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Identifying the differentially expressed circRNAs and mRNAs in the subacute stage of spinal cord injury in mice by RNA-sequencing

ABSTRACT: Spinal cord injury (SCI) is a pathological condition that leading to serious nerve damage、disability and even death. Some scholars have divided the pathophysiology of SCI into primary mechanism and secondary mechanism. Secondary SCI can be divided into acute, subacute, and chronic stages, with the subacute stage lasting 2 days to 2 weeks after SCI. The subacute stage of SCI is a potentially critical time point for further study of SCI repair, which may play a significant role in the secondary SCI. Increasing evidence have revealed that circular RNAs (circRNAs) and mRNA are widely involved in the regulation of the pathological process of neurological diseases by sponging microRNAs (miRNAs). Nevertheless, the potential biological functions and regulatory mechanisms of circRNAs in the subacute stage of SCI remain unclear. We analyzed the expression and regulatory patterns of circRNAs and mRNAs in SCI mice models using RNA-sequencing. A total of 24 circRNAs and 372 mRNAs were identified to be differentially expressed, and found that circRNA02778, circRNA02370, and Ctsd were top up-regulated as well as circRNA05219 and Huwe1 were top down-regulated after SCI. Meanwhile, we identified multiple immune-related genes (Ptprc, Fcgr2b, Ccl2, Tlr7, Cxcl10, Cd28, Il10ra, Cd68, and Tlr3) related to SCI and found they were significantly upregulated after SCI, but the specific mechanisms by which they regulate SCI by mediating neuronal inflammatory and immune responses need to be further explored. These findings will contribute to elucidate the pathophysiology of SCI and provide effective therapeutic targets for SCI patients.

ORGANISM(S): Mus musculus

PROVIDER: GSE217797 | GEO | 2022/11/14

REPOSITORIES: GEO

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Project description:Purpose: Spinal cord injury (SCI) is a devastating neurological disease without effective treatment. To generate a comprehensive view of the mechanisms involved in SCI pathology, we applied RNA-sequencing (RNA-seq) technology to characterize the temporal changes in global gene expression after contusive SCI in mice. Method Part1: A total of 27 female C57BI/6J mice (10-16 weeks of age; 20-25g; The Jackson Laboratory, Bar Harbor, ME) were used with 9 mice in each of following groups: shame control, 2 and 7 days after SCI. The surgical procedure for SCI were described previously [PMID:23289019]. Briefly, after anesthetization with a mixed solution of ketamine (80 mg/kg, ip) and xylazine (10 mg/kg, ip), mice received a dorsal laminectomy at the 9th thoracic vertebral (T9) level to expose the spinal cord and then a moderate T9 contusive injury using an Infinite Horizons impactor (PrecisionSystems and Instrumentation) at 60 kdyn with the spinestabilized using steel stabilizers inserted under the transverse processes one vertebra above and below the injury [PMID:19196178] . The shame control mice received only a dorsal laminectomy without contusive injury. Afterwards, the wound was sutured in layers, bacitracin ointment(Qualitest Pharmaceuticals,Huntsville, AL) was applied tothe wound area, 0.1mL of a 20 mg/ml stock of gentamicin(ButlerSchein, Dublin, OH) was injected subcutaneously, and the animals recovered on a water-circulating heating pad. Then mice received analgesic agent, buprenorphin(0.05 mg/kg, SQ; Reckitt Benckise, Hull, England)twice a day for two days. Bladders were manually expressed until automatic voiding returned spontaneously, which generally was within 7 days. At 2 or 7 days after SCI, the mice were anesthetized again with ketamine and xylazine and perfused briefly with normal physical saline. The injured spinal cords were then dissected and three 0.5 mm pieces of spinal cord were cut in the injured epicenter. All spinal cords were immediately frozen in liquid nitrogen and processed for RNA isolation. The spinal cords from three mice were combined into one biological replicate for RNA extraction. Three biological replicates were used. Method Part2: RNA-Seq was performed on the polyadenylated fraction of RNA isolated from tissue samples of acute (2D) and subacute phase (7D) and normal tissues (control, denoted as CTR hereafter). Three biological replicates were used for each phase.150-300 ng total RNAs were used for each sequencing library. RNA samples were polyA selected and paired-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit as described in the TruSeq RNA Sample Preparation V2 Guide (Illumina).The samples were then sequenced using the Illumina HiSeq sequencer. More than 30 million 100bp paired-end reads were generated from each biological replicate. Method Part3: Read mapping and Transcriptome construction were done by using optimized pipeline which integrate Tophat followed by Cufflinks. Result: We sequenced tissue samples from acute and subacute phases (2 days and 7 days after injury) and systematically characterized the transcriptomes with the goal of identifying pathways and genes critical in SCI pathology. The top enriched functional categories include ‘inflammation response’, ‘neurological disease’, ‘cell death and survival’ and ‘nervous system development’. The top enriched pathways include LXR/RXR Activation and Atherosclerosis Signaling etc. Furthermore, we developed a systems-based analysis framework in order to identify key determinants in the global gene networks of the acute and sub-acute phases. Some candidate genes that we identified have been shown to play important roles in SCI, which demonstrates the validity of our approach. There are also many genes whose functions in SCI have not been well studied and can be further investigated by future experiments. We have also incorporated pharmacogenomic information into our analyses. Among the genes identified, the ones with existing drug information can be readily tested in SCI animal models. Conclusion: in this study we have described an example of how global gene profiling can be translated to screening genes of interest and generating new hypotheses. Additionally, the RNA-seq enables splicing isoform identification and the estimation of expression levels, thus providing useful information for increasing the specificity of drug design and reducing potential side effect. In summary, these results provide a valuable reference data resource for a better understanding of the SCI process in the acute and sub-acute phases. mRNA profiles of Acute/subacute phase Spinal Cord Injury sample from mice were generated by RNA-sequencing using Illumina HiSeq.

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Identifying the differentially expressed circRNAs and mRNAs in the subacute stage of spinal cord injury in mice by RNA-sequencing

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