Overexpression of long non-coding RNA LINC00158 inhibits neuronal apoptosis by promoting autophagy in spinal cord injury :Fuchuang Qin, Guorong He, Yu Sun, Guangning Chen, Qijian Yu, Xilie Ma, Chinese Journal of Physiology

ORIGINAL ARTICLE
Year : 2022 | Volume : 65 | Issue : 6 | Page : 282--289

Overexpression of long non-coding RNA LINC00158 inhibits neuronal apoptosis by promoting autophagy in spinal cord injury

Fuchuang Qin¹, Guorong He², Yu Sun¹, Guangning Chen¹, Qijian Yu³, Xilie Ma⁴,
¹ Department of Neurosurgery, Shulan (Anji) Hospital, Anji County, Huzhou 313300, Zhejiang, China
² Department of Neurosurgery, Xixi Hospital of Hangzhou, Hangzhou 310023, Zhejiang, China
³ Department of Neurosurgery, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China
⁴ Department of Osteology, Hangzhou Red Cross Hospital, Hangzhou 310004, Zhejiang, China

Correspondence Address:
Dr. Xilie Ma
Department of Osteology, Hangzhou Red Cross Hospital, No. 208 Huancheng East Road, Hangzhou 310004, Zhejiang
China

Abstract

Spinal cord injury (SCI) is a common central nervous system disease. It is reported that long non-coding RNA LINC00158 is involved in the process of SCI. The purpose of this study was to explore the biological role of LINC00158 in the SCI. First, we established a rat SCI model by surgical method and evaluated the motor function of rats by the Basso-Beattie-Bresnahan locomotor rating scale. The results showed that the expression of LINC00158 decreased and apoptotic cells increased in the SCI model rats. Meanwhile, we found the upregulated LC3-II/LC3-I, Beclin-1, and p62 in the SCI rats. Then, primary rat spinal cord neurons were exposed to oxygen/glucose deprivation (OGD) as an in vitro cell model of SCI. After OGD treatment, the expression of LINC00158 decreased significantly and the apoptosis of spinal cord neurons increased. OGD treatment resulted in upregulation of LC3-II/LC3-I and Beclin-1 and downregulation of p62 in primary spinal cord neurons, which could be eliminated by overexpression of LINC00158. 3-Methyladenine and chloroquine (autophagy inhibitor) reversed the inhibitory effect of LINC00158 overexpression on apoptosis of primary spinal cord neurons. In conclusion, this study demonstrated that LINC00158 overexpression repressed neuronal apoptosis by promoting autophagy, suggesting that LINC00158 may be a potential therapeutic target in the SCI.

How to cite this article:
Qin F, He G, Sun Y, Chen G, Yu Q, Ma X. Overexpression of long non-coding RNA LINC00158 inhibits neuronal apoptosis by promoting autophagy in spinal cord injury.Chin J Physiol 2022;65:282-289

How to cite this URL:
Qin F, He G, Sun Y, Chen G, Yu Q, Ma X. Overexpression of long non-coding RNA LINC00158 inhibits neuronal apoptosis by promoting autophagy in spinal cord injury. Chin J Physiol [serial online] 2022 [cited 2023 Dec 5 ];65:282-289
Available from: https://www.cjphysiology.org/text.asp?2022/65/6/282/360035

Full Text

Introduction

Spinal cord injury (SCI) is a common central nervous system (CNS) disease, and is one of the diseases with a high disability rate in humans. SCI can cause the severe and continuous motor and sensory dysfunction in patients, which severely affects human health.[1] The pathological process of SCI is divided into primary injury and secondary injury. Primary injury refers to a direct mechanical injury that cannot be avoided or interfered with due to compression, abrasion, or swelling. Secondary injuries mainly include nerve cell apoptosis, axon demyelination and severance, microglia activation, and glial scar formation.[2] Finding effective targets for the treatment or suppression of secondary damage can help promote functional recovery after SCI.

Accumulating studies have confirmed that long non-coding RNAs (lncRNAs) play a crucial role in the progression of SCI.[3],[4] Targeted regulation of the corresponding lncRNA expression can inhibit secondary injury and promote functional recovery after SCI, which is of great significance for the design of new molecular treatment programs. Wang et al. have carried out a large-scale screening of expression changes of lncRNA in SCI rats, showing that lncRNA-SCIR1 is downregulated in SCI rats.[5] SCIR1 silencing significantly promotes the proliferation and migration of astrocytes, thereby promoting the formation of scars at injured sites after SCI. Subsequently, many lncRNAs have been reported to be abnormally expressed in SCI, including DGCR5, SNHG5, NEAT1, CasC7, and ZNF667-AS1.[6],[7],[8],[9],[10] These lncRNAs affect the progression of SCI by regulating neuronal apoptosis, neural stem cell differentiation, and microglia activation. LINC00158 has been reported being abnormally expressed in SCI, suggesting that overexpression of LINC00158 may have a certain therapeutic effect on SCI.[11]

