坐骨神经损伤后长链非编码RNA的聚类分析及长链非编码RNA MX1对大鼠雪旺细胞迁移、增殖能力的影响

目的 探讨坐骨神经损伤后长链非编码RNA（LncRNA）差异表达的基因,以及LncRNA MX1对大鼠雪旺细胞迁移、增殖能力的影响。方法 选取10周龄无特定病原体级雄性SD大鼠24只,随机分为0 d（T0）、3 d（T1）、7 d（T2）和14 d（T3）4组,每组6只,建立坐骨神经损伤动物模型。分别于模型建立后第0、3、7、14天取相应组大鼠坐骨神经损伤处的残端组织提取RNA,制备RNA基因芯片进行Heatmap聚类分析,筛选出各个时间点发生差异表达的基因,并选择其中差异表达显著的基因LncRNA MX1。培养iCell-r030大鼠雪旺细胞,分为对照组、Control siRNA组（siRNA组）和LncRNA MX1 siRNA组（siRNA-MX1组）,使用相应试剂进行转染。转染后采用实时荧光定量聚合酶链反应（qRT-PCR）检测3组雪旺细胞的LncRNA MX1mRNA表达情况,采用Transwell小室检测雪旺细胞的迁移能力,采用5-乙炔基-2'-脱氧尿苷（EDU）实验检测雪旺细胞的增殖能力。结果 各组大鼠均造模成功,实验过程中无大鼠死亡。大鼠损伤坐骨神经组织LncRNA差异基因Heatmap聚类分析的热图显示,与T0组比：T1组共3 066个LncRNA基因发生差异性表达,其中1 634个LncRNA基因表达上调,1 432个LncRNA基因表达下调;T2组共2 498个LncRNA基因发生差异性表达,其中1 634个LncRNA基因表达上调,864个LncRNA基因表达下调;T3组3 567个LncRNA基因发生差异性表达,其中有1 643个LncRNA基因表达上调,1 924个 LncRNA基因表达下调。各组差异表达的LncRNA基因中LncRNA MX1的差异倍数数值较大,差异表达显著。大鼠雪旺细胞qRT-PCR结果显示,转染LncRNA MX1 siRNA后,siRNA-MX1组LncRNA MX1的相对表达量为1.0±0.2,低于对照组的2.3±0.2和siRNA组的2.2±0.2,差异有统计学意义（F=78.47,P<0.001）;而对照组和siRNA组组间差异无统计学意义（P>0.05）。迁移、增殖试验结果显示,siRNA-MX1组细胞迁移数量为（24.1±4.2）个、EDU阳性细胞与DAPI阳性细胞的比率为27.5%±2.8%,低于对照组的（50.3±7.8）个、44.1%±7.2%和siRNA组的（49.2±6.2）个、41.8%±7.0%,差异均有统计学意义（F=93.15、121.26,P值均<0.001）;而对照组和siRNA组间差异均无统计学意义（P值均>0.05）。结论 大鼠坐骨神经损伤后LncRNA MX1差异表达显著,下调LncRNA MX1在大鼠雪旺细胞中的表达可显著降低细胞的迁移、增殖能力。

Cluster analysis of long noncoding RNA after sciatic nerve injury and effect of long noncoding RNA MX1 on the migration and proliferation of rat Schwann cells

Objective This study was performed to investigate the differentially expressed genes of long noncoding RNA after sciatic nerve injury and effects of long noncoding RNA MX1 on the migration and proliferation of rat Schwann cells. Methods Twenty-four 10-week-old male SD rats without specific pathogens were randomly divided into 4 groups (6 rats per group), namely, 0 d (T0), 3 d (T1), 7 d (T2), and 14 d (T3). The rats were selected to establish animal models of sciatic nerve injury. Animal models of sciatic nerve injury were established. RNA was extracted from the residual tissue of the injured sciatic nerve on days 0, 3, 7, and 14 after surgery according to the time point of the different groups. RNA gene chips were prepared, and heatmap clustering analysis was performed to screen out the differentially expressed genes at each time point. The gene LncRNA MX1 with significant differentially expressed genes was selected. Icell-r030 rat Schwann cells were cultured and divided into the control group, control siRNA group(si RNA group), and LncRNA MX1 siRNA group(si RNA-MX1 group). Transfection was performed with corresponding reagents. After transfection, the expression of LncRNA MX1mRNA in the three groups of Schwann cells was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Migration ability of the Schwann cells was detected by Transwell chamber, and the proliferation ability of the Schwann cells was detected by 5-ethynyl-2'-deoxyuridine (EDU) assay. Results The rats in each group were modeled successfully, and no animals died during the experiment. Compared with the T0 group, a total of 3 066 differentially expressed LncRNA genes were found in the T1 group, including 1 634 upregulated and 1 432 downregulated LncRNA genes. A total of 2 498 LncRNA genes were differentially expressed in the T2 group, including 1 634 upregulated and 864 downregulated LncRNA genes. In the T3 group, 3 567 LncRNA genes were differentially expressed, among which 1 643 LncRNA genes were upregulated, and 1 924 were downregulated. Fold change value of LncRNA MX1 in the differentially expressed LncRNA genes in each group was larger, and the differentially expressed LncRNA MX1 was significant. qRT-PCR results of the rat Schwann cells showed that the relative expression level of LncRNA MX1 in the siRNA-MX1 group was 1.0±0.2 after transfection with LncRNA MX1 siRNA, which was lower than those of the control group (2.3±0.2) and siRNA group (2.2±0.2). The difference was statistically significant (F=78.47, P<0.001). No significant difference was found between the control group and the siRNA group (P>0.05). The results of migration and proliferation experiments showed that the number of cell migration in the siRNA-MX1 group was 24.1±4.2. The ratio of EDU positive cells to DAPI positive cells was 27.5%±2.8%, which was lower than those of the control group (50.3±7.8; 44.1%±7.2%) and siRNA group (49.2±6.2; 41.8%±7.0%). The differences were statistically significant (F=93.15, 121.26, all P values<0.001). No significant difference was found between the control group and the siRNA group (all P values>0.05). Conclusion The differential expression of LncRNA MX1 after sciatic nerve injury in rats is significant. Downregulation of LncRNA MX1 expression in the rat Schwann cells can significantly reduce cell migration and proliferation.