MicroRNA-92a 对大鼠脊髓损伤后 运动功能恢复的影响

The mechanism of miR-92a promoting locomotor functional recovery in rats after spinal cord injury

摘要:

目的 分析microRNA-92a（miR-92a）对脊髓损伤大鼠运动功能恢复的影响及可能的作用机 制。方法 第一步，复制大鼠脊髓损伤模型，将30 只大鼠分为假手术组和脊髓损伤组，筛选目标miRNA。 第二步，分析miR-92a 对脊髓损伤大鼠的影响，将剩余54 只大鼠分为假手术组、对照组（脊髓损伤+miR- 92a mimics negative control）和实验组（脊髓损伤+miR-92a mimics）。采用BBB 评分评估大鼠运动功能， TUNEL 染色检测细胞凋亡，qRT-PCR 和Western blotting 检测相关基因和蛋白的表达，双荧光素酶验证人 第10 号染色体缺失的磷酸酶（PTEN）与miR-92a 的靶向关系。结果 ①脊髓损伤组与假手术组大鼠第1、 3、7、14、21 和28 天的BBB 评分在不同时间、不同组间及变化趋势上有差异（P <0.05），假手术组BBB 评 分高于脊髓损伤组（P <0.05）。脊髓损伤组与假手术组大鼠细胞凋亡率、Caspase-3、Bax、Bcl-2 比较，差异 有统计学意义（P <0.05）。脊髓损伤组与假手术组大鼠miR-92a、miR-132、miR-128、miR-107、miR- 202、miR-451、miR-17 表达水平比较，差异有统计学意义（P <0.05）。②假手术组、对照组、实验组大鼠 第7、14 和28 天的miR-92a 表达水平在不同时间、不同组间及变化趋势上有差异（P <0.05），实验组miR- 92a 表达水平最高（P <0.05）。假手术组、对照组、实验组大鼠第1、3、7、14、21 和28 天BBB 评分在不同 时间、不同组间及变化趋势上有差异（P <0.05），假手术组BBB 评分最高（P <0.05）。3 组大鼠细胞凋亡率、 Caspase-3、Bax、Bcl-2 比较，差异有统计学意义（P <0.05）。③对照组与实验组大鼠PTEN-WT 荧光活性比较， 差异有统计学意义（P <0.05）；两组PTEN-MUT 荧光活性比较，差异无统计学意义（P >0.05）。对照组与实 验组大鼠PTEN mRNA 和蛋白相对表达量比较，差异有统计学意义（P <0.05）。PTEN 与miR-92a 表达水平 呈负相关（P <0.05）。结论 大鼠脊髓损伤后miR-92a 表达下调。过表达miR-92a 可促进大鼠运动功能恢复， 抑制细胞凋亡。

Abstract:

Objective To investigate the effect of miR-92a on locomotor functional recovery in rats after spinal cord injury and explore the related mechanisms. Methods First, the model of spinal cord injury in rats was established to screen the target miRNA with 30 rats divided into sham operation group and spinal cord injury group. Second, the effects of miR-92a on spinal cord injury in rats were analyzed, where 54 rats were divided into sham operation group, control group (spinal cord injury + miR-92a mimics negative control) and experimental group (spinal cord injury + miR-92a mimics). Then BBB score was used to evaluate the motor function of rats, TUNEL staining was used to detect apoptosis, qRT-PCR and Western blotting were used to determine the expression of related genes and proteins, and dual luciferase was used to verify the targeting relationship between phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and miR-92a. Results ① The BBB scores of the rats in the spinal cord injury group and the sham operation group on the 1st, 3rd, 7th, 14th, 21st and 28th day were all different (P < 0.05), and the BBB scores in the sham operation group were higher than those in the spinal cord injury group (P < 0.05). There were significant differences in apoptosis rate, Caspase-3, Bax and Bcl-2 expression between the spinal cord injury group and the sham operation group (P < 0.05). The expression of miR-92a, miR-132, miR-128, mir-107, miR- 202, miR-451 and miR-17 in spinal cord injury group was significantly different from that in sham operation group (P < 0.05). ② The expression of miR-92a in sham operation group, control group and experimental group at 7th, 14th and 28th day was compared, and it was found to be different among groups and altered at different time points with distinct changing trends (P < 0.05), in which that of experimental group was the highest (P < 0.05). The BBB scores of these three groups on the 1st, 3rd, 7th, 14th, 21st and 28th day were also compared, and those of experimental group were the highest (P < 0.05), with the changing trend of BBB scores in three groups being statistically significant (P < 0.05). The apoptosis rate and the expression of Caspase-3, Bax and Bcl-2 in sham operated group, control group and experimental group were significantly different (P < 0.05). ③ The difference of PTEN-WT but not PTEN-MUT fluorescence intensity between the control group and the experimental group was statistically significant (PTEN-WT, P < 0.05; PTEN-MUT, P > 0.05 ). The expression of PTEN protein and mRNA in the control group was significantly higher than that in the experimental group (P < 0.05), and PTEN expression was negatively correlated with miR-92a expression (P < 0.05). Conclusions The expression of miR-92a was down-regulated after spinal cord injury, while overexpression of miR-92a could facilitate locomotor functional recovery in rats and suppress apoptosis.