微小RNA-21对脊髓缺血再灌注损伤后神经元细胞凋亡的影响

The anti-apoptosis effect of micro RNA-21 on the neurons after ischemia reperfusion injury in spinal cord

目的 :探讨微小RNA-21(miR-21)能否抑制FASL的表达进而减少脊髓缺血再灌注损伤后神经元细胞的凋亡。方法:通过慢病毒载体转染PC-12细胞,测定最佳的感染复数(MOI)。分别转染不同的慢病毒载体建立并验证上调miR-21、下调miR-21和对照的PC-12细胞体系,经过氧糖剥夺以及复糖复氧处理模拟脊髓损伤后缺血再灌注损伤过程。利用Hoechst/PI染色技术检测上调以及下调miR-21对PC-12细胞凋亡情况的影响;利用q RT-PCR技术以及Western blot技术检测上调以及下调miR-21表达对PC-12细胞中FASL表达水平的影响。在293T细胞中,通过质粒转染技术分别建立miR-21/miR-21阴性对照和FASL野生型/FASL突变型/FASL阴性对照的共转染体系,利用双荧光素酶实验验证miR-21和FASL的靶向关系。结果:MOI=20为最佳的感染复数。在该条件下进行慢病毒转染72h后,荧光显微镜下可观测到绝大多数PC-12细胞中有明显的绿色荧光,通过q RT-PCR技术检测miR-21的水平发现:上调组的miR-21水平较对照组明显提高(P<0.05);但是下调组miR-21水平和对照组相比却没有明显变化(P>0.05)。转染三种不同慢病毒的PC-12细胞经过氧糖剥夺及复氧处理后,Hoechst/PI双染法检测结果证实上调组细胞凋亡较对照组明显减少(P<0.05),而下调组的细胞凋亡比例较对照增高(P<0.05)。另外,氧糖剥夺及复氧处理后,三组PC-12细胞中FASL表达水平均较处理前增高,而上调组的FASL水平较对照组均下降(P<0.05),下调组的FASL水平较对照组有所升高(P<0.05)。双荧光素酶实验结果发现miR-21在FASL野生型(3′UTR-WT)组间有显著差异(P<0.05),而miR-21在FASL对照组以及FASL突变型组间无显著差异(P>0.05)。结论 :miR-21能够通过靶向抑制FASL表达,减少脊髓缺血再灌注损伤后神经元的凋亡。

Objectives： To explore the anti-apoptotic role of micro RNA-21（miR-21） in neurons after is- chemia reperfusion injury via suppressing the expression of FASL. Methods： The lentiviral vectors were used to overexpress（＋miR-21 group） or inhibit（-miR-21 group） the expression of miR-21 in PC-12 ceils, and the best multiplicity of infection（MOI） was selected. Substantially, the cells were suppressed oxygen and glucose deprivation （OGD） and reoxygenation to imitate the process of spinal cord injury. The level of apoptosis was measured by Hoechst/PI double fluorescence staining. The qRT-PCR and Western blot technologies were used to detect the transcription and expression of FASL. In 293T cells, co-transfection system of miR-21/miR-21- NC（negative control） and FASL-WT（wild type）/FASL-MU（mutant）/FASL-NC was established via plasmid trans- fection, and the target relation of miR-21 and FASL was investigated by dual-luciferase reporter assay. Results： The best MOI was 20, when a large majority of PC-12 cells was detected with GFP green fluorescence under fluorescence microscope after lentiviral transfection for 72h. The results of qRT-PCR showed that the ＋ miR-21 group had higher level of miR-21, while the -miR-21 group had lower level of miR-21 compared to the control group （P〈0.05）. After subjected to OGD and reoxygenation, the results of Hoechst/PI revealed that PC-12 cells in the ＋miR-21 group had lower apoptotic rate, while cells in the -miR-21 group had higher apoptotic rate than that in the control group （P〈0.05）. In PC-12 cells, the expression of FASL after OGD was higher than the normal cells in all the three groups. The expression in the ＋miR-21 group was lower than that in the control group, which was higher in the -miR-21 group（P〈O.05）, no matter pre or post OGD. Besides, in 293T cells, there was significant difference of miR-21 in FASL wild type（P〈0.05）, while there was no difference of miR-21 in FASL negative control and mutant type. Conclusions： miR-21 is able to reduce neurons apoptosis via suppressing the expression of FASL, which is a direct target gene of miR-21.