LINC00707 Silencing Alleviates Inflammation and Oxidative Stress in LPS-stimulated BV2 Microglia via the miR-145-5p/TRAF6 Axis in Spinal Cord Injury

Summary

Plasma LINC00707 expression is elevated in spinal cord injury and promotes microglia-mediated inflammation and oxidative stress through the miR-145-5p/TRAF6 signaling axis. Silencing LINC00707 attenuates these pathological responses, highlighting its potential as a therapeutic target.

Abstract

Spinal cord injury (SCI) causes lifelong disability, profoundly reduces well-being, and imposes a substantial socioeconomic burden. This study investigated the role of LINC00707 in SCI through regulation of the miR-145-5p/TRAF6 signaling axis. Plasma samples from 30 healthy subjects and 75 patients with SCI were analyzed for LINC00707, miR-145-5p, and TRAF6 expression using RT-qPCR. Associations between plasma LINC00707 levels and pro-inflammatory factors were assessed in patients with SCI. BV2 microglial cells were exposed to 100 ng/mL LPS, and inflammation- and oxidative stress-related factors were measured. Molecular interactions were assessed using a dual-luciferase reporter assay. Logistic regression analysis was used to identify factors associated with LINC00707 expression. Plasma LINC00707 and TRAF6 levels were increased in patients with SCI, whereas miR-145-5p levels were reduced. Plasma LINC00707 levels were positively correlated with plasma inflammatory factor levels. LINC00707 acted as a molecular sponge for miR-145-5p, and TRAF6 was a target gene of miR-145-5p. Inhibition of LINC00707 decreased inflammatory factors, pro-oxidative stress substances, and TRAF6 mRNA levels in BV2 cells. Inhibition of miR-145-5p produced the opposite effects. These findings suggest that elevated LINC00707 expression is associated with SCI severity and that LINC00707 knockdown may alleviate microglia-mediated inflammation and oxidative stress by regulating the miR-145-5p/TRAF6 signaling axis.

Introduction

Spinal cord injury (SCI) is a traumatic condition that damages the spinal cord, a critical component of the central nervous system. SCI directly disrupts neural conduction pathways between the spinal cord and the brain, causing major impairments in motor, sensory, and autonomic function^1,2. Epidemiological studies have reported an annual incidence of approximately 5 cases per 100,000 individuals, with disease-related mortality reaching up to 16.7%^3,4. A Canadian survey estimated the lifetime rehabilitation cost for each patient with SCI to be as high as US$336,000⁵. The high incidence, mortality, and substantial costs associated with diagnosis, treatment, and rehabilitation make SCI a major clinical challenge that requires improved therapeutic strategies.

The pathological progression of SCI consists of two stages: primary and secondary injury. Primary injury results from traumatic mechanical compression or disruption of spinal cord tissue, whereas secondary injury is mediated by pathological processes, including inflammation and oxidative stress^6,7. Under physiological conditions, microglia-mediated neuroinflammation contributes to central nervous system homeostasis^8,9,10. Following SCI, however, microglia become excessively activated and release large quantities of pro-inflammatory mediators^11,12. These mediators elicit excessive production of reactive oxygen species (ROS), causing neuronal damage and death and aggravating secondary spinal cord injury^13,14. Therefore, suppressing excessive microglial activation and interrupting the inflammatory cascade represent promising therapeutic strategies for promoting neurological recovery and improving outcomes following SCI.

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs longer than 200 nucleotides that lack protein-coding potential¹⁵. Initially regarded as transcriptional "noise," lncRNAs are now recognized as important regulators of diverse physiological and pathological processes¹⁶. Recent studies have demonstrated that lncRNAs play critical roles in neural injury. Long intergenic non-protein-coding RNA 707 (LINC00707) has emerged as an important regulator of nervous system disorders. Previous studies have reported that LINC00707 aggravates traumatic brain injury by enhancing microglia-mediated inflammation¹⁷. In addition, Zhu et al. demonstrated that silencing LINC00707 significantly attenuated lipopolysaccharide (LPS)-induced inflammation and apoptosis in PC12 cells¹⁸. These findings provide a preliminary theoretical basis for exploring the involvement of LINC00707 in SCI. However, whether LINC00707 regulates microglia-mediated inflammation during SCI remains to be elucidated.

LncRNAs act as molecular sponges through the competitive endogenous RNA (ceRNA) mechanism, sequestering microRNAs (miRNAs) and regulating downstream target gene expression¹⁹. This regulatory pattern is widely involved in the pathogenesis of various diseases. Notably, miR-145-5p is significantly downregulated in SCI tissues²⁰ and during LPS-induced injury²¹, and its overexpression effectively attenuates microglial inflammatory and oxidative stress responses²². Tumor necrosis factor receptor-associated factor 6 (TRAF6) also plays a pivotal role in SCI pathology^23,24,25. Nevertheless, whether LINC00707, miR-145-5p, and TRAF6 exert coordinated regulatory functions in SCI remains to be elucidated.

In the present study, LINC00707 expression was significantly upregulated in LPS-stimulated microglial cells. Therefore, we hypothesized that LINC00707 may act as a pro-inflammatory regulator in microglia and that its knockdown may attenuate the neuroinflammatory response, thereby ameliorating the pathological progression of SCI. This study investigated whether LINC00707 regulates microglial inflammatory responses and contributes to SCI pathogenesis.

Protocol

This study was approved by the Ethics Committee of Zhangjiakou First Hospital (approval No. 2024-KY-18). All participants were informed of the study objectives and potential risks and provided written informed consent before enrollment. Venous blood samples were collected from healthy controls during routine physical examinations and from patients with SCI immediately after hospital admission. Laboratory measurements were performed by the hospital clinical laboratory. The research tools used in this protocol are listed in the Table of Materials.

1. Subjects
A total of 75 patients with SCI were recruited from Zhangjiakou First Hospital between January 2023 and January 2025. The inclusion criteria were SCI confirmed by computed tomography (CT) or magnetic resonance imaging (MRI) and a time from injury to hospital admission of ≤ 12 h. The exclusion criteria were a history of spinal cord or brain trauma or surgery within the previous 6 months; severe cardiovascular disease, including myocardial infarction or arrhythmia; pregnancy or lactation; and malignant tumors, impaired liver or kidney function, severe cerebrovascular disease, or infectious diseases.

