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

English Keywords:Spinal cord injury  Neuron  miR-21  Apoptosis  FASL

English Abstract:

  【Abstract】 Objectives: To explore the anti-apoptotic role of micro RNA-21(miR-21) in neurons after ischemia 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 cells, 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 transfection, 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<0.05), 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.