Association of Serum lncRNA CASC11 with Injury Severity and Inflammation in Spinal Cord Injury

Summary

In spinal cord injury (SCI) patients, CASC11 upregulation is associated with injury severity. In vitro, CASC11 downregulation alleviated apoptosis and inflammation, suggesting an association with the miR-130b-5p/SPP1 axis. These results suggest CASC11’s potential role as a biomarker candidate in SCI pathogenesis.

Abstract

Spinal cord injury (SCI) is a severe central nervous system trauma. This single-center, observational and in vitro study investigated the diagnostic potential of CASC11 and its possible regulatory mechanism in SCI using clinical serum samples and lipopolysaccharide-stimulated cell models. CASC11, miR-130b-5p, and SPP1 levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Cellular functions and targeting relationships were assessed via cell counting kit-8 (CCK-8), flow cytometry, western blot, enzyme-linked immunosorbent assay (ELISA), dual-luciferase reporter, and RNA immunoprecipitation (RIP) assays. CASC11 was highly expressed in SCI and associated with inflammation as a potential diagnostic biomarker. In lipopolysaccharide (LPS)-treated PC-12 cells, silencing CASC11 alleviated suppressed cell activity, apoptosis, and inflammation, which was reversed by miR-130b-5p inhibitor. These findings suggest that CASC11 is associated with SCI severity, while in vitro data indicate its involvement in inflammatory and apoptotic responses through the CASC11/miR-130b-5p/SPP1 axis. This study is limited by its single-center design and lack of in vivo validation.

Introduction

Spinal cord injury (SCI) denotes a temporary or permanent systemic disorder arising from spinal cord damage caused by external forces or diseases, resulting in impairment of motor, sensory, reflex, and autonomic nervous system functions^1,2. Patients frequently encounter lifelong physical functional deficits and complications, imposing considerable suffering and burdens on individuals and families. The etiology of SCI is broadly classified into traumatic and non-traumatic origins³. Traumatic injuries, predominantly resulting from spinal fractures due to external forces such as traffic accidents, falls from height, and sports-related incidents, represent a growing challenge in the clinical management of SCI⁴. Meanwhile, the secondary injury cascade initiated after the primary injury, especially the dysregulation of the inflammatory response and neuronal apoptosis, are regarded as crucial factors contributing to the continuous deterioration of neurological function. Recent studies have highlighted the critical role of neuroinflammation, regeneration, and functional recovery in the pathophysiology and repair of SCI⁵. As a central secondary response, neuroinflammation plays a dual role in the process of tissue injury and repair. Given the limited regenerative capacity of the injured spinal cord, a synergistic therapeutic approach combining anti-inflammatory treatment, cell transplantation, rehabilitation, and molecular interventions is required⁶. While a range of diagnostic and therapeutic strategies have been established for SCI, current clinical practice is predominantly reliant on imaging examinations⁷. However, these techniques are restricted by their high requirements for advanced equipment, strict patient prerequisites, and substantial financial expenses⁸. Consequently, there is an urgent and unmet need for a convenient, accurate, and non-invasive laboratory biomarker. The identification of such a marker would allow for the early diagnosis of SCI without disrupting conventional treatment protocols, thus facilitating the development of more timely and effective therapeutic interventions.

Long non-coding RNA (lncRNA) represent a major category of functional RNA molecules that, despite lacking protein-coding potential, are integral to a wide array of biological functions, including the regulation of inflammation and apoptosis⁹. LncRNAs act as molecular sponges for microRNAs (miRNAs), thereby effectively impeding these miRNAs from binding to and silencing their targeted mRNAs¹⁰. For instance, lncRNA MAGI2-AS3 mediates miR-223-3p to accelerate fracture healing¹¹. Targeting the lncRNA OIP5-AS1/miR-128-3p/Nrf2 network presents a promising therapeutic approach for improving recovery in SCI¹². CASC11, characterized by an 872-bp transcript located on chromosome 8q24.21, is recognized as a molecule with dysregulated expression in various human cancers¹³. CASC11 exhibits positive expression in patients with postmenopausal osteoporosis and has the ability to differentiate affected individuals from healthy controls¹⁴. Moreover, CASC11 displays a similar upregulation pattern and therapeutic potential in patients with fractures¹⁵. Based on these reports, CASC11 may regulate inflammatory responses and cell activity. Despite these insights, the expression pattern and biological role of CASC11 in SCI remain completely unknown. Given that hyperinflammation and apoptosis are major drivers of secondary SCI, we hypothesized that CASC11 may be a candidate biomarker and regulatory factor in SCI. This study investigated the clinical association and biomarker potential of CASC11 in SCI by analyzing patient cohorts and in vitro cell models (Figure 1). Furthermore, we explored a possible molecular mechanism of CASC11, including its potential role in modulating the inflammatory response subsequent to SCI.

