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 cytokines tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and interleukin-6 (IL-6) (P < 0.001).

Diagnostic potential of elevated CASC11 in SCI

CASC11 levels in serum were markedly elevated in all SCI patient groups relative to controls. This elevation was most pronounced in individuals diagnosed with CSCI (Figure 2A). ROC curve analysis revealed that CASC11 exhibited discriminative capacity in distinguishing SCI patients from the control group, with an AUC of 0.891, sensitivity of 76.17%, and specificity of 86.67%. Furthermore, CASC11 showed potential as a biomarker candidate in differentiating patients with NNF from those with ISCI or CSCI (AUC=0.853, sensitivity=65.80%, specificity=93.46%). NNF group is a critical clinical threshold for identifying the transition to severe neurological deficits. Notably, CASC11 also showed promise for distinguishing injury severity between ISCI and CSCI groups (AUC=0.799, sensitivity=63.27%, specificity=90.53%, P < 0.001; Figure 2B and Table 2). Additionally, the positive correlation was identified between CASC11 expression and levels of the inflammatory mediators TNF-α (r=0.706), IL-1β (r=0.632), and IL-6 (r=0.662) within the CSCI patient group, though not indicative of a causal relationship (Figure 2C).

Induction of PC-12 and BV-2 cells by LPS

PC-12 and BV-2 cells were treated with different concentrations of LPS, and it was confirmed that 5 µg/mL was the threshold concentration for significant induction of inflammatory injury and suppression of cellular activity (Figure 3A). Finally, 5 µg/mL LPS was selected for subsequent cell assays. Following LPS exposure, RT-qPCR analysis revealed approximately two-fold increased CASC11 expression in PC-12 and BV-2 cells. CASC11 levels were suppressed by approximately 50% upon CASC11 knockdown under this condition (Figure 3B). Assessment of the biological functions in LPS-stimulated cells revealed that while LPS exposure diminished cell viability and promoted apoptosis, the subsequent knockdown of CASC11 rescued the observed loss in cell viability (Figure 3C,D). In BV-2 cells, similar results were observed, where LPS exposure also suppressed cell viability and promoted apoptosis, and CASC11 knockdown similarly rescued the LPS-induced apoptotic phenotype and viability loss (Figure 3B-D).

Negative regulation of miR-130b-5p by CASC11

A potential interaction between wt-CASC11 and miR-130b-5p was detected via lncRNASNP2, which predicted the existence of complementary binding sequences (Figure 4A). miR-130b-5p mimic obviously reduced the luciferase activity in the wt-CASC11 group, whereas miR-130b-5p inhibitor elevated it. Conversely, neither had a significant impact on the mut-CASC11 group (Figure 4B). Significant enrichment of CASC11 and miR-130b-5p was observed in the Ago2-based RIP assays compared to the IgG control, offering direct evidence for a specific interaction between them (Figure 4C). Moreover, miR-130b-5p expression was markedly lower in SCI patients than in controls, with expression decreasing to the clinical severity of the injury (Figure 4D). CASC11 and miR-130b-5p expression in CSCI group was inversely proportional (Figure 4E, r=-0.668).

Regulation of apoptosis and inflammatory factors by silencing CASC11 and miR-130b-5p

Silencing CASC11 counteracted the LPS-induced downregulation of miR-130b-5p, and this effect was abolished by miR-130b-5p inhibitor (Figure 5A). The cytoprotective effect conferred by CASC11 knockdown in LPS-treated PC-12 and BV-2 cells was mediated via miR-130b-5p. This was substantiated by the finding that inhibition of miR-130b-5p annulled the rescue of cell proliferation and suppression of apoptosis otherwise achieved by CASC11 silencing (Figure 5B, C). Moreover, LPS induced pro-apoptotic molecular profile, characterized by elevated Bax/Cleaved caspase-3 and reduced Bcl-2. This profile was reversed upon CASC11 knockdown, indicating its protective function. Nevertheless, this reversal was effectively counteracted by co-transfection with miR-130b-5p inhibitor (Figure 5D). Meanwhile, the concentrations of inflammatory factors TNF-α, IL-1β and IL-6 were activated by LPS treatment. Knockdown of CASC11 alleviated the inflammatory response in PC-12 and BV-2 cells, yet the effect of CASC11 was partially offset by miR-130b-5p (Figure 5E).

