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Molecular mechanism of microRNA-21 promoting Schwann cell proliferation and axon regeneration during injured nerve repair.

ABSTRACT: 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.

SUBMITTER: Ning XJ

PROVIDER: S-EPMC7549677 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:After peripheral nerve injury, intraperitoneal injection of folic acid improves axon quantity, increases axon density and improves electromyography results. However, the mechanisms for this remain unclear. This study explored whether folic acid promotes peripheral nerve injury repair by affecting Schwann cell function. Primary Schwann cells were obtained from rats by in vitro separation and culture. Cell proliferation, assayed using the Cell Counting Kit-8 assay, was higher in cells cultured for 72 hours with 100 mg/L folic acid compared with the control group. Cell proliferation was also higher in the 50, 100, 150, and 200 mg/L folic acid groups compared with the control group after culture for 96 hours. Proliferation was markedly higher in the 100 mg/L folic acid group compared with the 50 mg/L folic acid group and the 40 ng/L nerve growth factor group. In Transwell assays, the number of migrated Schwann cells dramatically increased after culture with 100 and 150 mg/L folic acid compared with the control group. In nerve growth factor enzyme-linked immunosorbent assays, treatment of Schwann cell cultures with 50, 100, and 150 mg/L folic acid increased levels of nerve growth factor in the culture medium compared with the control group at 3 days. The nerve growth factor concentration of Schwann cell cultures treated with 100 mg/L folic acid group was remarkably higher than that in the 50 and 150 mg/L folic acid groups at 3 days. Nerve growth factor concentration in the 10, 50, and 100 mg/L folic acid groups was higher than that in the control group at 7 days. The nerve growth factor concentration in the 50 mg/L folic acid group was remarkably higher than that in the 10 and 100 mg/L folic acid groups at 7 days. In vivo, 80 μg/kg folic acid was intraperitoneally administrated for 7 consecutive days after sciatic nerve injury. Immunohistochemical staining showed that the number of Schwann cells in the folic acid group was greater than that in the control group. We suggest that folic acid may play a role in improving the repair of peripheral nerve injury by promoting the proliferation and migration of Schwann cells and the secretion of nerve growth factors.

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Molecular mechanism of microRNA-21 promoting Schwann cell proliferation and axon regeneration during injured nerve repair.

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