At present, the functional recovery after nerve injury is not satisfactory in clinical practice. The aim of this study was to explore the molecular mechanism of miR-21 promoting Schwann cells (SC) proliferation and axon regeneration after peripheral nerve injury, providing a theoretical basis for injured nerve repair. Nerve injury models were constructed to determine the expression of miR-21 in the injured nerve by Quantitative Real-Time PCR (qRT-PCR). After miR-21 over-expression SC (mimic-miR-21) group, control SC (control-miR-21) group and blank SC (RSC96) group were constructed, SC proliferation was determined by CCK-8, cell cycle was analysed by flow cytometry, dorsal root ganglion neuron (DRGn) axon regeneration was observed after DRGn was cultured with SCs for 7 days, the expressions of TGFβI, TIMP3, EPHA4 as well as apoptosis-related proteins caspase-3 and caspase-9 were detected by qRT-PCR and Western blot in the three groups, respectively. Target genes were confirmed by dual-luciferase reporter gene assay. The expressions of TGFβI, TIMP3 and EPHA4 were assessed by immunofluorescence in vivo. qRT-PCR indicated that miR-21 expression was significantly higher in the model group than in the sham operation and blank groups. SC proliferation index (PI) was significantly higher, the apoptosis rate was significantly lower, the axon was significantly longer, and mRNA and protein expressions of TGFβI, TIMP3, EPHA4 as well as apoptosis-related proteins caspase-3 and caspase-9 were significantly lower in the mimic-miR-21 group than in the control-miR-21 and RSC96 groups. The double luciferase assay confirmed that TGFβI, TIMP3 and EPHA4 were potential target genes of miR-21. In vivo immunofluorescence also indicated that expressions of TGFβI, TIMP3, EPHA4 were lower in the mimic-miR-21 group than in the control-miR-21 and RSC96 groups. We conclude that during injured peripheral nerve repair, miRNA-21 plays an important role in promoting SC proliferation and axon regeneration by regulating TGFβI, TIMP3 and EPHA4 target genes.

目前，神经损伤后的功能恢复在临床上并不令人满意。这项研究的目的是探索miR-21促进周围神经损伤后雪旺细胞（SC）增殖和轴突再生的分子机制，为损伤的神经修复提供理论依据。构建神经损伤模型，以通过实时定量PCR（qRT-PCR）确定miR-21在受伤神经中的表达。构建miR-21过表达SC（mimic-miR-21）组，对照组SC（control-miR-21）组和空白SC（RSC96）组，用CCK-8测定SC增殖，分析细胞周期流式细胞仪检测SCs培养7d后，观察到背根神经节神经元轴突再生，TGFβI，TIMP3，EPHA4的表达三组分别通过qRT-PCR和Western blot检测细胞凋亡相关蛋白caspase-3和caspase-9。通过双荧光素酶报告基因测定来确认靶基因。通过体内免疫荧光评估TGFβ1，TIMP3和EPHA4的表达。qRT-PCR表明，模型组中的miR-21表达明显高于假手术组和空白组。TGFβI，TIMP3，EPHA4的SC增殖指数（PI）显着更高，凋亡率显着降低，轴突显着更长，mRNA和蛋白质表达mimi-miR-21组的凋亡相关蛋白caspase-3和caspase-9明显低于对照组miR-21和RSC96组。双重荧光素酶测定法证实TGFβ1，TIMP3和EPHA4是miR-21的潜在靶基因。体内免疫荧光法还表明，mimic-miR-21组中TGFβI，TIMP3，EPHA4的表达低于对照组miR-21和RSC96组。我们得出结论，在受伤的周围神经修复过程中，miRNA-21通过调节TGFβI，TIMP3和EPHA4靶基因在促进SC增殖和轴突再生中起重要作用。