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MiR-181d-5p Promotes Neurite Outgrowth In PC12 Cells Via PI3K/Akt Pathway

Posted on:2019-04-02Degree:DoctorType:Dissertation
Country:ChinaCandidate:L M ShenFull Text:PDF
GTID:1364330545971678Subject:Surgery
Abstract/Summary:PDF Full Text Request
Spinal cord injury(SCI)is the most serious complication of Spinal fractures,resulting in the loss of movement and sensory function in the following segments.Spinal cord injury not only causes serious physical and psychological harm to the patient,but also imposes a huge economic burden on the whole society.The treatment and recovery of spinal cord injury is a worldwide problem,and there is no effective treatment.Accumulating evidence indicates that mi RNAs dysregulate in spinal cord injury and participate in the pathophysiology of post-injury.Through regulating its target genes involved in related signaling pathways,mi RNAs play crucial role in neural development,neural stem cells proliferation and differentiation,repair and reconstruction after injury of neurons.It may be an effective strategy for the treatment of SCI through endogenous regulation of mi RNAs to promote the function recovery of spinal cord.Aim: To explore the role and concrete mechanism of mir-181d-5p on axonal growth of nerve cells.Methods: 1.In this study we constructed a mouse model of SCI,extracted RNA from injured spinal cord tissue for the use of microarray assay.Mi R-181d-5p and PI3K/Akt pathway selected from mi RNA microarray assay results were chosen for our further study;2.Firstly lentivirus(LV-mi R-181d-5p and LV-sh-GFP)were generated.To demonstrate whether mi R-181d-5p can promote neurite outgrowth in PC12 cells via PI3K/Akt signaling pathway,we performed function analysis of mi R-181d-5p with LV-mi R-181d-5p and LV-sh-GFP to infect PC12 cells.PC12-sh-GFP cells as a control,we assessed the role of overexpression mi R-181d-5p on PC 12 cells;3.Via point mutation and dual-luciferase reporter we demonstrated the concrete binding site of mir-181d-5p on the 3 'UTR of PTEN m RNA;4.Through cell immunofluorescence the effect of overexpression of mir-181d-5p on neurite outgrowth of PC12 cells was assessed.The downstream changes of PI3K/AKT pathway were detected by RT-PCR and Western blot.Result:Through microarray assay analysis we totally found 262 significantly expressed mi RNAs and 2973 target genes in SCI and observed that their expression dynamically changed from the initial injury to 1 day,3 days,1 week,and 3 weeks post-injury.Via functional and pathway analysis of m RNA we also obtain 332 significant functionofm RNA and 41 related significant pathway.Finally mi RNA-m RNA-network was created which can reveal the relationship between mi RNA and m RNA.Here,we provided enough evidences that the overexpression of mi R-181d-5p significantly decreased the expression of PTEN,up-regulated p-Akt expression,increased neurite outgrowth related proteins(GAP-43 and NF-200)and synaptic vesicle-related proteins(Synapsin and PSD95)and then promoted neurite outgrowth in PC12 cells.Furthermore we confirmed that mi R-181d-5p could directly target to the 3'-UTR of PTEN m RNA through dual-luciferase report assay.Conclusion: Our study supports that aberrant expression of mi RNAs is involved in the pathogenesis of SCI,mi R-181d-5p plays an important role in neurite growth in PC12 cells via PI3K/Akt signaling pathway and may be a candidate target for the treatment of SCI in the future.
Keywords/Search Tags:miRNA, neurite outgrowth, miR-181d-5p, PTEN, Akt
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