组织、BC细胞系（T47D、MCF7和MDA-MB-231）和人正常乳腺上皮细胞MCF-10A中的表达水平。采用Western blot实验检测细胞焦亡相关蛋白NLRP3、Caspase-1和GSDMD的表达水平。ELISA法检测焦亡相关炎性因子IL-18和IL-1β的表达水平。ChIP实验用于测定HDAC3与miR-625的互作用关系。双荧光素酶报告实验验证miR-625和ASF1B的靶向关系。 结果 与癌旁正常组织和MCF-10A细胞比较,BC组织和细胞中的HDAC3和ASF1B表达升高,miR-625表达降低（均 P<0.05）。与si-NC组比较,si-HDAC3组细胞NLRP3、Caspase-1和GSDMD的蛋白表达水平升高,细胞培养上清液中IL-18和IL-1β的浓度升高（均 P<0.05）。HDAC3通过结合miR-625的启动子区域抑制其表达（ P<0.05）。与si-HDAC3+miR-NC组比较,si-HDAC3+miR-625 inhibitor组细胞中NLRP3、Caspase-1、GSDMD、IL-18和IL-1β的表达减少（均 P<0.05）。ASF1B被证实为miR-625的靶基因,si-HDAC3+pcDNA3.1-ASF1B组细胞中的焦亡相关因子水平显著低于si-HDAC3+pcDNA3.1-NC组。 结论 HDAC3通过抑制miR-625上调ASF1B的表达,进而抑制BC细胞焦亡。
Objective To investigate the effects of histone deacetylase 3 (HDAC3) on the pyroptosis of breast cancer (BC) cells via regulating miR-625/anti-silencing function 1B (ASF1B) and its mechanism. Methods The expression level of HDAC3, miR-625 and ASF1B in BC tissue, adjacent normal tissue, BC cell lines (T47D, MCF7 and MDA-MB-231) and human normal breast epithelial cell MCF-10A was detected by qRT-PCR. The expression level of cell pyroptosis related protein NLRP3, Caspase-1 and GSDMD was detected by Western blot. The expression level of IL-18 and IL-1βwere detected by ELISA. ChIP experiment was used to determine the interaction between HDAC3 and miR-625. The dual luciferase reporter assay was used to verifiy the targeted regulation between miR-625 and ASF1B. Results Compared with adjacent normal tissue and MCF-10A cells, the expression of HDAC3 and ASF1B was increased and the expression of miR-625 was decreased in BC tissue and cells (all P<0.05) . Compared with si-NC group, the protein expression level of NLRP3, Caspase-1 and GSDMD in si-HDAC3 group was increased, and the concentration of IL-18 and IL-1β in cell culture supernatant was increased (all P<0.05) . HDAC3 inhibited the expression of miR-625 by binding to the promoter region of miR-625 ( P<0.05) . Compared with si-HDAC3+miR-NC group, The expression of NLRP3, Caspase-1, GSDMD, IL-18 and IL-1β in si-HDAC3+miR-625 inhibitor group was decreased (all P<0.05) . ASF1B was confirmed as a target gene of miR-625, the level of pyroptosis related factors in si-HDAC3+pcDNA3.1-ASF1B group was significantly lower than that in si-HDAC3 + pcDNA3.1-NC group. Conclusion HDAC3 up regulates the expression of ASF1B by inhibiting miR-625, and then inhibits BC cell pyroptosis.