According to the American Spinal Injury Association (ASIA) neurological classification standards²⁶, 75 patients with SCI were classified into the normal neurological function (n = 21), incomplete SCI (n = 36), and complete SCI (n = 18) groups. The distribution of injury sites was as follows: cervical, 36.0%; thoracic, 30.7%; and lumbar, 33.3%. 30 healthy individuals matched to the study population were randomly recruited to serve as the control group. Baseline clinical information was collected for all participants. The inclusion criteria for healthy individuals were no history of spinal cord or brain trauma, surgery, or neurological disorders; no history of chronic inflammatory diseases, autoimmune disorders, or malignancies; no severe cardiovascular, hepatic, or renal disease; no acute or chronic infection within the previous 4 weeks; no pregnancy or lactation; and age and sex matched to the SCI group.

The Functional Independence Measure (FIM) was used to assess functional independence in daily activities. The maximum FIM score is 126. Tactile sensation, acmesthesia, and motor scores were used to evaluate sensory and motor function. The maximum tactile sensation and acmesthesia scores were 112, and the maximum motor score was 100. Lower scores indicate more severe neurological impairment.

2. Cell culture, model construction, and transfection
Immortalized mouse microglial BV2 cells were obtained from the Cell Resource Center of the Shanghai Institute of Biotechnology. Cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 U/mL). Cells were maintained at 37 °C in a humidified incubator containing 5% CO₂.

For model construction, BV2 cells were stimulated with 0, 10, 20, 50, 100, 150, or 200 ng/mL lipopolysaccharide (LPS) for 24 h. The optimal LPS concentration was selected based on the experimental results.

For transfection, BV2 cells were seeded in 6-well plates and transfected once they reached the appropriate confluence. A transfection reagent was used at 1.5 µL/well according to the manufacturer’s instructions. After 6 h, the culture medium was replaced with fresh medium, and the cells were cultured for an additional 24 h. The LINC00707 knockdown construct, corresponding negative control, miR-145-5p oligonucleotide, and corresponding control oligonucleotide were each used at a concentration of 50 nM per well. All transfection experiments were independently repeated at least 5 times, with 3 parallel replicates per independent experiment. Data were statistically analyzed using the results of at least 5 independent experiments.

3. ELISA
The culture supernatants of transfected and LPS-stimulated BV2 cells were harvested by centrifugation at 12,000 × g for 10 min, and the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) were measured using the corresponding ELISA kits. Briefly, the assay was performed by sequentially incubating with a biotinylated antibody, an HRP-streptavidin conjugate, and a TMB substrate according to the manufacturer's instructions. Absorbance was recorded at 450 nm using a microplate reader. Cytokine levels were determined from standard curves. Each sample was measured in triplicate, and 6 independent experiments were performed.

4. ROS measurement
Intracellular ROS levels in LPS-stimulated BV2 cells were measured using a reactive oxygen species assay kit. Briefly, cells were incubated with 10 µmol/L DCFH-DA at 37 °C for 20 min. After incubation, the supernatant was discarded, and the cells were washed 3 times with phosphate-buffered saline (PBS) to remove excess extracellular probe. Fluorescence intensity was measured using a microplate reader at excitation and emission wavelengths of 488 nm and 525 nm, respectively. Relative ROS levels were calculated based on fluorescence intensity. All samples were analyzed in triplicate, and the entire experiment was independently repeated at least 5 times.

5. Detection of malondialdehyde, superoxide dismutase, and glutathione
Treated BV2 cells were collected, and cell homogenates were prepared. Samples were centrifuged at 12,000 × g for 10 min at 4 °C to obtain the supernatant. Commercial detection kits for malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were used according to the manufacturer's instructions to determine their levels in the cell supernatants. All samples were analyzed in triplicate, and the entire experiment was independently repeated at least 5 times.

6. Dual-luciferase reporter assay
The 3' untranslated region (3' UTR) fragments of LINC00707 and TRAF6 containing the predicted miR-145-5p binding sites, together with the corresponding mutant sequences, were cloned into the pmirGLO luciferase reporter vector to generate wild-type (WT-LINC00707 and WT-TRAF6) and mutant (MUT-LINC00707 and MUT-TRAF6) reporter constructs. BV2 cells were co-transfected with the reporter constructs and miR-145-5p oligonucleotides and cultured for 24 h. The cells were then collected, and firefly and Renilla luciferase activities were measured using a microplate reader.

7. RT-qPCR
Total RNA was extracted using an RNA extraction reagent. RNA quality was assessed using the OD₂₆₀/OD₂₈₀ ratio, with values between 1.8 and 2.0 considered acceptable. Approximately 1 µg of total RNA was reverse transcribed into cDNA using a reverse transcription kit. Gene expression was quantified by RT-qPCR using a SYBR Green PCR master mix. Reverse transcription was performed at 42 °C for 15 min, followed by reverse transcriptase inactivation at 95 °C for 3 min. The synthesized cDNA was stored at −20 °C until analysis. Quantitative real-time PCR was performed using pre-denaturation at 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing and extension at 60 °C for 15 s. Fluorescence signals were collected at the end of each annealing and extension step. Melting curve analysis was performed at 95 °C for 15 s, followed by 60 °C for 1 min. Relative expression levels were calculated using the 2⁻^ΔΔCt method. GAPDH or 5S was used as the internal reference for gene expression normalization. The primer sequences were as follows: LINC00707, forward 5'-CCAACAGGGTATCAGAATTCTC-3' and reverse 5'-TGCTGACAATAGCCATTAGG-3'; miR-145-5p, forward 5'-GCCGAGGTCCAGTTTTCCCA-3' and reverse 5'-CTCAACTGGTGTCGTGGA-3'; TRAF6, forward 5'-ATTACAACTGAGGTGCTTCT-3' and reverse 5'-GCATCTGGTTCTGTTATAGGA-3'; GAPDH, forward 5'-ATCCCATCACCATCTTCCAGG-3' and reverse 5'-CGCCCCACTTGATTTTGG-3'; and 5S, forward 5'-TCGTCTGATCTCGGAAGCTAA-3' and reverse 5'-AAGCCTACAGCACCCGGTAT-3'.