In recent years, the study of autophagy in SCI has gradually become a hot spot. The role of autophagy in SCI has two sides.[12] On the one hand, autophagy induces autophagic cell death and participates in the occurrence of apoptosis. On the other hand, autophagy promotes the metabolism of damaged and denatured proteins and inhibits apoptosis. The degree of the activation of early autophagy has a decisive effect on the repair after SCI. Appropriately increased autophagy levels after SCI promote the metabolism of damaged and denatured proteins and inhibit cell apoptosis. However, excessive activation of autophagy may trigger autophagic cell death. It is currently unclear whether LINC00158 can inhibit SCI-induced neuronal apoptosis by regulating autophagy. Thus, this study will detect the expression of LINC00158 in SCI rats and further investigate the effect of LINC00158 on autophagy and neuronal apoptosis in SCI.

Materials and Methods

Animals

Male Sprague-Dawley (SD) rats (6-7 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). All rats were housed under SPF conditions with 12-h light/dark and fed with food and water. The rat use and care protocol was authorized by the Ethics Committee of Shulan (Hangzhou) Hospital (No. 21467).

Spinal cord injury rat model

SCI rat model was established as the previous protocol described.[13] In briefly, SD rats were anesthetized with intraperitoneal injection of 1% pentobarbital (5 mg/100 g). Anesthetized rats were kept in prone position on the console. Laminectomy was performed to thoroughly expose the spinal cord at the thoracic (T) 9-T10 spinous process. The exposed spinal cord was subjected to impact trauma by compression at an interval of 12.5 mm to produce severe injury. The muscle and skin were sutured in layers. The success of SCI model was determined by the lower limb paralysis, tail swinging, and spinal cord hematoma formation. Sham-operated rats were only subjected to laminectomy as control. After the SCI surgery, the urinary bladder of rats was subjected to manual massage twice per day until autonomous bladder voidance reflex recovered.

After modeling, SD rats were euthanized with 10% chloral hydrate (10 mg/kg) by intraperitoneal injection. The spinal cord centered at the injury epicenter with 10 mm was harvested at 1, 3, and 7 days after the surgery for further use.

Behavioral assessment

The Basso–Beattie–Bresnahan (BBB) locomotor rating scale was carried out to evaluate the locomotor function of rats at 1, 3, 7, 14, 21, and 28 days after the surgery as previously described.[14] In brief, rats were placed on the platform for 4 min, and the limb exercise of hindlimbs were scored and observed by three independent observers blinded to the experimental groups. Rats were assigned to the following three categories according to the BBB scores (0–21 points): (1) Early stage (BBB score 0–7): Rats exhibited isolated joint movements with little or no hindlimb movement; (2) Intermediate stage (BBB score 8–13): Rats with occasional uncoordinated stepping; (3) Late stage (BBB score 14–21): Rats were capable of hindlimb coordination, equilibrium, and stepping.

TUNEL staining

Colorimetric TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China) was used to examine apoptosis in the spinal cord tissues. Spinal cord tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Following dewaxing and hydration, the paraffin sections of spinal cord tissues were obtained. Sections were incubated with Biotin-dUTP at 37°C in darkness for 1 h. Then, sections were stained with Streptavidin-horseradish peroxidase (HRP) and diaminobenzidine (DAB) successively. Finally, the sections were observed under an optical microscope (Olympus, Tokyo, Japan).

Primary spinal cord neuron culture

Primary spinal cord neurons were separated from the spinal cords of 15-day-old SD rat embryos as described in the previous study.[15] Briefly, spinal cords were dissected from rat embryos; the meninges and attached dorsal root ganglia were removed. Spinal cords were cut into pieces and digested with 0.125% trypsin (Sigma-Aldrich, MO, USA) at 37°C for 30 min. Subsequently, spinal cords were ground to single neurons using a pipette. The neuron suspension was seeded into 0.01% poly-d-lysine-coated 24-well plates for cell adhesion. Neurons were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Invitrogen, CA, USA), containing 10% fetal bovine serum (Gibco), 5% horse serum (Gibco), and 1% penicillin/streptomycin (Sangon-Biotech, Shanghai, China) at 37°C and 5% CO2. Ara-C (5 μM, Sigma-Aldrich) was used to treat neurons on the third cultured day and incubated for additional 24 h to purify the neurons. The cellular morphology of primary spinal cord neurons was observed under an optical microscope.