SPP1 as a downstream target of miR-130b-5p

SPP1 was identified and validated as a direct downstream target of miR-130b-5p. Initially, this was achieved through TargetScan prediction (Figure 6A), and subsequently confirmed via dual-luciferase reporter assay. Specifically, overexpression of miR-130b-5p led to the suppression of the luciferase activity of the wt-SPP1 reporter, while the inhibition of miR-130b-5p resulted in its enhancement. This regulatory effect was abolished when the predicted binding site in SPP1 was mutated (Figure 6B).  SPP1 mRNA was significantly upregulated in the serum of SCI patients compared to control group (Figure 6C). Subsequently, Pearson correlation analysis revealed that miR-130b-5p could inversely regulate the expression of SPP1 mRNA in CSCI group (Figure 6D, r=-0.638).

Effects of the miR-130b-5p/SPP1 axis on apoptosis and inflammatory factors

Overexpression of miR-130b-5p suppressed LPS-induced SPP1 expression in PC-12 and BV-2 cells, whereas co-transfection with ov-SPP1 restored SPP1 expression (Figure 7A). In LPS-treated PC-12 and BV-2 cells, miR-130b-5p mimic exerted a protective effect by increased cell viability and reduced apoptosis, while SPP1 overexpression partially reversed these protective effects (Figure 7B,C). Furthermore, LPS induced the upregulation of the pro-apoptotic proteins Bax and cleaved caspase-3 and the downregulation of the anti-apoptotic protein Bcl-2. miR-130b-5p mimic reversed these apoptotic changes, whereas SPP1 overexpression counteracted this effect (Figure 7D). Similarly, LPS increased the secretion of TNF-α, IL-1β, and IL-6, while miR-130b-5p mimic attenuated these inflammatory responses, further confirming its protective role (Figure 7E). Co-transfection with ov-SPP1 partially reversed the anti-inflammatory and protective effect of miR-130b-5p mimic in PC-12 and BV-2 cells, demonstrating that ov-SPP1 compromises the rescue effect of miR-130b-5p.

Data Availability:

The data that support the findings of this study are available in Supplemental Table S1.

Figure 1: Overview of the clinical and in vitro study design. Clinical serum samples were collected from 90 healthy controls and 300 patients with SCI, including patients with normal neurological function (NNF; n = 107), incomplete spinal cord injury (ISCI; n = 95), and complete spinal cord injury (CSCI; n = 98). Serum levels of CASC11, miR-130b-5p, and SPP1 were measured and compared across groups. In parallel, PC-12 and BV-2 cells were used for lipopolysaccharide (LPS)-induced in vitro assays, followed by transfection, CCK-8, flow cytometry, apoptotic marker, inflammatory cytokine, dual-luciferase reporter, and RNA immunoprecipitation (RIP) analyses. Abbreviations: CCK-8 = cell counting kit-8; CSCI = complete spinal cord injury; ISCI = incomplete spinal cord injury; LPS = lipopolysaccharide; NNF = normal neurological function; RIP = RNA immunoprecipitation; SCI = spinal cord injury. Please click here to view a larger version of this figure.

Figure 2: Discriminative capacity of CASC11 in SCI severity stratification.  (A) CASC11 is highly expressed in SCI patients (NNF/ISCI/CSCI vs control; One-way ANOVA with Tukey’s post-hoc test). (B) The ability of CASC11 to distinguish clinical subgroups was evaluated by ROC curve analysis. (C) CASC11 was positively correlated with the levels of inflammatory factors (Pearson correlation analysis). Abbreviation: SCI = spinal cord injury; NNF = normal neurological function; ISCI = incomplete spinal cord injury; CSCI = complete spinal cord injury; ANOVA = analysis of variance; ROC = receiver operating characteristic. Please click here to view a larger version of this figure.

Figure 3: LPS induction and CASC11 silencing transfection in PC-12 and BV-2 cells. (A) Cell viability was downregulated after treatment with different concentrations of LPS (5/10 µg/mL vs 0 µg/mL). (B) Effect of CASC11 knockdown on CASC11 expression under LPS stimulation (LPS vs control; LPS+si-CASC11 vs LPS). (C,D) LPS exposure suppressed cellular activity and accelerated apoptosis, whereas silencing CASC11 demonstrated a protective function (LPS vs control; LPS+si-CASC11 vs LPS). One-way ANOVA with Tukey’s post-hoc test. Each group consisted of three technical replicates and three biological replicates. Abbreviation: LPS = lipopolysaccharide; ANOVA = analysis of variance. Please click here to view a larger version of this figure.

