摘要
目的探讨mmu-miR-672-5p调控大鼠胎盘绒毛膜滋养层细胞凋亡的分子机制。方法过表达或敲低mmu-miR-672-5p后,检测大鼠胎盘绒毛膜滋养层细胞的凋亡水平。过表达或敲低mmu-miR-672-5p后,通过RNA-seq检测mmu-miR-672-5p调控的基因。设定阈值后,通过siRNA敲低相关基因后,检测大鼠胎盘绒毛膜滋养层细胞的凋亡水平。通过荧光素酶报告实验验证mmu-miR-672-5p是否靶向此基因。过表达此基因后,通过免疫印迹检测CLEAVED-CAS3的表达水平,以及大鼠胎盘绒毛膜滋养层细胞的凋亡水平。结果过表达mmu-miR-672-5p后,大鼠胎盘绒毛膜滋养层细胞的凋亡水平下降(P<0.05);敲低mmu-miR-672-5p后,大鼠胎盘绒毛膜滋养层细胞的凋亡水平上升(P<0.05)。分别敲低和过表达mmu-miR-672-5p后,高通量测序发现大鼠胎盘绒毛膜滋养层细胞中多种基因的表达被调节,包括Zfp229、Zfp503、Slc4a5、Stk24、Tmem106b、Bax、Adgrb3、Tmem63b、Pmp22、Mroh2a、Dsn1、Pramef25、Naaladl2、Clk2、Stx16、Usp28、Clint1、Jph4、Msl2、Krtap8-1、Pkp2、Mllt3、Rai14。敲低上述基因后,发现仅敲低Bax时,大鼠胎盘绒毛膜滋养层细胞的凋亡水平下降(P<0.05)。过表达Bax后,CLEAVED-CAS3的表达水平上升,大鼠胎盘绒毛膜滋养层细胞的凋亡水平上升(P<0.05)。过表达mmu-miR-672-5p后,Bax的mRNA和蛋白水平均下降(P<0.05);敲低mmu-miR-672-5p后,Bax的mRNA和蛋白水平均上升(P<0.05)。此外,mmumiR-672-5p靶向Bax的3端非编码区(P<0.05)。同时过表达mmu-miR-672-5p和Bax后,大鼠胎盘绒毛膜滋养层细胞的凋亡水平无显著变化(P>0.05);同时敲低mmu-miR-672-5p和Bax后,发现大鼠胎盘绒毛膜滋养层细胞的凋亡水平无显著变化(P>0.05)。结论mmu-miR-672-5p通过靶向Bax的3端非编码区,降低了Bax的表达水平,随后抑制了大鼠胎盘绒毛膜滋养层细胞的凋亡。
Objective To investigate the molecular mechanism of mmu-miR-672-5p in regulating apoptosis of placental chorionic trophoblast cells in rat.Methods Apoptosis of placental chorionic trophoblast cells was detected after overexpression or knockdown of mmu-miR-672-5p.After overexpression or knockdown of mmu-miR-672-5p,the expression of genes regulated by mmu-miR-672-5p was detected by RNA-seq.After setting the threshold and knocking down relevant genes by siRNA,the level of placental chorionic trophoblast cell apoptosis was determined.mmu-miR-672-5p targeting particular genes was verified by luciferase reporter assays.After overexpression of the gene of interest,the expression level of cleaved caspase 3 and the level of placental chorionic trophoblast cell apoptosis was determined by western blotting.Results After overexpression of mmu-miR-672-5p,the level of placental chorionic trophoblast cell apoptosis decreased(P<0.05).After mmu-miR-672-5p was knocked down,the level of placental chorionic trophoblast cell apoptosis was increased(P<0.05).After knockdown or overexpression of mmu-miR-672-5p,high-throughput sequencing reveled the differential regulation of multiple genes in placental chorionic trophoblast cells,including Zfp229、Zfp503、Slc4a5、Stk24、Tmem106b、Bax、Adgrb3、Tmem63b、Pmp22、Mroh2a、Dsn1、Pramef25、Naaladl2、Clk2、Stx16、Usp28、Clint1、Jph4、Msl2、Krtap8-1、Pkp2、Mllt3、Rai14 After knocking down the above genes,only knockdown of Bax caused a decrease in the level of placental chorionic trophoblast cell apoptosis(P<0.05).After overexpression of Bax,the level of cleaved caspase 3 and the level of the placental chorionic trophoblast cell apoptosis increased(P<0.05).After overexpression of mmu-miR-672-5p,both the mRNA and protein levels of Bax decreased(P<0.05).After mmu-miR-672-5p was knocked down,both mRNA and protein levels of Bax increased(P<0.05).In addition,mmu-miR-672-5p targeted the 3′-non-coding region of Bax(P<0.05).After simultaneous overexpression of mmu-miR-672-5p and Bax,there was no significant change in the level of placental chorionic trophoblast cell apoptosis(P>0.05).After simultaneously knocking down mmu-miR-672-5p and Bax,there was no significant change in the level of placental chorionic trophoblast cell apoptosis(P>0.05).Conclusions mmu-miR-672-5p decreased Bax expression by targeting its 3′-non-coding region,and subsequently inhibited the apoptosis of placental chorionic trophoblast cells.
作者
陈琳
周知
马宁
周璟
CHEN Lin;ZHOU Zhi;MA Ning;ZHOU Jing(Hainan Provincial Medical Center for Women and Children Reproductive Medicine,Haikou 570206,China)
出处
《中国比较医学杂志》
CAS
北大核心
2021年第4期70-76,共7页
Chinese Journal of Comparative Medicine
基金
海南省重点研发计划(ZDYF2017086)。
