摘要
目的探讨帕金森病(PD)中含有Tudor和KH结构域蛋白(TDRKH)对长散布核元件-1(LINE-1)活性与细胞凋亡的调控作用。方法利用GEO2R筛选PD患者(PD组)和健康对照者(HC组)黑质区差异表达基因,并与PIWI相互作用RNA(piRNA)相关基因取交集。采用1-甲基-4-苯基吡啶离子(MPP^(+))处理SH-SY5Y细胞构建PD模型。通过慢病毒介导的特异性shRNA稳定敲低TDRKH。使用RT-qPCR和Western blot法检测TDRKH及LINE-1的mRNA和蛋白表达水平;流式细胞术分析shCtrl+MPP^(+)组与shTDRKH+MPP^(+)组细胞凋亡情况。结果生物信息学分析发现,PD组与HC组有两个基因显著重叠:HENMT1和TDRKH。与0 mmol/L组比较,随着MPP^(+)浓度的升高,TDRKH mRNA及蛋白表达量下降(P<0.01)。与shCtrl组比较,shTDRKH组LINE-1各区域的转录本(5’UTR、ORF1、ORF2)及ORF1p蛋白表达均升高(P<0.01)。与shCtrl+MPP^(+)组比较,shTDRKH+MPP^(+)组BCL-2/BAX比值下降、Cleaved Caspase-3表达升高及凋亡率增加(P<0.01)。结论在PD中,TDRKH表达下调可能通过削弱piRNA通路对转座子的抑制功能,导致LINE-1异常激活,进而诱发多巴胺能神经元凋亡。
Objective To investigate the regulatory role of Tudor and KH domain-containing protein(TDRKH)in long interspersed nuclear element-1(LINE-1)activity and cell apoptosis in Parkinson’s disease(PD).Methods GEO2R was used to screen differentially expressed genes in the substantia nigra between PD patients(PD group)and healthy controls(HC group),which were then intersected with PIWI-interacting RNA(piRNA)-related genes.SH-SY5Y cells were treated with 1-Methyl-4-phenylpyridinium(MPP^(+))to establish a PD cell model.Lentivirus-mediated stable knockdown of TDRKH was performed using the specific shRNA.RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of TDRKH and LINE-1.Flow cytometry was used to analyze cell apoptosis in the shCtrl+MPP^(+)group and shTDRKH+MPP^(+)group.Results Bioinformatics analysis revealed that there were two genes that were significantly overlapping between the PD group and the HC group:HENMT1 and TDRKH.Compared with the 0 mmol/L group,as the concentration of MPP^(+)increased,the expression levels of TDRKH mRNA and protein decreased(P<0.01).Compared with the shCtrl group,the shTDRKH group showed significantly increased expression of LINE-1 transcripts in various regions(5’UTR,ORF1,ORF2)and ORF1p protein(P<0.01).Compared with the shCtrl+MPP⁺group,the shTDRKH+MPP⁺group exhibited a decreased BCL-2/BAX ratio,increased Cleaved Caspase-3 expression,and elevated apoptotic rate detected by flow cytometry(P<0.01).Conclusion In PD,the downregulation of TDRKH may weaken the inhibitory function of the piRNA pathway on transposons,leading to abnormal activation of LINE-1 and subsequent induction of dopaminergic neuron apoptosis.
作者
刘博语
时晓龙
杨倩
LIU Boyu;SHI Xiaolong;YANG Qian(Department of Experimental Surgery,Tangdu Hospital,Air Force Medical University,Shaanxi Province,Xi’an 710038,China)
出处
《中国医药导报》
2026年第6期59-65,共7页
China Medical Herald
基金
陕西省重点研发计划项目(2023-ZDLSF-52)。