Figure 4: Sponge relationship between CASC11 and miR-130b-5p. (A) Prediction of complementary sites between CASC11 and miR-130b-5p. (B,C) Luciferase activity (miR-130b-5p mimic/inhibitor vs control) and RIP assays (Ago2 vs IgG) verification of the direct targeting between CASC11 and miR-130b-5p (n=3 biological replicates). (D) miR-130b-5p is downregulated in the serum of SCI patients (NNF/ISCI/CSCI vs control). (E) miR-130b-5p is negatively mediated by CASC11 (Pearson correlation analysis). One-way ANOVA with Tukey’s post-hoc test. Abbreviation: RIP = RNA immunoprecipitation; SCI = spinal cord injury; NNF = normal neurological function; ISCI = incomplete spinal cord injury; CSCI = complete spinal cord injury; ANOVA = analysis of variance. Please click here to view a larger version of this figure.

Figure 5: Regulation of CASC11 and miR-130b-5p on the biological functions of PC-12 and BV-2 cells. (A) Transfection efficiency of miR-130b-5p by RT-qPCR assays. (B-E) miR-130b-5p inhibitor reversed the effects of silencing CASC11 on cell proliferation, apoptosis, apoptotic indicators, and inflammatory factors after LPS intervention. (LPS vs control; LPS+si-CASC11 vs LPS; LPS+si-CASC11+miR-130b-5p inhibitor vs LPS+si-CASC11). One-way ANOVA with Tukey’s post-hoc test. Each group consisted of three technical replicates and three biological replicates. Abbreviation: RT-qPCR = real-time quantitative polymerase chain reaction; LPS = lipopolysaccharide; ANOVA = analysis of variance. Please click here to view a larger version of this figure.

Figure 6: SPP1-targeting mechanism of miR-130b-5p. (A) Bioinformatic prediction of the binding site for miR-130b-5p within the SPP1 sequence. (B) Identification of SPP1 as a downstream target for miR-130b-5p via luciferase activity assay (n=3 biological replicates; miR-130b-5p mimic/inhibitor vs control).  (C) SPP1 mRNA is enhanced in the serum of SCI groups compared to control group (NNF/ISCI/CSCI vs control). (D) miR-130b-5p directly targets and inhibits SPP1 (Pearson correlation analysis). One-way ANOVA with Tukey’s post-hoc test. Abbreviation: SCI = spinal cord injury; NNF = normal neurological function; ISCI = incomplete spinal cord injury; CSCI = complete spinal cord injury; ANOVA = analysis of variance. Please click here to view a larger version of this figure.

Figure 7: Regulation of the miR-130b-5p/SPP1 axis on the biological functions of PC-12 and BV-2 cells. (A) SPP1 levels were quantified by RT-qPCR. (B-E) LPS exposure inhibited cell viability and accelerated apoptosis and inflammation, whereas miR-130b-5p mimic rescued cell function, an effect reversed by SPP1 overexpression. (LPS vs control; LPS+miR-130b-5p mimic vs LPS; LPS+miR-130b-5p mimic+ov-SPP1 vs LPS+miR-130b-5p mimic). One-way ANOVA with Tukey’s post-hoc test. Each group consisted of three technical replicates and three biological replicates. Abbreviation: RT-qPCR = real-time quantitative polymerase chain reaction; LPS = lipopolysaccharide; ANOVA = analysis of variance. Please click here to view a larger version of this figure.