8. Statistical analysis
Statistical analyses were performed using statistical software. The normality of the data distribution was assessed using the Shapiro-Wilk test, with P > 0.05 indicating a normal distribution. Normally distributed data were presented as the mean ± SD, whereas non-normally distributed data were presented as the median (P25, P75). For continuous variables with a normal distribution, comparisons between 2 groups were performed using Student's t-test, and comparisons among multiple groups were performed using one-way ANOVA followed by Tukey's post hoc test. For non-normally distributed data, the Mann-Whitney U test was used for 2-group comparisons, and the Kruskal-Wallis test was used for multiple-group comparisons. The chi-square test was used to analyze categorical variables between groups. Univariate and multivariate logistic regression analyses were used to evaluate clinical factors associated with LINC00707 expression. Continuous variables were dichotomized at the cohort median (high-value group = 1; low-value group = 0). Variables with P < 0.05 in the univariate logistic regression analysis were entered into the multivariate logistic regression analysis. Results were presented as odds ratios (ORs) with 95% confidence intervals (CIs), where OR > 1 indicated a positive association and OR < 1 indicated a negative association. All cell phenotype experiments included at least 4 independent biological replicates, with 3 technical replicates per biological replicate. P < 0.05 was considered statistically significant.

Results

Plasma LINC00707 levels were elevated in patients with SCI
The baseline clinical characteristics of 30 healthy subjects and 75 patients with SCI were analyzed. Compared with healthy subjects, patients with SCI had markedly lower tactile sensation, acmesthesia, motor, and Functional Independence Measure (FIM) scores, indicating impaired sensory and motor function. In addition, serum levels of pro-inflammatory cytokines were significantly higher in patients with SCI than in healthy controls (Table 1, P < 0.001), suggesting an enhanced inflammatory response.

Items	HS	SCI	P-value
	(n=30)	(n=75)
Age (years)	45.83±10.06	46.17±9.92	0.786
BMI (kg/m2)	22.82±1.57	23.94±1.29	0.69
Gender (man, n)	18	41	0.619
Smoking (n)	11	28	0.949
Drinking (n)	14	33	0.804
ASIA Classification of SCI (n)	/
Complete SCI	/	18
Incomplete SCI	/	36
Normal neural function	/	21
The level of SCI injury (n, %)	/
Cervical vertebra	/	27 (36.0%)
Thoracic vertebra	/	23 (30.7%)
Lumbar vertebra	/	25 (33.3%)
Tactile sensation score	/	53.42±22.12
Acmesthesia score	/	53.39±24.68
Exercise score	/	49.07±18.74
Functional Independence Measure score	/	71.33±19.41
IL-1β (pg/mL)	1.47±0.52	52.21±11.32	<0.001
IL-6 (pg/mL)	2.14±0.97	88.47±23.52	<0.001
TNF-α (pg/mL)	2.84±1.13	61.09±18.24	<0.001
CRP (mg/L)	1.42±0.63	62.67±21.85	<0.001
Abbreviation: HS, Healthy subject; SCI, Spinal Cord Injury; BMI, Body mass index; ASIA, American Spinal Injury Association; FIM, Functional independence measure; IL, Interleukin; TNF, Tumor necrosis factor; CRP, C-reactive protein.

Table 1: Comparison of baseline clinical characteristics between healthy subjects and patients with spinal cord injury. This table compares demographic characteristics, neurological function scores, injury-related variables, and inflammatory marker levels between healthy subjects and patients with spinal cord injury. Abbreviations: HS, healthy subjects; SCI, spinal cord injury; ASIA, American Spinal Injury Association; FIM, Functional Independence Measure; IL, interleukin; TNF, tumor necrosis factor; CRP, C-reactive protein.

Plasma LINC00707 expression was quantified using RT-qPCR. Compared with healthy subjects, patients with SCI had significantly increased plasma LINC00707 expression (Figure 1A, P < 0.001). Receiver operating characteristic (ROC) curve analysis provided preliminary evidence that LINC00707 expression might have some discriminatory value in distinguishing patients with SCI from healthy subjects (Figure 1B). Furthermore, plasma LINC00707 expression increased with increasing clinical severity of SCI (Figure 1C, P < 0.001). Correlation analysis showed that plasma LINC00707 expression was positively associated with IL-1β (r = 0.635, P < 0.001), IL-6 (r = 0.612, P < 0.001), TNF-α (r = 0.675, P < 0.001), and C-reactive protein (CRP; r = 0.611, P < 0.001) levels (Figure 1D–G). These findings suggest that elevated LINC00707 expression is associated with inflammatory responses in patients with SCI.

Figure 1: Plasma LINC00707 expression and its correlations with plasma inflammatory factors. (A) Plasma LINC00707 expression was higher in patients with spinal cord injury (SCI) than in healthy subjects. Statistical analysis was performed using an independent-samples t-test. (B) Receiver operating characteristic (ROC) curve analysis provided an initial indication that LINC00707 expression may be useful for distinguishing patients with SCI from healthy subjects. (C) Plasma LINC00707 expression increased with increasing SCI severity according to the American Spinal Injury Association (ASIA) classification. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. (D–G) Plasma LINC00707 expression was positively correlated with inflammatory factor levels. Statistical analysis was performed using Pearson correlation analysis. *P < 0.01 and **P < 0.001. Healthy subjects, n = 30; patients with SCI, n = 75. Data are presented as the mean ± SD. Please click here to view a larger version of this figure.

Factors associated with LINC00707 expression were analyzed using univariate and multivariate logistic regression
Univariate and multivariate logistic regression analyses were performed to identify factors associated with LINC00707 expression. Univariate analysis showed that the ASIA classification of SCI (OR = 3.912, 95% CI: 1.792–8.537, P < 0.001), FIM score (OR = 0.281, 95% CI: 0.108–0.729, P = 0.009), and CRP level (OR = 4.600, 95% CI: 1.724–12.271, P = 0.002) were associated with LINC00707 expression. After adjustment for potential confounding variables, multivariate logistic regression showed that the ASIA classification of SCI (OR = 2.598, 95% CI: 1.088–6.203, P = 0.032), FIM score (OR = 0.303, 95% CI: 0.102–0.902, P = 0.032), and CRP level (OR = 3.202, 95% CI: 1.024–10.015, P = 0.045) were independent factors associated with LINC00707 expression (Table 2). A more severe ASIA classification and elevated CRP levels were associated with higher LINC00707 expression, whereas a higher FIM score was associated with lower LINC00707 expression. These associations further support the possibility that LINC00707 is involved in the pathological process of SCI.