Protocol

Enrollment of subjects

Patients with acute spinal trauma (n = 300) treated in the hospital from January 2023 to May 2025 were selected and classified into the complete spinal cord injury (CSCI) group (grade A, n = 98), incomplete spinal cord injury (ISCI) group (grades B, C, and D; n = 95), and normal neurological function (NNF) group (grade E, n = 107) according to the American Spinal Injury Association Impairment Scale (AIS classification). For patients in the NNF group, radiological evidence of SCI, such as edema, contusion, or compression, was confirmed by magnetic resonance imaging (MRI), but no sensory or motor deficits were demonstrated. Another 90 healthy participants recruited during the same period were selected as the control group. Inclusion criteria were: (1) diagnosis of SCI confirmed by imaging (MRI or CT), in accordance with established clinical management guidelines¹⁶; (2) trauma sustained within 24 h prior to hospital admission; and (3) adult participants with complete clinical data. Exclusion criteria were: (1) history of prior surgery related to fracture or SCI; (2) severe coronary artery disease or significant cardiopulmonary disease; (3) acute severe craniocerebral injury; and (4) malignant tumors, cardiovascular/cerebrovascular diseases, or immune diseases. Approval for this study was obtained from the Ethics Committee of The Fourth People’s Hospital of Shenzhen, and informed consent was obtained from the subjects. The procedures used in this study adhered to the tenets of the Declaration of Helsinki.

Collection of specimens

Venous blood samples (5 mL) were collected from participants upon admission using serum separator tubes within 24 h post injury. The blood was allowed to coagulate at room temperature for 30 min and was then centrifuged at 3,000 × g for 10 min at 4 °C. The supernatant serum was carefully separated, aliquoted into RNase-free microtubes, and stored at -80 °C until further analysis.

Cell culture and LPS induction

Rat pheochromocytoma cells (PC-12) and murine microglial cells (BV-2) were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum at 37 °C. To establish in vitro inflammatory injury models, PC-12 and BV-2 cells were exposed to lipopolysaccharide (LPS) at concentrations of 0, 1, 5, and 10 µg/mL for 12 h¹⁷. Each concentration was tested in three independent biological replicates.

RT-qPCR

Total RNA was isolated from the samples using a phenol-chloroform extraction protocol, followed by precipitation with isopropanol. RNA purity was assessed by spectrophotometry, and samples with an A_{260/280 nm} ratio within the 1.8–2.1 range were used for downstream analysis. RNA was reverse-transcribed into cDNA according to the reverse transcription kit protocol, and the resulting cDNA was used as the template for RT-qPCR reactions with a SYBR Green premix on a PCR instrument. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference gene for CASC11 and SPP1 mRNA, and small nuclear RNA (U6) was used as the reference gene for miR-130b-5p. For serum samples, synthetic cel-miR-39 was spiked into the serum before RNA extraction to control for variations in RNA recovery. The primers were as follows: CASC11 forward 5'-ACCCTATGGAGAACCGAGAC-3' and reverse 5'-GAGGACCAACTCAGTAGGAAAT-3'; miR-130b-5p forward 5'-ATCCATGGTTGAGCTTCCCG-3' and reverse 5'-TAGTGCAACCTCGTCAGAGC-3'; and SPP1 mRNA forward 5'-GTTAAACAGGCTGATTCTGG-3' and reverse 5'-CATGGTCATCATCATCTTCA-3'. Expression was calculated using the 2^-ΔΔCt method. Each RT-qPCR reaction was performed in triplicate technical replicates with three biological replicates.

Transfection

Silencing CASC11 (si-CASC11), miR-130b-5p mimic/inhibitor, SPP1 overexpression plasmid (ov-SPP1), and their respective negative controls (NC) were synthesized and transfected into PC-12 and BV-2 cells using a transfection reagent. Transfection efficiency was assessed 48 h post transfection in triplicate biological replicates.

Proliferation and apoptosis assay

For the cell viability assay, cells were seeded into 96-well plates at 5 × 10³ cells per well and cultured for 24 h to permit attachment. After this incubation, 10 µL of CCK-8 reagent was dispensed into each well, followed by incubation for 2 h at 37 °C. Optical density was then recorded at 450 nm with a microplate reader. For apoptosis detection, cells were detached with trypsin, rinsed twice with phosphate-buffered saline (PBS), and suspended in binding buffer to a final concentration of 1 × 10⁶ cells/well. Next, 100 µL of the suspension was placed into a 5 mL flow cytometry tube, followed by the addition of 5 µL Annexin V-FITC and 5 µL propidium iodide (PI) staining solution. Samples were mixed gently and kept for 15 min at room temperature protected from light. Binding buffer was then added to a final additional volume of 400 µL per tube, and apoptotic cells were quantified by flow cytometry. All conditions were analyzed using three technical replicates in three independent biological experiments.

Dual-luciferase activity assay

The downstream targets of CASC11 and miR-130b-5p were predicted through bioinformatics websites (lncRNASNP2 and TargetScan). Fragments containing the wild-type (wt) or mutant (mut) complementary sequences were synthesized and cloned into a dual-luciferase reporter vector to construct wt and mut reporter plasmids. PC-12 cells were seeded and co-transfected with miR-130b-5p mimic/inhibitor and the constructed wt/mut plasmids using a lipid-based transfection reagent. After 48 h, luciferase activity was quantified to assess the interaction. Each group consisted of three technical replicates and three biological replicates.