Items	Control (n=90)	SCI (n=300)			P
		NNF (n=107)	ISCI (n=95)	CSCI (n=98)
Sex (female/male)	36/54	35/72	32/63	40/58	0.526
Age (year)	44.11±9.92	44.61±8.19	45.88±8.83	45.34±11.17	0.547
BMI (kg/m2)	22.73±4.16	23.19±4.83	22.75±4.02	22.60±5.03	0.834
Length of stay (day)	/	25.65±5.07	30.59±7.15	40.84±9.79	<0.001
Etiology of injury					0.602
Traffic accident (n, %)	/	64 (59.81%)	47 (49.47%)	58 (59.18%)
Fall (n, %)	/	16 (14.95%)	22 (23.16%)	19 (19.39%)
Object hit (n, %)	/	19 (17.76%)	22 (23.16%)	15 (15.31%)
Sports (n, %)	/	8 (7.48%)	4 (4.21%)	6 (6.12%)
ASIA grade					<0.001
A (n, %)	/	/	/	98 (100.00%)
B (n, %)	/	/	31 (32.63%)	/
C (n, %)	/	/	37 (38.95%)	/
D (n, %)	/	/	27 (28.42%)	/
E (n, %)	/	107 (100.0%)	/	/
Time of injury					0.888
< 72 h (n, %)	/	65 (60.75%)	57 (60.00%)	62 (63.27%)
72 h - 1 month (n, %)	/	42 (39.25%)	38 (40.00%)	36 (36.73%)
TNF-α (pg/mL)	93.69±6.48	106.13±11.15	118.93±7.52	138.87±10.92	<0.001
IL-1β (pg/mL)	164.93±16.91	175.82±15.54	184.49±28.75	199.01±18.77	<0.001
IL-6 (pg/mL)	101.45±14.84	118.53±11.04	131.36±28.05	151.89±9.41	<0.001

Table 1: Comparison of basic data of subjects. Abbreviations: SCI = spinal cord injury; NNF = normal neurological function; ISCI = incomplete spinal cord injury; CSCI = complete spinal cord injury; BMI = body mass index; TNF-α = Tumor Necrosis Factor-α; IL-1β = Interleukin-1β; IL-6 = Interleukin-6. Data was collected from January 2023 to May 2025.

	Control vs. SCI	NNF vs. ISCI/CSCI	ISCI vs. CSCI
AUC	0.891	0.853	0.799
95% CI	0.850 - 0.931	0.811 - 0.894	0.736 - 0.863
Cut-off value	1.235	1.305	1.495
Sensitivity (%)	76.17%	65.80%	63.27%
Specificity (%)	86.67%	93.46%	90.53%
P value	＜0.001	＜0.001	＜0.001

Table 2: Diagnostic capabilities of lncRNA CASC11. Abbreviations: ROC = receiver operating characteristic; AUC = Area under the curve; CI = confidence interval; SCI = spinal cord injury; ISCI = incomplete spinal cord injury; CSCI = complete spinal cord injury.

Supplemental Table S1: Raw data supporting the clinical and in vitro analyses. The file contains the individual serum expression values of CASC11, miR-130b-5p, and SPP1 in the Control, NNF, ISCI, and CSCI groups; clinical data used for baseline comparisons; sensitivity and specificity calculations used for ROC analysis; and replicate-level data from PC-12 and BV-2 cell assays, including LPS induction, transfection, cell viability, apoptosis, apoptotic protein expression, and inflammatory cytokine measurements. Abbreviations: CSCI = complete spinal cord injury; ISCI = incomplete spinal cord injury; LPS = lipopolysaccharide; NNF = normal neurological function; ROC = receiver operating characteristic.Please click here to download this file.

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 injury cascade, which is characterized by intense neuroinflammation, edema, and infiltration of inflammatory cells. This cascade, in conjunction with the stress of traumatic pain, gradually disrupts neurological function^20,21. The early post-injury phase is characterized by a high risk of clinical deterioration, underscoring the essential role of timely medical intervention in halting disease progression.

The realm of clinical research on spinal trauma and nerve injury is undergoing rapid transformation, offering novel perspectives into the underlying mechanisms of the disease. One of the most compelling recent insights is the discovery that a vast number of lncRNAs are abnormally expressed in SCI²². For instance, lncRNA Airsci was downregulated SCI and promoted functional recovery in SCI rats by reducing inflammatory responses, as demonstrated in Zhang et al.'s study²³. In the report by Wang et al., SNHG3 levels were enhanced in the serum of patients with SCI and the diagnostic ability of patients with CSCI, which may inhibit the inflammatory response by mediating miR-139-5p²⁴. Clinically, we reported that CASC11 is upregulated in SCI, especially in CSCI, and serves as a potential biomarker for reflecting injury severity. Simultaneously, the onset of SCI is associated with significant elevation in inflammatory markers. Notably, in patients with CSCI, these markers TNF-α, IL-1β and IL-6 are positively correlated with the level of CASC11. This finding implies that elevated CASC11 may serve as an indicator of exacerbated inflammation subsequent to SCI. In previous studies concerning CASC11, it has been observed that CASC11 is also overexpressed in plasma samples from patients with small cell lung cancer and exhibits a positive correlation with the inflammatory cytokine TGF-β1²⁵. In vitro, we modeled SCI-related inflammatory injury in vitro using LPS induction. Similarly, LPS exposure markedly suppressed the proliferation of PC-12 cells and accelerated apoptosis²⁶. However, silencing CASC11 restored cell viability.