Items	Univariate regression analysis			Multivariate regression analysis
	OR	95% CI	P-value	OR	95% CI	P-value
Age	1.176	0.475-2.914	0.725
Gender	1.303	0.524-3.240	0.57
ASIA Classification of SCI	3.912	1.792-8.537	<0.001	2.598	1.088-6.203	0.032
ASIA classification of SCI	0.588	0.497-1.480	0.582
Tactile sensation score	0.85	0.343-2.105	0.725
Acmesthesia score	0.854	0.345-2.119	0.734
Exercise score	0.392	0.153-1.006	0.051
FIM score	0.281	0.108-0.729	0.009	0.303	0.102-0.902	0.032
IL-1β	1.181	0.475-2.935	0.72
IL-6	1.453	0.585-3.608	0.42
TNF-α	1.304	0.526-3.233	0.567
CRP	4.6	1.724-12.271	0.002	3.202	1.024-10.015	0.045
Abbreviation: OR, Odds Ratio; CI, confidence interval; ASIA, American Spinal Injury Association; FIM, Functional independence measure; IL, Interleukin; TNF, Tumor necrosis factor; CRP, C-reactive protein.

Table 2: Logistic regression analysis of factors associated with LINC00707 expression. This table presents the univariate and multivariate logistic regression analyses used to identify clinical factors associated with LINC00707 expression in patients with spinal cord injury. Results are presented as odds ratios with 95% confidence intervals and corresponding P values. Abbreviations: OR, odds ratio; CI, confidence interval; ASIA, American Spinal Injury Association; FIM, Functional Independence Measure; IL, interleukin; TNF, tumor necrosis factor; CRP, C-reactive protein.

LINC00707 knockdown alleviated inflammation and oxidative stress in LPS-stimulated BV2 cells
BV2 cells were treated with increasing concentrations of LPS (0, 10, 20, 50, 100, 150, or 200 ng/mL). RT-qPCR analysis showed that LINC00707 expression was significantly increased following treatment with 100, 150, and 200 ng/mL LPS (Figure 2A, P < 0.001, n = 6). Based on these findings and previous reports²⁷˒²⁸, 100 ng/mL LPS was selected to establish the in vitro SCI model. Compared with the control group, LINC00707 expression was significantly elevated in LPS-stimulated BV2 cells. Transfection with si-LINC00707 effectively suppressed LINC00707 expression (Figure 2B, P < 0.001, n = 6).

The effects of LINC00707 knockdown on inflammation and oxidative stress were subsequently evaluated. LPS stimulation significantly increased the secretion of IL-1β (Figure 2C, P < 0.001, n = 5), IL-6 (Figure 2D, P < 0.001, n = 5), and TNF-α (Figure 2E, P < 0.001, n = 5), as well as intracellular ROS levels (Figure 2F, P < 0.001, n = 6) and MDA content (Figure 2G, P < 0.001, n = 5). In contrast, LPS stimulation significantly reduced the levels of the antioxidant markers SOD (Figure 2H, P < 0.001, n = 6) and GSH (Figure 2I, P < 0.001, n = 5). Silencing LINC00707 significantly reduced pro-inflammatory cytokine secretion and pro-oxidative stress marker levels, while restoring antioxidant levels in LPS-stimulated BV2 cells. These findings indicate that LINC00707 knockdown attenuates LPS-induced inflammatory responses and oxidative stress in BV2 cells.

Figure 2: Effects of LINC00707 knockdown on inflammation and oxidative stress in LPS-stimulated BV2 cells. BV2 cells were stimulated with 100 ng/mL lipopolysaccharide (LPS). (A) LINC00707 expression was measured by RT-qPCR in BV2 cells treated with different concentrations of LPS. (B) LINC00707 expression was increased in the LPS group and reduced following transfection with si-LINC00707. (C–E) ELISA showed that the secretion of pro-inflammatory factors was increased in the LPS group and reduced following LINC00707 knockdown. (F–G) Reactive oxygen species (ROS) levels and malondialdehyde (MDA) content were increased in the LPS group and reduced following LINC00707 knockdown. (H–I) Superoxide dismutase (SOD) activity and reduced glutathione (GSH) levels were decreased in the LPS group and increased following LINC00707 knockdown. **P < 0.001; ns, not significant. n ≥ 5. Statistical analyses were performed using one-way ANOVA followed by Tukey’s HSD post hoc test. Data are presented as the mean ± SD. Please click here to view a larger version of this figure.

LINC00707 functions as a molecular sponge for miR-145-5p
To investigate the molecular mechanism underlying the biological function of LINC00707, the ENCORI database was used to predict potential target miRNAs. Bioinformatic analysis identified complementary binding sequences between LINC00707 and miR-145-5p (Figure 3A). Dual-luciferase reporter assays showed that luciferase activity in the WT-LINC00707 construct was significantly decreased by the miR-145-5p mimic and increased by the miR-145-5p inhibitor. In contrast, neither treatment affected luciferase activity in the MUT-LINC00707 construct (Figure 3B, P < 0.001, n = 4), confirming the direct interaction between LINC00707 and miR-145-5p.

RT-qPCR analysis showed that plasma miR-145-5p expression was significantly decreased in patients with SCI compared with healthy subjects (Figure 3C, P < 0.001). In addition, LINC00707 expression was negatively correlated with miR-145-5p expression (Figure 3D, P < 0.001), and plasma miR-145-5p levels progressively decreased with increasing SCI severity (Figure 3E, P < 0.001). In vitro, miR-145-5p expression was significantly reduced in LPS-stimulated BV2 cells compared with control cells, whereas LINC00707 knockdown restored miR-145-5p expression (Figure 3F, P < 0.001, n = 6). These findings suggest that LINC00707 functions as a competing endogenous RNA (ceRNA) by sequestering miR-145-5p and thereby regulating its expression during the pathological progression of SCI.

