| Spinal cord injury,caused by direct or indirect external factors,can result in various motor,sensory and sphincter dysfunctions in the corresponding segment of the injury.After spinal cord injury,fibroblasts accumulated in the lesion proliferate actively and secrete extracellular matrix such as type ? collagen(Collagen I)to form fibrotic scars.Fibrotic scars and glial scars can prevent inflammatory cells spreading to surrounding tissues and extending injury size in the early stages of spinal cord injury,however,they also act as dense physical and chemical barriers in the late stages of spinal cord injury,hindering the regeneration and repair of neural tissues.As the most abundant microRNA(miRNA)in the central nervous system(CNS),miR-124 plays an important role in physiological and pathological processes such as nervous system development,cell differentiation,metabolism and repair.The study has found that the miRNA expression profile in the injury site changed significantly when spinal cord injury occurred,among which miR-124 was one of the most significantly down-regulated miRNAs.It has not been reported about miR-124 expression in fibroblasts in fibrotic scars and whether it inhibits the formation of fibrotic scars in spinal cord injury.The purpose of this article is to explore the effects and mechanism of highly expressed miR-124 on the proliferation and expression of Collagen ? of fibroblasts isolated and purified from the spinal cord injury site.This research provides scientific basis and research data for the application of exogenous miR-124 to inhibit fibrotic scars formation and promote spinal cord repair,and brings new research clue for the application of miR-124 in the treatment of spinal cord injuryIn this study,utilizing rat models of spinal cord injury,the following studies were performed:? Tissue immunofluorescence analysis was applied to analyze the expression of fibroblast markers in rat spinal cord injury site and qRT-PCR analysis was conducted to detect the expression of miR-124 after spinal cord injury;? A method for the isolation and purification of fibroblasts from rat spinal cord injury site was established.Fibroblasts were identified by cell immunofluorescence analysis,osteogenic and adipogenesis inducting experiments.Artificial overexpression of miR-124 induced fibroblasts differentiating into neurons in neural induction medium The expression level of miR-124 was determined by qRT-PCR and neural differentiation was identified by cell immunofluorescence.? After transfection with the optimal transfection concentration of miR-124-3p mimic,the effect of miR-124 on the proliferation of fibroblasts was detected by CCK-8,and the effect of miR-124 on the protein expression of Collagen I in fibroblasts was detected by Western blot?Combine RT-PCR,qRT-PCR,Western blot with luciferase assay to determine whether PTBP1 is the target gene of miR-124;? After interfering the expression of PTBP1 in fibroblasts by si-PTBP1,the mRNA expression of Collagen I was analyzed by qRT-PCR,and the protein expression of Cyclin D1 and Collagen I was analyzed by Western blotResults:(1)Tissue immunofluorescence results showed that a hollow lesion was formed in the center of the spinal cord injury site.Fibrotic scars characterized by Collagen I and Fibronectin and glial scars characterized by GFAP were identified forming around the hollow lesion,fibrotic scars also highly expressed PTBP1.qRT-PCR results confirmed that the expression of miR-124 in the injured spinal cord tissue was significantly reduced,while the mRNA expression levels of Collagen I,Sox9,and PTBP1 were significantly increased after spinal cord injury(2)The fibroblasts were isolated and purified from rat spinal cord injury site.The morphology of primary fibroblasts was spindle-shaped and fibroblast-like.At cell immunofluorescence analysis,the cultured cells positvely expressed fibroblast markers,such as Collagen I,Fibronectin,Vimentin,PDGFR-?,NG-2,while astrocyte marker GFAP and oligodendrocyte marker Sox 10 showed negative expression.The results proved that the purified cells were fibroblasts with a purity of about 90%.Additionally,fibroblasts highly expressed PTBP1.After 28 days of osteogenesis induction,no calcium salt deposition was found in alizarin red staining.After 21 days of adipogenesis induction,no fat droplets were observed through oil red O staining.These results proved that the obtained fibroblasts did not have osteogenic and adipogenic differentiation characteristics.After 7 days of neurogenic induction,the cells showed slender protrusions.Cell immunofluorescence confirmed the expression of DCX and MAP-2 proteins,which proved that fibroblasts could be transdifferentiated into neurons under the combined action of miR-124 and small molecular compounds;qRT-PCR results confirmed that the expression of miR-124 in fibroblasts derived from the spinal cord injury site was significantly reduced(3)It was detected by qRT-PCR that after 25,50,100 nM miR-124-3p transfection,the miR-124 content in the fibroblasts increased by about 81,119,163 fold in order;CCK-8 results confirmed that the proliferation of fibroblasts was significantly reduced after overexpression of miR-124;Western blot results showed that the protein expression levels of Collagen I and Sox9 in fibroblasts were significantly reduced after overexpression of miR-124(P<0.05)(4)The results of RT-PCR,qRT-PCR and Western blot showed that the gene and protein expression levels of PTBP1 were significantly down-regulated(P<0.001)after overexpression of miR-124 in the fibroblasts.The 3'UTR sequence of PTBP1 mRNA was predicted to have the binding site of miR-124 by TargetScanHuman website.Combined with luciferase assay,it was confirmed that PTBP1 was the target gene of miR-124 in the 293T cells.Western blot results showed that the protein expression levels of Cyclin D1 were significantly reduced(P<0.001)after knocking down PTBP1 with siRNA.qRT-PCR and Western blot results showed that the gene(P<0.05)and protein(P<0.01)expression levels of Collagen I were significantly increased after knocking down PTBP1 with siRNAConclutions:(1)After the spinal cord injury in rats,fibrotic scars formed by fibroblasts are present at the center of the injury sites;(2)Overexpression of miR-124 inhibits the proliferation of fibroblasts derived from the spinal cord injury site,suggesting that miR-124 might reduce the formation of fibrotic scars and promote the repair of spinal cord injury;(3)miR-124 might reduce the proliferation of fibroblasts derived from the spinal cord injury site by inhibiting the protein expression of its target gene PTBP1,however,the inhibition of PTBP1 does not reduce the expression of Collagen I.It is speculated that miR-124 may reduce the expression of Collagen I in fibroblasts derived from the spinal cord injury site through other mechanisms. |
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