MicroRNAs (miRNAs) function as crucial regulatory factors, assuming central roles in the pathophysiology of diverse diseases. Recent research indicates that miR-130b-5p participates in regulating key processes such as apoptosis and inflammatory responses across multiple tissues—processes central to spinal cord injury²⁷. In this study, miR-130b-5p was identified as a downstream site of CASC11 and was found to be poorly expressed in SCI. Our findings are in accordance with those of Wang et al., who also reported downregulation of miR-130b-5p in the microglia-based SCI models²⁸. Our experimental results demonstrated that LPS-induced injury resulted in a significant increase in cellular apoptosis and inflammation. Conversely, the knockdown of CASC11 effectively counteracted these effects, suppressing inflammation and restoring cell viability. However, the protective function of CASC11 was nullified when co-treated with miR-130b-5p inhibitor. Collectively, these findings suggest that CASC11 confers a protective effect against LPS-induced injury by regulating miR-130b-5p. Nevertheless, it must be acknowledged that the actual clinical application of miR-130b-5p remains extremely challenging. For instance, the blood-brain barrier, as the natural protective barrier of the central nervous system, strictly restricts the entry of exogenous macromolecules, presenting a significant obstacle for the delivery of miRNA-based therapeutics²⁹. Moreover, since a single miRNA may regulate hundreds of different target genes, transfection of miR-130b-5p may have unexpected effects on targets or pathways, namely off-target effects³⁰. This further imposes requirements on the administration mode and drug dosage control. In the context of neuroprotection research, recent studies have highlighted strategies such as remote ischemic preconditioning (RIC), a process that involves restoring blood flow after transient limb ischemia, as a possible approach to mitigate secondary injury³¹. Additionally, mechanisms related to inflammation have also attracted much attention, such as apolipoproteins (APOLs) and their role in cell activation³². It is thus speculated that targeting the CASC11/miR-130b-5p/SPP1 axis in combination with strategies such as RIC or APOL modulation could provide a multifaceted approach to neuroprotection after SCI.

In this report, SPP1 was identified as a downstream target of miR-130b-5p and found to be negatively regulated by it. SPP1 was enriched in microglia following SCI in the latest research and provided direction for functional recovery in patients³³. Further in vitro assays showed that LPS exposure inhibited cell viability and accelerated apoptosis and inflammation, miR-130b-5p mimic rescued cell function, and this protective effect was reversed by SPP1 overexpression. These results support that the CASC11/miR-130b-5p/SPP1 axis may be involved in the inflammatory response in SCI.

The limited size of the clinical cohort and its single-center origin restrict the generalizability of the findings regarding CASC11 as a potential biomarker. Meanwhile, ROC analysis only reflected the ability of CASC11 to distinguish patients with different degrees of SCI, but its diagnostic effect needs to be further verified in independent cohorts and multivariate models. Furthermore, cellular models cannot fully replicate the complexity of the spinal cord microenvironment, and the lack of in vivo validation in animal models means that the translational medical significance of the CASC11/miR-130b-5p/SPP1 axis in physiological conditions remains to be confirmed. To address these limitations, future studies will utilize in vivo rat models of SCI to validate our findings and further elucidate the complexity of this regulatory network.

In summary, CASC11 levels were upregulated in SCI patients compared to healthy controls, with its expression correlating with the severity of the injury. Mechanistically, downregulation of CASC11 alleviated apoptosis and inflammation in association with the miR-130b-5p/SPP1 axis. CASC11 is associated with SCI severity and inflammatory response and may represent a candidate biomarker and regulatory factor requiring further validation.

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