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.

KEYWORDS:

• Microrna-21
• Schwann cell
• axon regeneration
• peripheral nerve injury

Abbreviation

miR-21	=	microRNA-21
SC	=	Schwann cell
DRGn	=	dorsal root ganglion neuron
PI	=	proliferation index; A: absorbance
CCND1	=	cyclin D1
ECM	=	extracellular matrix;.

Disclosure statement

No potential conflict of interest was reported by the authors.

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Additional information

Funding

This study was supported by the National Natural Science Foundation of China [No.81571202] and Guangdong Provincial Scientific and Technological Funds [No.2014B090901043].