Figure 3. LINC00707 functions as a molecular sponge for miR-145-5p. (A) The ENCORI database predicted complementary binding sequences between LINC00707 and miR-145-5p. (B) A dual-luciferase reporter assay confirmed the interaction between LINC00707 and miR-145-5p. Statistical analysis was performed using two-way ANOVA followed by Tukey’s HSD post hoc test. (C) RT-qPCR showed that plasma miR-145-5p expression was decreased in patients with SCI. Statistical analysis was performed using an independent-samples t-test. (D) LINC00707 expression was negatively correlated with miR-145-5p expression in patients with SCI. Statistical analysis was performed using Pearson correlation analysis. (E) Plasma miR-145-5p expression decreased with increasing SCI severity according to the ASIA classification. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. (F) miR-145-5p expression was decreased in the LPS group, increased following transfection with si-LINC00707, and reduced following transfection with the miR-145-5p inhibitor. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. P < 0.05, *P < 0.01, and **P < 0.001; ns, not significant. Healthy subjects, n = 30; patients with SCI, n = 75; cell experiments, n ≥ 4. Data are presented as the mean ± SD. Please click here to view a larger version of this figure.

miR-145-5p inhibition enhanced inflammation and oxidative stress in LPS-stimulated BV2 cells
To investigate the role of miR-145-5p, BV2 cells were transfected with a miR-145-5p inhibitor or the corresponding negative control. The levels of inflammatory cytokines and oxidative stress markers were subsequently evaluated. Compared with the inhibitor negative control group, miR-145-5p inhibition significantly increased the secretion of IL-1β (Figure 4A, P < 0.001, n = 5), IL-6 (Figure 4B, P < 0.001, n = 5), and TNF-α (Figure 4C, P < 0.001, n = 5). In addition, intracellular ROS levels (Figure 4D, P < 0.001, n = 6) and MDA content (Figure 4E, P < 0.001, n = 5) were significantly increased, whereas SOD (Figure 4F, P < 0.001, n = 6) and GSH (Figure 4G, P < 0.001, n = 5) levels were significantly decreased. These findings indicate that inhibition of miR-145-5p reverses the protective effects of LINC00707 knockdown on LPS-induced inflammation and oxidative stress in BV2 cells.

Figure 4: Effects of miR-145-5p inhibition on inflammation and oxidative stress in BV2 cells. (A–C) ELISA showed that the secretion of pro-inflammatory factors was significantly increased following transfection with the miR-145-5p inhibitor. (D–E) Reactive oxygen species (ROS) levels and malondialdehyde (MDA) content were significantly increased following transfection with the miR-145-5p inhibitor. (F–G) Superoxide dismutase (SOD) activity and glutathione (GSH) levels were significantly reduced following transfection with the miR-145-5p inhibitor. **P < 0.001. n ≥ 5. Statistical analyses were performed using one-way ANOVA followed by Tukey’s HSD post hoc test. Data are presented as the mean ± SD. Please click here to view a larger version of this figure.

TRAF6 was identified as a target gene of miR-145-5p
The miRDB database predicted TRAF6 as a potential target of miR-145-5p. Complementary binding sequences between the 3′ UTR of TRAF6 and miR-145-5p were identified (Figure 5A). Dual-luciferase reporter assays showed that luciferase activity in the WT-TRAF6 construct was significantly decreased by the miR-145-5p mimic and increased by the miR-145-5p inhibitor. In contrast, neither treatment affected luciferase activity in the MUT-TRAF6 construct (Figure 5B, P < 0.001, n = 4), confirming that TRAF6 is a direct target of miR-145-5p.

RT-qPCR analysis showed that plasma TRAF6 mRNA expression was significantly increased in patients with SCI (Figure 5C, P < 0.001). TRAF6 expression was negatively correlated with miR-145-5p expression (Figure 5D, P < 0.001), and plasma TRAF6 mRNA levels increased with SCI severity (Figure 5E, P < 0.001). In vitro, TRAF6 expression was significantly increased in LPS-stimulated BV2 cells compared with the control group but was significantly reduced following LINC00707 knockdown (Figure 5F, P < 0.001, n = 6). Conversely, inhibition of miR-145-5p restored TRAF6 expression (Figure 5G, P < 0.001, n = 6). These findings indicate that LINC00707 regulates TRAF6 expression through the miR-145-5p signaling pathway.

Figure 5: TRAF6 is a target gene of miR-145-5p. (A) The miRDB database predicted complementary binding sequences between TRAF6 and miR-145-5p. (B) A dual-luciferase reporter assay confirmed the targeting relationship between TRAF6 and miR-145-5p. Statistical analysis was performed using two-way ANOVA followed by Tukey’s HSD post hoc test. (C) RT-qPCR showed that plasma TRAF6 expression was elevated in patients with SCI. Statistical analysis was performed using an independent-samples t-test. (D) TRAF6 expression was negatively correlated with plasma miR-145-5p expression in patients with SCI. Statistical analysis was performed using Pearson correlation analysis. (E) Plasma TRAF6 expression increased with increasing SCI severity according to the ASIA classification. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. (F) TRAF6 expression was increased in LPS-stimulated BV2 cells and reduced following LINC00707 knockdown. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. (G) TRAF6 expression was increased following inhibition of miR-145-5p. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. P < 0.05, *P < 0.01, and **P < 0.001; ns, not significant. Healthy subjects, n = 30; patients with SCI, n = 75; cell experiments, n ≥ 4. Data are presented as the mean ± SD. Please click here to view a larger version of this figure.

DATA AVAILABILITY:
The datasets generated and/or analyzed in the current study are available in the Zenodo repository: https://doi.org/10.5281/zenodo.21470454.

Discussion

Spinal cord injury (SCI) is a severe traumatic disorder of the central nervous system that results in impaired or complete loss of neural conduction through the spinal cord, leading to lifelong motor, sensory, and autonomic dysfunction, as well as profound physiological and psychological consequences²⁹˒³⁰. The severity of these impairments generally increases with the extent of SCI. In the present study, plasma LINC00707 expression was elevated in patients with SCI and positively correlated with circulating pro-inflammatory cytokine levels, suggesting a potential role for LINC00707 in SCI-associated inflammation and oxidative stress. Based on these clinical findings, we further investigated the regulatory role of LINC00707 in inflammation and oxidative stress in LPS-treated BV2 cells in the context of SCI pathogenesis.