For oxygen/glucose deprivation (OGD) treatment, primary spinal cord neurons were maintained in glucose-free DMEM and placed in a hypoxic chamber of a Ruskin Bugbox Plus (Ruskinn Technology, Ltd., Cardiff, UK) at 37°C with 95% N2 and 5% CO2 for 4 h. Primary spinal cord neurons were cultured in normal DMEM under normal conditions as control. Primary spinal cord neurons were treated with 5 mM 3-methyladenine (3-MA, MedChem Express, Monmouth Junction, NJ, USA) to inhibit autophagy.

Cell transfection

Full length of LINC00158 was subcloned into the vector pcDNA3.1, generating the vector pcDNA3.1-LINC00158 (GeneChem, Shanghai, China). The empty vector pcDNA3. 1-NC served as control. The vectors pcDNA3.1-LINC00158 and pcDNA3.1-NC were packaged into lentivirus particles (LV-LINC00158 and LV-NC). Primary spinal cord neurons were infected with LV-LINC00158 and LV-NC using 5 μg/mL polybrene. After 48 h of infection, the primary spinal cord neurons were collected for further use.

Quantitative real-time polymerase chain reaction

The total RNA from spinal cord tissues and primary spinal cord neurons was extracted using TRIzol reagent (Invitrogen) as the protocol described. RNA integrity was examined by 1.5% agarose gel electrophoresis. Complementary DNA was generated applying TaqMan Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression of LINC00158 was estimated by quantitative real-time polymerase chain reaction (qRT-PCR) using SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 7500 sequence detection system (Thermo Fisher Scientific). Glyceraldehyde-3-phosphate dehydrogenase was served as reference gene for normalization. The results were analyzed using 2−ΔΔCT method for quantification.

Western blotting

The total protein from spinal cord tissues and primary spinal cord neurons was extracted using Total Protein Extraction Kit (Solarbio). BCA Protein Assay Kit (Solarbio) was used to estimate the concentration of proteins. Protein samples were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis protein electrophoresis and then transferred onto polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk at room temperature for 2 h. Subsequently, the membranes were incubated with the primary antibodies, LC3 (#14600-1-AP; 1:1000; Proteintech, Wuhan, China), Beclin-1 (#11306-1-AP; 1:1000; Proteintech), or p62 (#13916-1-AP; 1:1000; Proteintech) at 4°C for 12 h. HRP-conjugated immunoglobulin G antibody (#SA00001-2; 1:5000; Proteintech) was incubated with the membranes at room temperature for 2 h. β-actin antibody (#20536-1-AP; 1:5000; Proteintech) was used as a reference protein for normalization. The data were analyzed by ImageJ (National Institutes of Health, Bethesda, MD, USA) software.

Flow cytometry

Flow cytometry was performed to assess apoptosis of primary spinal cord neurons using Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China). Primary spinal cord neurons were collected by centrifugation and then washed with phosphate buffer saline for several times. Neurons were mixed with 100 μL 1 × binding buffer. The cell suspension was incubated with 5 μL Annexin V-FITC and 10 μL PI staining solution at darkness for 15 min. Subsequently, the cell suspension was mixed with 400 μL 1 × Binding Buffer and put on ice. Cell apoptosis was determined by flow cytometry (BD Biosciences, San Jose, CA, USA) in an hour.

Statistical analysis

Each assay was performed three times. All data were reported as mean ± standard deviation. SPSS 22.0 statistical software (IBM, Armonk, NY, USA) was used for statistical analysis. Two-tailed Student's t-test and one-way ANOVA were used to analyze the statistical difference. P < 0.05 was considered as a significant difference.

Results

LINC00158 was downregulated and neuronal apoptosis was increased in spinal cord injury rats

To explore the underlying mechanism of LINC00158 in SCI, we constructed SCI rat model. The general locomotor recovery of SCI was evaluated by BBB score. In [Figure 1]a, 3 days after the surgery, the motor function of SCI rats began to gradually recover. At 28 days after operation, the BBB score of SCI rats was the highest; but, it was still lower than that of the Sham rats. Subsequently, we compared the expression of LINC00158 between SCI rats and Sham rats by qRT-PCR. LINC00158 was significantly downregulated in the spinal cord tissues of SCI rats at 1, 3, and 7 days after operation [Figure 1]b. Moreover, results of TUNEL staining uncovered that apoptotic cells were increased in the spinal cord tissues of SCI rats at 1, 3, and 7 days after operation with respect to Sham rats [Figure 1]c and [Figure 1]d. Thus, these data suggested that LINC00158 was downregulated, and neuronal apoptosis was increased in SCI rats.{Figure 1}