RNA immunoprecipitation (RIP) assay

RIP was performed using an RNA-binding protein immunoprecipitation kit. Cells were lysed with lysis buffer and incubated with anti-Ago2 antibody or normal IgG antibody. RNA-protein complexes were enriched using magnetic beads. After washing, bound proteins were removed, and immunoprecipitated RNA was isolated. The enrichment levels of CASC11 and miR-130b-5p were quantified by RT-qPCR. Each group consisted of three technical replicates and three biological replicates.

Western blot

Protein lysates were prepared from PC-12 and BV-2 cells using cell lysis buffer, and the total protein content was measured with a BCA assay. For each sample, 30 µg of protein was loaded onto 12% SDS-PAGE gels and transferred to PVDF membranes after electrophoretic separation. The membranes were blocked for 2 h at room temperature in 5% skim milk diluted in TBST and then incubated overnight at 4 °C with primary antibodies against Bcl-2, Bax, cleaved caspase-3, and GAPDH at a dilution of 1:1,000. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein signals were analyzed using image analysis software, and band densities were used to assess the expression of apoptosis-related proteins. The western blot experiments included three biological replicates, with three technical replicates for each group.

ELISA

The concentrations of inflammatory cytokines TNF-α, IL-1β, and IL-6 in patient serum and PC-12 and BV-2 cell supernatants were quantified using ELISA kits according to the manufacturer’s instructions. Samples were incubated in precoated wells, and detection antibodies were added for further incubation. Enzyme conjugates were added and incubated. After washing, substrates were added for color development, and the reactions were terminated. Absorbance was measured at 450 nm using a microplate reader and converted to inflammatory factor concentrations based on the provided standard curve. Each ELISA assay was performed in triplicate technical replicates across three independent biological replicates.

Statistical analysis

Continuous variables were reported as the mean ± standard deviation (SD), and categorical variables were presented as numbers and percentages (n, %). Comparisons between two groups were performed using Student’s t-test. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was applied, followed by Tukey’s post-hoc test. The ability of CASC11 to discriminate among clinical groups was evaluated using receiver operating characteristic (ROC) curve analysis. Associations between two variables were examined by Pearson correlation analysis. For cell-based experiments, each group contained three independent biological replicates, and each measurement was repeated in three technical replicates. Statistical significance was defined as P < 0.05.

Results

Basic data of the subjects

The baseline characteristics of patients in the Control, NNF, ISCI, and CSCI groups are presented in Table 1. Statistical analysis revealed no significant inter-group differences in variables, such as sex, age, body mass index (BMI), etiology of injury, and time of injury (P > 0.05). Significant differences among the four groups were observed for length of stay, ASIA grade, and the concentrations of the pro-inflammatory c...

Discussion

SCI is a devastating condition of the central nervous system that frequently results in significant deficits in sensation, motor control, and autonomic function, thus substantially diminishing a patient’s quality of life and long-term prognosis¹⁸. Patients may present with functional impairment and sensory loss following spinal trauma, and imaging studies (such as MRI) can be employed to assess the occurrence of SCI¹⁹. The onset of SCI triggers a deleterious secondary...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCA protein assay kit	Yeasen, China	P0010
CCK-8 solution	Sigma-Aldrich, USA	96992
Dual-luciferase reporter vector	Promega, USA	E133A	pmirGLO vector
Dulbecco’s Modified Eagle Medium	ATCC, Manassas, VA, USA	30-2002
ELISA kit for IL-1β	R&D Systems, USA	DY401
ELISA kit for IL-6	R&D Systems, USA	HS600B
ELISA kit for TNF-α	R&D Systems, USA	DTA00C
Fetal bovine serum	Gibco, Grand Island, NY, USA	10099141
Flow cytometer	BD FACSCalibur, USA	FACS101
Image analysis software	NIH, Bethesda, MD, USA	version 1.44p	ImageJ software
Isopropanol	Beijing Chemical Factory, China	32064
Lipid-based transfection reagent	Invitrogen, Carlsbad, CA, USA	L3000008	Lipofectamine 3000
Lipopolysaccharide	Sigma-Aldrich, USA	L2880
Lysis buffer	Beyotime, China	P0013D	RIPA lysis buffer
Microplate reader	Reagen, China	RNE90002
PC-12 and BV-2 cells	ATCC, Manassas, USA
Phenol-chloroform reagent	Thermo, USA	15596026	Trizol reagent
Reverse transcription kit	Takara, Japan	RR047A	PrimeScript RT Kit
RNA-binding protein immunoprecipitation kit	Millipore, USA	17-700	Magna RIP Kit
RNase-free microtubes	Axygen, Corning, NY, USA	MCT-150-C-S
Serum separator tubes	Terumo, Tokyo, Japan	VP-AS109K60
Statistical analysis software	San Diego, USA	version 9.0	GraphPad Prism
SYBR Green premix	Takara, Japan	RR420A	SYBR Premix Ex Taq