LINC00707 has been implicated in a variety of biological processes through the regulation of multiple signaling pathways³¹. In addition to regulating cell proliferation, apoptosis, migration, and invasion, LINC00707 has been shown to participate in inflammatory responses and oxidative stress¹⁸˒³²˒³³. Previous studies have reported elevated LINC00707 expression in lung³⁴, breast³⁵, cervical³⁶, and liver cancers³⁷, where it promotes disease progression by regulating downstream molecular targets. In addition, silencing LINC00707 promotes the osteogenic differentiation of bone marrow mesenchymal stem cells through regulation of miR-103a-3p³⁸. Zou et al. further demonstrated that LINC00707 promotes LPS-induced inflammatory responses in MRC-5 cells, supporting its pro-inflammatory role³³. Consistent with these findings, the present study showed that LINC00707 expression was significantly increased in LPS-stimulated BV2 cells, whereas LINC00707 knockdown reduced the production of pro-inflammatory cytokines and oxidative stress markers. These findings further support the involvement of LINC00707 in inflammatory and oxidative stress responses.

LINC00707 has also been reported to function as a competing endogenous RNA (ceRNA) by regulating miR-145-5p in several pathological conditions, including rheumatic heart disease³⁹, bladder cancer⁴⁰, and osteogenic differentiation⁴¹. Previous studies have demonstrated that miR-145-5p exerts protective effects in SCI by suppressing inflammation and oxidative stress⁴²˒⁴³ and by promoting angiogenesis and neural repair⁴⁴˒⁴⁵. Consistent with these observations, the present study showed that plasma miR-145-5p expression was decreased in patients with SCI and in LPS-stimulated BV2 cells. LINC00707 was further confirmed to interact directly with miR-145-5p, and silencing LINC00707 restored miR-145-5p expression. Moreover, inhibition of miR-145-5p reversed the protective effects of LINC00707 knockdown on inflammatory responses and oxidative stress. These findings suggest that LINC00707 may contribute to SCI progression by regulating miR-145-5p-mediated inflammatory and oxidative stress responses.

Previous studies have demonstrated that TRAF6 plays an important role in diseases of the central nervous system⁴⁶. Evidence also indicates that inhibiting TRAF6 can significantly improve SCI⁴⁷˒⁴⁸. The present study further identified TRAF6 as a direct target of miR-145-5p. TRAF6 expression was significantly increased in patients with SCI and in LPS-stimulated BV2 cells. In addition, LINC00707 knockdown reduced TRAF6 expression, whereas inhibition of miR-145-5p restored TRAF6 expression. These findings suggest that LINC00707 may regulate TRAF6 expression through miR-145-5p during SCI.

This study suggests that LINC00707 may affect microglia-mediated inflammatory responses and oxidative stress injury during SCI by regulating the miR-145-5p/TRAF6 signaling axis. This finding is consistent with a previous report by Zhu et al., who demonstrated that LINC00707 knockdown alleviated LPS-induced inflammation and apoptosis in PC-12 cells; however, the underlying mechanism in their study involved the miR-30a-5p/Neurod1 axis¹⁸. Collectively, these findings suggest that LINC00707 may not act through a single miRNA target but rather through distinct miRNA networks in a cell-type-dependent manner, thereby contributing to the pathological processes of SCI.

This study has several limitations. First, only TRAF6 mRNA expression was evaluated, whereas protein expression was not examined. Future studies should include protein-level validation, such as Western blot analysis, to further characterize the regulatory relationship among LINC00707, miR-145-5p, and TRAF6. Second, this study was conducted exclusively at the cellular level using an LPS-stimulated BV2 microglial model, without in vivo validation. Given the complexity of the SCI microenvironment, the current findings may not fully recapitulate the pathophysiological conditions in vivo. Whether LINC00707 exerts similar pro-inflammatory effects in animal models of SCI remains to be confirmed. Future studies will establish SCI animal models to validate the regulatory role of LINC00707 and its underlying molecular mechanisms. Third, although our results indicate that LINC00707 acts through the miR-145-5p/TRAF6 signaling pathway to influence inflammation, it remains unknown whether LINC00707 functions solely as a sponge for specific miRNAs or also binds directly to certain proteins. Further studies are required to clarify these mechanistic details. Fourth, the ROC curve analysis was based on data from a single medical center and a relatively small number of patients. This may have introduced bias, making the findings preliminary. Larger multicenter studies with more patients are needed to confirm the diagnostic potential of the identified biomarkers.

The findings of this study suggest that LINC00707 may represent a potential exploratory plasma biomarker and therapeutic target for SCI. However, translating these findings into clinical applications will require several additional steps. First, the potential discriminatory value of plasma LINC00707 should be evaluated in larger multicenter cohorts, independent validation cohorts, and relevant trauma or inflammatory control groups. Clinical assessment and radiological examination remain the primary approaches for diagnosing SCI. Second, the therapeutic potential of targeting LINC00707 should be evaluated in animal models to assess its efficacy and safety. Ultimately, modulating LINC00707/miR-145-5p/TRAF6 signaling may offer a novel approach to mitigating neuroinflammation and oxidative stress in patients with SCI, although substantial work remains to bridge the gap between bench and bedside.