Autophagy was increased in spinal cord injury rats

The expression of autophagy-related proteins in rats was detected by Western blotting. Results of [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d uncovered that the expression of LC3-II/LC3-I, Beclin-1, and p62 was increased and then reduced in the spinal cord tissues with prolongation of SCI. These findings indicated that autophagy was increased in SCI rats.{Figure 2}

LINC00158 overexpression reduced apoptosis in OGD-treated primary spinal cord neurons

Next, we investigated the biological role of LINC00158 in SCI in vivo. Primary spinal cord neurons were isolated from the spinal cords of rat embryos. The morphology of neurons was pleomorphic [Supplementary Figure 1]. Primary spinal cord neurons were subjected to OGD treatment to mimic the SCI conditions. QRT-PCR data showed that LINC00158 was severely downregulated in the OGD-treated primary spinal cord neurons [Figure 3]a. Furthermore, LINC00158 was overexpressed in primary spinal cord neurons. The expression of LINC00158 was upregulated in the primary spinal cord neurons in the presence of LV-LINC00158 [Figure 3]b. We performed CCK8 assay and flow cytometry to estimate the impact of LINC00158 overexpression on cell viability and apoptosis of primary spinal cord neurons. OGD treatment decreased cell viability and caused an increase of apoptosis in primary spinal cord neurons. LINC00158 overexpression notably increased cell viability and reduced apoptotic cells in OGD-treated primary spinal cord neurons [Figure 3]c, [Figure 3]d, [Figure 3]e. In addition, we also detected the expression of apoptosis-related proteins (Bax and cleaved caspase-3), and the Western blot results showed that OGD treatment increased the expression of Bax and cleaved caspase-3, but overexpression of LINC00158 significantly reversed the effect of OGD on the the expression of apoptosis-related proteins [Figure 3]f, [Figure 3]g, [Figure 3]h. Thus, these data suggested that LINC00158 overexpression reduced apoptosis in OGD-treated primary spinal cord neurons.{Figure 3}[INLINE:1]

LINC00158 overexpression suppressed apoptosis of oxygen/glucose deprivation-treated primary spinal cord neurons by promoting autophagy

The influence of LINC00158 overexpression on autophagy of primary spinal cord neurons was assessed by Western blotting. OGD treatment led to an upregulation of LC3-II/LC3-I and Beclin-1 and a downregulation of p62 in primary spinal cord neurons. LINC00158 upregulation further enhanced the expression of LC3-II/LC3-I and Beclin-1 and repressed p62 expression in OGD-treated primary spinal cord neurons [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d. In addition, primary spinal cord neurons were treated with 3-MA or chloroquine to inhibit autophagy. The results of flow cytometry revealed that LINC00158 overexpression reduced apoptotic cells in OGD-treated primary spinal cord neurons, which was effectively abolished by 3-MA or chloroquine treatment [Figure 4]e, [Figure 4]f, [Figure 4]g, [Figure 4]h. Taken together, these data confirmed that LINC00158 overexpression suppressed OGD-induced apoptosis in primary spinal cord neurons by promoting autophagy.{Figure 4}

Discussion

Accumulating studies have confirmed that lncRNAs play a crucial role in the progression of SCI. Wang et al. have used high-throughput RNA sequencing to identify lncRNA expression profiles in the lesion epicenters of spinal tissues after acute traumatic SCI.[16] This study confirmed that lncRNA ENSMUST00000195880 is abnormally expressed in acute traumatic SCI, and lncRNA ENSMUST00000195880 participates in the pathophysiology of ATSCI by targeting miR-21a-5p. LncRNA NEAT1 silencing reduces neuronal apoptosis and attenuates lesions in the spinal cord by sponging miR-29b and thus represses regeneration after SCI.[17] LncRNA Airsci enhances the inflammatory response through activation of the NF-κB signaling pathway, thereby alleviating the injury of spinal cord tissues and promoting motor function recovery in SCI rats.[18] The previous studies have reported that LINC00158 is associated with severe asthma and rheumatoid arthritis.[19],[20] However, the biological role of LINC00158 in SCI has not been reported. In these data, we found that the expression of LINC00158 was deceased in SCI rats, suggesting that LINC00158 was closely associated with SCI.

