摘要
神经管畸形(neural tube defects,NTDs)是一种由细胞增殖凋亡紊乱引起的先天缺陷性疾病。本室前期利用RNA-Seq技术,发现小鼠神经管畸形胚胎脑组织中DNA损伤诱导转录因子-4(DNA damage-inducible transcript 4,Ddit4)表达水平增加,但其在神经管畸形中的作用尚不清楚。本文拟探索Ddit4过表达对海马神经元HT-22细胞增殖和凋亡的影响及相关机制,从而为后续研究Ddit4在小鼠神经管畸形中的作用奠定基础。本研究首先根据小鼠Ddit4序列构建真核表达载体pEX-3-Ddit4,限制性酶切分析和测序结果表明pEX-3-Ddit4构建成功。qRT-PCR和Western印迹检测显示转染pEX-3-Ddit4后,HT-22细胞中DDIT4的表达水平明显增加(P<0.01)。CCK-8和Western印迹结果显示,Ddit4过表达导致HT-22细胞增殖下降(P<0.01)。流式细胞仪结果显示,转染pEX-3-Ddit4后,G0/G1期细胞比例增加、S期比例下降(P<0.01),细胞凋亡率增加(P<0.05)。Western印迹检测发现,转染pEX-3-Ddit4能够显著上调切割胱天蛋白酶3(cleaved caspase3)水平(P<0.05),表明Ddit4过表达促进HT-22细胞凋亡。细胞免疫荧光及Western印迹结果显示,过表达Ddit4显著降低细胞β-联蛋白(β-catenin)、T细胞因子4、细胞周期蛋白D1和C-myc的表达水平(P<0.05),核内β-联蛋白减少,提示Ddit4过表达抑制了Wnt/β-catenin信号通路。过表达Ddit4的同时加入Wnt/β-catenin信号通路激动剂LiCl处理细胞可逆转上述现象,表明Ddit4过表达可能通过Wnt/β-catenin信号通路对HT-22细胞发挥抗增殖和促凋亡作用。
Neural tube defects(NTDs)are congenital defect diseases caused by cell proliferation and apoptosis disorders.Using RNA-Seq assays,we found the increased expression of DNA damage-inducible transcript 4(Ddit4)in embryonic brain tissues from NTD fetuses.In this study,we intend to explore the effects of Ddit4 overexpression on the proliferation and apoptosis of HT-22 cells and related mechanisms to lay the foundation for the study of the role of Ddit4 in NTDs.According to the mouse Ddit4 sequence,we constructed the eukaryotic expression vector pEX-3-Ddit4.The results of restriction enzyme analysis and sequencing showed that the eukaryotic expression vector pEX-3-Ddit4 was successfully constructed.qRT-PCR and Western blotting results showed that the expression level of Ddit4 in HT-22 cells was significantly increased after transfection of PEX-3-Ddit4(P<0.01).CCK-8 and Western blotting results showed that Ddit4 overexpression decreased the proliferation of HT-22 cells(P<0.01).Flow cytometry showed that Ddit4 overexpression increased the proportion of cells in the G0/G1 phases(P<0.001)and decreased the proportion of cells in the S phase(P<0.01),in addition,the percentage of apoptosis cell was increased(P<0.05).Western blotting showed that pEX-3-Ddit4 transfection could significantly enhance the expression of cleaved caspases 3 in HT-22 cells(P<0.05),which indicates that Ddit4 overexpression promotes the apoptosis of HT-22 cells.Immunofluorescence staining and Western blotting showed Ddit4 overexpression downregulated the expression ofβ-catenin,TCF4,cyclin D1 and C-myc(P<0.01)and inhibited the accumulation ofβ-catenin in the nucleus,suggesting that Ddit4 overexpression inhibits the Wnt/β-catenin signaling pathway.The changes above can be reversed when pEX-3-Ddit4-transfected HT-22 cells were treated with LiCl,a Wnt/β-catenin pathway agonist,indicating that Ddit4 overexpression could inhibit proliferation and promote apoptosis of HT-22 cells by regulating the Wnt/β-catenin signaling pathway.
作者
王宇飞
孙雨晴
张凯丽
宋美燕
李佳琪
张娟
王磊
吴雪梅
赵虹
刘志贞
WANG Yu-Fei;SUN Yu-Qing;ZHANG Kai-Li;SONG Mei-Yan;LI Jia-Qi;ZHANG Juan;WANG Lei;WU Xue-Mei;ZHAO Hong;LIU Zhi-Zhen(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Shanxi Key Laboratory of Birth Defect and Cell Regeneration,Key Laboratory of Cellular Physiology(Shanxi Medical University),Ministry of Education,Taiyuan 030000,China;Department of Cell Biology and Genetics,Shanxi Medical University,Taiyuan 030000,China;Department of Neurology,General Hospital of Taiyuan Iron&Steel(Group)Co.,Ltd.,Taiyuan 030000,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2021年第7期927-936,共10页
Chinese Journal of Biochemistry and Molecular Biology
基金
山西省自然基金面上项目(No.201901D111184)
国家青年基金(No.81300487)
山西省“1331工程”重点学科建设计划经费项目(No.1331KSC)
山西省回国留学人员科研资助项目(2016-051)资助。