Overall, plasma LINC00707 and TRAF6 expression were increased, whereas miR-145-5p expression was decreased, in patients with SCI and in LPS-stimulated BV2 cells. LINC00707 knockdown attenuated inflammatory responses and oxidative stress through the miR-145-5p/TRAF6 signaling axis, suggesting that this regulatory pathway may represent a potential therapeutic target for SCI.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tube	KIRGEN	KG2211	Polypropylene snap-cap tube, DNase/RNase-free, non-pyrogenic, maximum centrifugal force 20,000 × g, for RNA/protein sample storage and centrifugation.
6-Well Cell Culture Plate	Corning	3516	TC-treated polystyrene plate with a flat bottom, sterile, and individually wrapped; well volume is 16.8 mL with a growth area of 9.5 cm
Blood Collection Needle	Becton, Dickinson and Company	367283	BD Vacutainer Single-Use Blood Collection Needle. Double-ended needle design, with one end for venipuncture and the other connecting to the holder and vacuum tube for multi-tube collection. Silicone-coated to minimize patient discomfort during insertion.
Blood Collection Tubes	Becton, Dickinson and Company	367605	BD Vacutainer Evacuated Blood Collection Tubes. Different color-coded stoppers indicate various additives, corresponding to specific laboratory tests.
BV2 immortalized mouse microglial cell line	Cell Resource Center of the Shanghai Institute of Biotechnology	RRID: CVCL_0182	Immortalized murine microglia cell line that stably expresses microglia specific markers (Iba1, CD11b, F4/80). Upon LPS stimulation, BV2 cells secrete pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and accumulate intracellular ROS, MDA to mimic in vivo microglial inflammatory activation after spinal cord injury. Cells are cultured in high-glucose DMEM supplemented with 10% FBS and 1× penicillin-streptomycin, maintained at 37 °C with 5% CO2 humidified incubator; passaged when reaching 70–80% confluence to avoid overactivation.
Cell Malondialdehyde (MDA) Assay Kit	Nanjing Jiancheng Bioengineering Institute	A003-4-2	Microplate-based colorimetric kit for quantitative detection of cellular malondialdehyde, a marker of lipid peroxidation. MDA reacts with thiobarbituric acid (TBA) under heating to form red adducts; absorbance is detected at 532 nm by microplate spectrophotometer. Optimized for cell lysate samples, high-throughput 96-well operation, linear quantification range covers low to moderate lipid oxidation levels in microglia.
CO2 incubator	Thermo Scientific	3111	Humidified water-jacketed CO2 incubator with 184 L inner volume, IR CO2 sensor, HEPA air filtration system to minimize microbial contamination. Maintains stable culture environment at 37 °C, 5% CO2 and saturated humidity for long-term adherent cell culture.
Dual Luciferase Reporter Gene Assay Kit	Beyotime Biotechnology	RG028	This dual-luciferase kit enables sequential quantitative detection of firefly luciferase and Renilla luciferase in cell lysates. Firefly luciferase signal is first measured using luciferin substrate; the subsequent Renilla detection reagent contains a potent quencher that eliminates >99.9% residual firefly luminescence, ensuring pure Renilla signal readout with coelenterazine substrate. The kit includes dedicated cell lysis buffer for adherent cell lysis, compatible with 96-well microplate luminometer. Firefly reagent is pre-mixed liquid for direct use; luminescence values of firefly and Renilla are recorded sequentially to calculate normalized relative luciferase activity (Firefly/Renilla ratio) for ceRNA target validation.
Dulbecco's Modified Eagle Medium	Gibco	11995065	DMEM is a widely used basal medium for supporting the growth of many different mammalian cells. This formulation contains high glucose, L-glutamine, phenol red, and sodium pyruvate. It requires supplementation, commonly with 10% Fetal Bovine Serum (FBS), and a 5–10% CO2 environment to maintain physiological pH.
Fetal Bovine Serum	EVERY GREEN	13011-8611	Premium grade FBS, low endotoxin, mycoplasma & bacteriophage negative, supplemented to DMEM at 10% v/v to support cell growth.
GraphPad Prism Version 9	GraphPad Software, San Diego, CA, USA	NA	Scientific graphing and statistical analysis software specialized for in vitro cell experimental data. All raw qPCR, ELISA, oxidative stress, dual-luciferase luminescence absorbance values were imported into Prism 9 to conduct normality verification, parametric/nonparametric group comparisons, generate all quantitative figures, calculate mean ± SD, exact P values and 95% CIs, and export high-resolution (300 dpi) figures meeting journal publication standards. Original .prism project source files of all graphs are deposited in the public data repository as required.
IBM SPSS Statistics Version 26.0	IBM Corporation, Armonk, NY, USA	NA	Comprehensive statistical software package used for clinical sample baseline data sorting, normality testing (Shapiro-Wilk test), independent/unpaired t-test, one-way ANOVA with Tukey’s multiple comparison correction, Mann-Whitney U nonparametric test, and calculation of exact P-values and 95% confidence intervals for all human and cellular quantitative datasets. All clinical patient demographic and laboratory data were processed and analyzed within this platform.
IL-1β SimpleStep ELISA Kit	Abcam	ab255730	This one-wash sandwich colorimetric ELISA kit adopts proprietary SimpleStep technology, completing the whole detection procedure within 90 min. It quantitatively measures rat interleukin-1β in cell culture supernatant, serum, EDTA/citrate/heparin plasma samples. The detection sensitivity reaches 26.58 pg/mL, with linear quantification range of 54.69–3500 pg/mL. Pre-coated breakable 8-well strip microplate is included. Absorbance signals are detected at 450 nm via microplate reader to calculate IL-1β concentration.
IL-6 SimpleStep ELISA Kit	Abcam	ab234570	A sandwich colorimetric SimpleStep ELISA kit optimized for quantitative detection of rat interleukin-6 (IL-6) in cell culture supernatant, serum, citrate/EDTA/heparin plasma samples. Adopts one-step incubation and single-wash workflow to shorten total assay time to 90 min, with a detection sensitivity of 43 pg/mL and linear quantification range from 125 pg/mL to 8000 pg/mL. Pre-coated breakable 8-well strip microplate is included; absorbance values are read at 450 nm using a microplate spectrophotometer for final IL-6 concentration calculation.
LightCycler Multiwell Plate 96, white	Roche LifeScience, Switzerland	4729692001	Half-skirted white polypropylene 96-well reaction plates specially optimized for the LightCycler 96 real-time PCR instrument. The white well walls reduce background fluorescence interference and enhance signal reflection to improve detection sensitivity for low-abundance transcripts. Uniform thin-wall design guarantees consistent thermal conductivity across all wells, compatible with reaction volumes ranging from 10 to 50 μL. DNase/RNase-free and pyrogen-free for RNA-derived cDNA amplification assays.
