Differentially Expression Of Long Non-coding RNA In Chronic Spinal Cord Injury Rat

Objective To prepare a stable CSCI(Chronic Spinal Cord Injury)model by implanting polyurethane polymer sheets into the space between the C5 vertebral plate and spinal dura mater on the left dorsolateral side of SD rats,and to obtain the differentially expressed profile of lnc RNA after CSCI by microarray sequencing technology.It is our aim to find key lnc RNA associated with CSCI and to reveal the intrinsic pathogenesis of CSCI at the transcriptional level so that new theoretical basis and strategy for the diagnosis and treatment of CSCI will be discovered.Methods Thirty-two SD rats were randomly divided into either the CSCI group or the CON group.Rats in the CSCI group underwent laminectomy left of the C6,and then polyurethane polymer sheets were placed into the space between the C5 vertebral plate and spinal dura mater on the left dorsolateral side.Polyurethane polymer sheets,which expanded to induce persistent compression of the spinal cord over time and gradually reached the maximum volume,were utilized to prepare the CSCI model.Rats in the CON group only underwent laminectomy the same as the CSCI group.The motor function of both groups was evaluated and recorded daily postoperative using the BBB motor assessment scale.The C5 spinal cord segments were collected and then suffering microarray sequencing analysis on the 28 th day after cervical MRI.Furthermore,q RT-PCR was performed to verify these high-throughput results.Finally,the significant differentially expressed lnc RNA was deeply explored by mainstream bioinformatics analysis technology,and a ce RNA pathway was constructed and confirmed by q RT-PCR.Results The BBB scores showed that the CSCI group exhibited a significant decrease to almost 16 the first few days,and then gradually increased to about 17.5 on the 7th day after surgery and maintained.However,the CON group was maintained at a level of about 20.5 though a slight decrease on the first day.There were significant differences in BBB scores between the two groups at each time point,and the statistical significance was obvious(P<0.05).The MRI showed that the polyurethane polymer sheets in the CSCI group were expanded and stable on the left dorsolateral side of the C5 spinal cords.There was no significant change in the T2-weighted transverse for the C5 spinal cords of the CON group,while the C5 spinal cords of the CSCI group were significantly compressed on the left dorsolateral side with significantly low signals in the T2-weighted transverse and the T1,T2 weighted sagittal.No abnormal signals such as edema and hemorrhage were observed in the spinal cords of the two groups.Microarray sequencing showed that a total of 68842 RNAs were found,which were 2872 lnc RNAs,24753 m RNAs and 15365 pre-mi RNAs.Among them,there were 2746 differentially expressed RNAs.Compared with the CON group,1266 lnc RNAs(738 up-regulated and 528 down-regulated),847 m RNAs(487 up-regulated and 360 down-regulated)and 633 pre-mi RNAs(383 up-regulated and 250 down-regulated)were significantly changed in the CSCI group(P < 0.05).Among them,17 lnc RNAs(13 up-regulated and 4 down-regulated),18 m RNAs(13 up-regulated and 5 down-regulated)and 10 pre-mi RNAs(7 up-regulated and 3 down-regulated)were found deregulated by more than 2-fold.In addition,these differentially expressed lnc RNA and m RNA were widely distributed on all rat chromosomes.Bioinformatics revealed the results of GO enrichment analysis,which showed that the most significant enriched molecular function of up-regulated genes in the CSCI group were cysteine-type endopeptidase activity involved in apoptotic process,identity protein binding,double-stranded RNA binding,GTPase activity and TAP1 binding;cell component were extracellular matrix,extracellular space,proteinaceous extracellular matrix,AIM2 inflammasome complex and cytosol;biological process were cellular response to interferon-beta,defense response to virus response to virus,innate immune response and response to lipopolysaccharide.The most significant enriched molecular function of down-regulated genes in the CSCI group were olfactory receptor activity,fibroblast growth factor receptor binding,growth factor activity,G-protein coupled receptor activity and bombesin receptor activity;cell component were integral component of membrane,plasma membrane,Z disc and perinuclear region of cytoplasm;biological process were detection of chemical stimulus involved in sensory perception of smell,G-protein coupled receptor signaling pathway,bombesin receptor signaling pathway,localization within membrane and regulation of sprouting angiogenesis.The results of KEGG pathway enrichment analysis indicated that the most important pathways for up-regulated genes in the CSCI group include NOD-like receptor signaling pathway,Herpes simplex infection,TNF signaling pathway,Influenza A and Measles;the most important pathways for down-regulated genes in the CSCI group include Olfactory transduction,Melanoma,Steroid biosynthesis,Sphingolipid metabolism and Cytokine-cytokine receptor interaction.In addition,further classification of pathways showed that top classification mainly included Human Diseases,Organismal Systems,Environmental Information Processing,Cellular Processes and Metabolism;middle classification mainly included Infectious diseases,Immune system,Immune diseases,Cancers and Signal transduction.Based on the calculated correlation coefficient values,we finally constructed a co-expression network among 519 differentially expressed lnc RNAs and 562 differentially expressed m RNAs and confirmed 787 positively co-expressed lnc RNA-m RNA pairs and 740 negatively co-expressed lnc RNA-m RNA pairs.Among them,787 positively co-expressed lnc RNA-m RNA pairs consisted of 344 differentially expressed lnc RNAs and 433 differentially expressed m RNAs,while 740 negatively co-expressed lnc RNA-m RNA pairs involved 321 differentially expressed lnc RNAs and 388 differentially expressed m RNAs.In addition,102 pairs(6.68%)of the 1527 lnc RNA-m RNA pairs were located on the same chromosomes,while the remaining 1425 pairs(93.32%)were located on the different chromosomes.Through microarray sequencing results and bioinformatics analysis,combined with q RT-PCR preliminary validation,we obtained a CSCI related ce RNA pathway: lnc RNA6032/mi R-330-3p/Col6a1.As was shown,q RT-PCR data were in good accordance with the microarray results.Besides,compared with the CON group,the expressions of lnc RNA6032 and Col6a1 were considerably higher in the CSCI group while the level of mi R-330-3p expression was markedly lower.Conclusions1.The rat CSCI model,prepared by polyurethane polymer sheets compression method is validated by BBB behavioral evaluation and MRI can be used in CSCI related experimental research.2.After CSCI,the genome will change significantly.As for this experiment,the expression profiles of lnc RNA,m RNA and pre-mi RNA in rats after CSCI have changed significantly.3.Microarray sequencing technology provides a new opportunity to study the diagnosis,treatment and prognosis of CSCI.4.Bioinformatics technology can use a network-based functional analysis tool to process large data sets and generate a gene network for searching signaling pathways to achieve a systematic analysis of transcriptional changes after CSCI,facilitating our understanding of lnc RNA regulation.5.Lnc RNA can further elucidate the molecular pathogenesis and regulation mechanism of CSCI at the transcriptional level,and provide new theoretical basis and strategy for the diagnosis and treatment of CSCI and the development of molecular target drugs.6.The q RT-PCR confirmed that the data of microarray sequencing was reliable,and it was initially shown that lnc RNA6032 may up-regulate the expression of Col6a1 by competitive binding to mi R-330-3p,thus playing a critical role in CSCI.7.Further research is needed to fully understand and clarify the functional of lnc RNA in CSCI and its repair,and to actively develop lnc RNA-based therapies to improve nerve regeneration.