Autophagy is a key cellular pathway that plays a crucial role in cellular survival under stress conditions, and it is one of the major catabolic mechanisms in eukaryotes for the degradation of cytoplasmic constituents through the autophagosomal-lysosomal pathway. Previously, many landmark studies have shown that autophagy plays a significant role in chronic CNS disorders such as amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's dementia. Findings from these studies have sparked considerable interest into investigating the role of autophagy in acute CNS injuries like SCI, and a growing body of evidence suggests that autophagy plays a critical role in SCI as well.[21],[22] The expression of autophagy-related proteins, LC3-II/LC3-I, Beclin-1, and p62 was increased and then reduced in SCI rats. Increased levels of autophagy after SCI promote the metabolism of damaged and denatured proteins and inhibit cell apoptosis, which may be a selfprotection mechanism of cells. Meanwhile, OGD treatment promoted the expression of LC3-II/LC3-I and Beclin-1 and repressed p62 expression in primary spinal cord neurons. The expression of p62 levels in the in vivo (SCI) and in vitro (OGD) studies was different. In vivo, the ratio of LC3-II/LC3-I and the expression of Beclin-1 first increased and then decreased after SCI modeling. On the 7th day of SCI modeling, there was no significant difference with Sham group. The expression of p62 first increased and then decreased, which was significantly higher than that of Sham group by the 7th day after SCI modeling. In vivo results showed that after SCI modeling, autophagy was activated in the injured spinal cord of rats; but with the extension of modeling time, autophagy returned to normal. In vitro, LC3-II/LC3-I ratio, and Beclin-1 expression were significantly increased in OGD-treated rat neurons, while p62 expression was significantly decreased. The results in vitro showed that OGD induced autophagy in neurons. The results of LC3-II/LC3-I ratio and Beclin-1 expression in vitro and in vivo were consistent, indicating that SCI modeling and OGD treatment induced autophagy. This is consistent with the existing literature reports.[23],[24],[25] p62 is a ubiquitin binding protein, which is closely related to the ubiquitination of proteins, and participates in a variety of cell signal transduction regulation and autophagy processes. In the process of autophagy, p62 binds to ubiquitinated proteins and forms a complex with LC3-II protein located on the inner membrane of autophagosome, which is degraded in autophagy lysosome. Therefore, when autophagy occurs, p62 protein is continuously degraded in the cytoplasm; when autophagy activity is weakened and autophagy function is defective, p62 protein will continuously accumulate in the cytoplasm. In fact, the increased expression of p62 in vivo may be due to the activation of autophagy in vivo immediately after SCI modeling in rats, resulting in a large accumulation of p62 and failure to degrade in time. With the extension of SCI modeling time, autophagy activity decreased gradually, and p62 protein accumulated in the cytoplasm. Therefore, the expression of p62 was significantly increased after SCI modeling, which can still indicate the increase of autophagy level in vivo after SCI modeling.

Apoptosis plays a crucial role in the progression of SCI, and many molecules participate in SCI development by regulating apoptosis. For instance, knockdown of MSK1 induces inflammatory response and neuronal apoptosis, thereby inhibiting the functional recovery after SCI.[26] Gao et al. have confirmed that miR-137 represses H2O2-induced inflammatory response and apoptosis of C8-D1A and C8-B4 cells by interacting with MK2 in SCI.[27] LncRNA TCTN2 overexpression enhances miR-216b/Beclin-1 pathway-dedicated autophagy to repress apoptosis of nerve cells, thereby ameliorating the progression of SCI.[28] The deficiency of LINGO-1 suppresses cell apoptosis, inflammatory response, and glial scar, thereby promoting nerve regeneration in SCI mice.[29] In our study, our data revealed that LINC00158 was downregulated in OGD-treated primary spinal cord neurons. Moreover, OGD treatment enhanced apoptosis of primary spinal cord neurons, which was abrogated by LINC00158 overexpression. Thus, these data confirmed that LINC00158 regulated apoptosis of primary spinal cord neurons to affect the development of SCI.

Moreover, LINC00158 overexpression repressed apoptosis of OGD-treated primary spinal cord neurons, which was abolished by 3-MA treatment. 3-MA is an autophagy inhibitor that specifically inhibits autophagy activity.[21] Therefore, these results demonstrated that LINC00158 overexpression suppressed apoptosis of OGD-treated primary spinal cord neurons by promoting autophagy.

Conclusion

Our data demonstrate that LINC00158 overexpression represses OGD-induced neuronal apoptosis by promoting autophagy. Thus, this study suggests that LINC00158 may be a potential therapeutic target in SCI.

Acknowledgment

This study was funded by the Basic Public Welfare Research Program of Zhejiang Province.

Financial support and sponsorship

This study was funded by the Basic Public Welfare Research Program of Zhejiang Province (grant number: GF19H090011).

Conflicts of interest

There are no conflicts of interest.

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