LipofectamineTM 3000 Transfection Reagent	Thermo Fisher Scientific	L3000015	Lipid nanoparticle transfection reagent paired with P3000 Enhancer, enables high-efficiency siRNA/plasmid transfection in BV2 microglia with low cytotoxicity.
lnRcute lncRNA First-Strand cDNA Synthesis Kit	TIANGEN	KR202-02	This kit is specially optimized for reverse transcription of long non-coding RNAs (lncRNAs) with low abundance, variable GC content and complex secondary structures. It contains independent gDNA removal buffer to eliminate genomic DNA contamination before reverse transcription, and specialized lnR-RT primer mix to enrich full-length lncRNA templates. The modified reverse transcriptase enables efficient synthesis of long-strand cDNA from total RNA input ranging from 10 ng to 2 ng.
lnRcute lncRNA qPCR Detection Kit (SYBR Green)	TIANGEN	FP402	A dedicated SYBR Green-based quantitative PCR premix for lncRNA quantification. The antibody-mediated hot-start DNA polymerase ensures high amplification specificity; H-competitor components relieve secondary structure interference of high-GC lncRNA templates. Compatible with cDNA generated from lnRcute lncRNA reverse transcription kit, and suitable for low-abundance lncRNA relative expression quantification .
miR-145-5p oligonucleotides	Generalbiol (Wuhan, China)	NA	Synthetic miR-145-5p mimic, inhibitor, and corresponding negative-control oligonucleotides used to modulate miR-145-5p activity in BV2 cells. Used in expression, inflammatory, oxidative stress, and dual-luciferase reporter assays.
miRcute Plus miRNA First-Strand cDNA Kit	TIANGEN	KR211-02	Adopts combined poly (A)-tailing and reverse transcription one-tube reaction to simplify experimental workflow. The enzyme mix contains E. coli poly (A) polymerase, RNase H-deficient reverse transcriptase and RNase inhibitor, which specifically targets mature single-stranded miRNA and avoids amplification interference from pre-miRNA with hairpin structures. Suitable for miRNA extracted from cell lysate, plasma, tissue samples.
miRcute Plus miRNA qPCR Detection Kit (SYBR Green)	TIANGEN	FP411	SYBR Green premix optimized for mature miRNA quantification, matched perfectly with miRcute reverse transcription products. The buffer system improves primer annealing efficiency for short miRNA amplicons, delivers stable fluorescence signals and high sensitivity for detecting weakly expressed miRNAs. U6 snRNA is commonly used as internal reference for normalization.
Penicillin-Streptomycin Solution (100×)	Beyotime Biotechnology	C0222	Sterile filtered mixture containing 10,000 U/mL penicillin and 10 mg/mL streptomycin; working dilution 1:100 in culture medium to prevent bacterial contamination.
Reactive Oxygen Species Assay Kit with Diluent	Beyotime Biotechnology	S0034M	This kit adopts DCFH-DA fluorescent probe to quantify intracellular reactive oxygen species (ROS). DCFH-DA penetrates cell membrane and is hydrolyzed by intracellular esterase to DCFH, which is oxidized by ROS to generate fluorescent DCF. Compatible with 96-well microplate and fluorescence microplate reader for high-throughput detection (Ex = 488 nm, Em = 525 nm). Equipped with dedicated probe diluent and Rosup positive control reagent to stabilize staining signals, suitable for adherent BV2 microglia oxidative stress detection without cell lysis.
Reduced Glutathione (GSH) Assay Kit	Nanjing Jiancheng Bioengineering Institute	A006-1-2	Colorimetric microplate kit for detecting reduced glutathione (GSH), the core intracellular antioxidant. GSH reacts with dithiodinitrobenzoic acid (DTNB) to produce yellow chromogenic product, absorbance detected at 405 nm on microplate reader. The kit adapts standard tube protocol to high-throughput 96-well microplate detection for cell lysate antioxidant analysis.
si-LINC00707	GenePharma (Shanghai, China)	NA	Small interfering RNA designed to reduce LINC00707 expression in BV2 cells. Used for loss-of-function experiments to assess the effects of LINC00707 knockdown on inflammatory responses, oxidative stress, miR-145-5p expression, and TRAF6 expression.
si-LINC00707 NC	GenePharma (Shanghai, China)	NA	Non-targeting small interfering RNA used as the negative control for si-LINC00707 transfection. Used to control for nonspecific effects of the transfection procedure and siRNA exposure.
SuperReal PreMix Plus (SYBR Green)	TIANGEN	4992777	Universal SYBR Green quantitative PCR premix for mRNA relative quantification, containing hot-start Taq polymerase, dNTPs, SYBR Green I and ROX reference dye for fluorescence normalization. Low non-specific amplification, stable amplification efficiency, widely applied for mRNA target gene quantification (TRAF6, GAPDH etc.) .
Thermo ScientificTM SorvallTM ST 8 Small Benchtop Centrifuge	Thermo Scientific	75200709	The classic choice for clinical laboratories, capable of handling up to 24 × 5/7 mL of blood collection tubes at one time. The R series is a refrigerated version, which can better protect samples that require low-temperature centrifugation.
TIANScript II First-Strand cDNA Synthesis Kit	TIANGEN	GKR107-02	Designed for general mRNA reverse transcription to generate long-length cDNA up to 12 kb. Equipped with both Oligo (dT) 15 and random hexamer primers for flexible priming strategies. The modified M-MLV reverse transcriptase efficiently reads through high-GC and complex secondary mRNA structures, with broad compatibility for downstream mRNA qPCR amplification.
TNF-α SimpleStep ELISA Kit	Abcam	ab236712	A rapid single-wash SimpleStep sandwich ELISA kit optimized for quantitative detection of rat tumor necrosis factor-α (TNF-α) in cell culture supernatant, serum and various plasma specimens. The total assay duration is 90 min, with ultra-high sensitivity of 1.04 pg/mL and linear detection range from 15.63 pg/mL to 1000 pg/mL. Equipped with detachable 8×12 well pre-coated plate; OD values at 450 nm are recorded for final TNF-α quantitative calculation.
Total Superoxide Dismutase (T-SOD) Assay Kit	Nanjing Jiancheng Bioengineering Institute	A001-3-2	WST-1 chromogenic microplate kit to detect total SOD enzymatic activity. SOD eliminates superoxide anion radicals and inhibits WST-1 reduction reaction. Absorbance recorded at 450 nm via microplate reader for activity calculation. Suitable for cell homogenate, serum and tissue samples, with stable color development and high repeatability for antioxidant capacity evaluation.
TRIzol Reagent	Thermo Fisher Scientific	15596018	Monophasic phenol-guanidine isothiocyanate reagent for one-step isolation of total RNA from cultured microglia cells; simultaneously extracts DNA and protein from identical samples.