Function And Mechanism Study Of Pp1 And MiR-6324 Genes In Spinal Cord Injury

Background:Spinal Cord Injury(SCI)is a highly morbid disease that can cause motor dysfunction and injury.SCI can not only cause dysfunction or loss of sensory and motor function below the plane of injury,but also cause dysfunction in multiple organs such as urinary system,respiratory system,circulatory system and digestive system.At present,the treatment of SCI mainly include drug treatment,hypothermia treatment,stem cell transplantation treatment and immunotherapy.Although the therapeutic methods and effects of SCI have made considerable progress and improvement over the past few decades,the long-term prognosis of SCI patients remains unsatisfactory,and thus it is urgent to explore new therapeutic strategies and methods.The emergence of Next Generation Sequencing(NGS)can comprehensively screen and identifydisease-related genes from the transcriptional level,laying a technical foundation for exploring disease mechanisms in the life sciences.In this study,preliminary functional and mechanistic studies were carried out by sequencing mRNA and microRNA and combining molecular biology to explore genes involved in SCI injury repair.MicroRNAs are a class of non-coding single-stranded RNA molecules of about 22 nucleotides in length encoded by endogenous genes,involved in important life processes such as reproductive development and tumor development by acting on target genes.Effects of microRNAs in SCI have been confirmed by a large number of scientific research documents,but the specific potential mechanism of action in this process is still unclear,and thus fiurther research and exploration experiments are urgently needed.Objective:To screen and identify potential genes involved in pathological changes of SCI,select key genes,and study their functions and mechanisms in the treatment and repair of SCI,so as to lay a theoretical foundation for exploring new target molecules for the treatment and repair of SCI,as well as for exploring new therapeutic pathways and methods for SCI.Methods:The role and mechanism of Ppl in SCI repair in rats1.To determine whether the SCI rat model was successful:30 SD rats were randomly divided into sham operation group(Sham group)and SCI group.The motor function of rats were evaluated by Basso Beattie Bresnahan(BBB)scoring system,slope test and electrophysiological test,and the changes of spinal nerve cell morphology were detected by immunofluorescence to confirm whether the SCI model rat was successfully constructed.2.The total RNA of spinal cord tissue in rats was extracted to analyze the mRNA expression profiles of Sham group and SCI group by transcriptome sequencing,and the accuracy of transcriptome sequencing data was verified by qPCR.3.The differential gene Ppl was picked and the synthetic siRNA was designed.The Ppl gene was silenced by transfected siRNA for 20 days in SCI model rats.After silencing Ppl gene,the motor function of rats was evaluated by BBB scoring system to observe the the silencing effect and repair changes of SCI model rats;qPCR,Western Blot and immunofluorescence assay were used to evaluate the repair effect of SCI,and the function and mechanism of Ppl gene in SCI repair were revealed.The role and mechanism of miR-6324 in SCI repair in rats1.The changes of SCI-related genes were observed.The successfully constructed SCI injury model animals were divided into two groups.The 1d group was sacrificed one day after the injury,and the 5d group was taken after 5 days of injury.The miRNAs expression profiles of Id and 5d groups were compared by miRNA sequencing and bioinformatics analysis,and the related gene miR-6324 was screened.We observed that PIK3CD,PPP3CA,PPP3CB,PPP3R1,CASP3 and ILIA were target genes of miR-6324.The expression levelsof these genes in the 1d group and 5d group were determined by qPCR.2.The miR-6324 enhancer or inhibitor was transfected into PC-12 cells,respectively,and the expression level of miR-6324 in the transfected cells was determined using qPCR After successful transfection,the expression level of caspase 3 in each group was measured by qPCR and Western Blot,and the viability of each group was determined.Bioinformatics was used to predict the miR-6324 target site and construct a reporter gene expression vector for PIK3CD 3'-UTR and its mutants.Fluorescent reporter gene technology was used to verify whether PIK3CD is a target of miR-6324,and the expression of PIK3CD was measured to explore the downstream pathway of miR-6324.3.To synthesize the siRNA of miR-6324,and transfected it into the SCI model rats.The motor function of the rats was evaluated by BBB scoring system,and the silencing effect and functional recovery after SCI were observed.Western Blot and immunofluorescence assay were used to evaluate the repair effect of SCI,and initially revealed the function and mechanism of miR-6324 in SCI repair.Results:1.Compared with Sham group,the behavioral,electrophysiological and morphological results of SCI group showed obvious spinal cord injury,indicating that the SCI model was successfully constructed.RNA sequencing results showed that 330 mRNAs were differentially expressed in SCI group and Sham group.Of these,238 mRNAs were up-regulated and 92 were down-regulated.The most significantly upregulated gene was Ppl,whose expression level was significantly higher than that of the control group(P<0.05),and the results of qPCR and Western Blot validation were consistent with the sequencing results.2.The SCI recovery effect in the Pp]-silenced group was significantly better than that in the non-silenced group.The behavioral and electrophysiological findings of the two groups,as well as the morphology and number of immunofluorescence-targeted neurons were also significantly different(all P<0.05).Quantitative PCR and Western Blot results showed that the expression of IL-6,IL-17 and IL-23 was significantly decreased after silencing Ppl gene expression,while the expression of p-STAT3 gene was significantly increased(all P<0.05),suggesting that the expression level of differential gene Ppl is closely related to the inflammatory response in the SCI lesion.3.MicroRNA sequencing and bioinformatics analysis at 1 and 5 days after SCI treatment showed that there were 18 miRNAs with different expression profiles among SCI related genes.Among them,there were 3 upregulated miRNAs(rno-miR-10b-5p,rno-miR-375-3p,and rno-miR-496-3p,respectively).There were 15 downregulated miRNAs(fold change>1.5)(rno-miR-122-5p,rno-miR-18a-5p,rno-miR-19a-3p,rno-miR-208a-3p,rno-miR-147,rno-miR-184,rno-miR-293-5p,rno-miR-199a-3p,and rno-miR-142a-3p,rno-miR-6324,rno-miR-199a-5p,rno-miR-3 120,rno-miR-214-3p,rno-miR-130b-5p,rno-miR-21-5p,respectively)),and among them rno-miR-6324 were most significantly down-regulated.4.The results of qPCR showed that the apoptosis of neural stem cells overexpressing miR-6324 was significantly higher than that of the control group(both P<0.05),the expression of Caspase 3 protein was up-regulated,and the cell proliferation ability was decreased(both P<0.05).The results of the prediction showed that miR-6324 targets PIK3CD gene,and the results of the dual luciferase reporter gene system show that miR-6324 and PIK3CD genes interact suggesting that miR-6324 may target PIK3CD gene to affect SCI repair.5.The efficacy of silencing MiR-6324 on rats with SCI showed that the behavioral,electrophysiological and morphological recovery of the miR-6324-silenced group was significantly better than that of the control group(all P<0.05),indicating low expression of miR-6324 has a good effect on the treatment and repair of SCI.Conclusions:1.Compared with the sham group,there was a significant change in spinal cord mRNA expression after SCI.2.Down-regulation of Ppl gene can reduce the inflammatory response in the injured area,involved in the SCI treatment and repair process.3.The expression of miRNAs in the spinal cord was significantly changed at 1 day and 5 days after SCI.4.miR-6324 is involved in SCI treatment and repair by targeting PIK3CD to mediate apoptosis.5.Ppl and miR-6324 are expected to be new target molecules for SCI treatment and repair,and can be used as a new SCI treatment.