Inhibition of TERC inhibits neural apoptosis and inflammation in spinal cord injury through Akt activation and p-38 inhibition via the miR-34a-5p/XBP-1 axis

2.1 Animal experiment

Twenty-four male Wistar rats (10–12 weeks old, and weighing around 230–250 g) were purchased from the Model Animals Institute of Nanjing University (Nanjing, China), and were maintained at 21–23°C, in 55 ± 5% humidity, under a 12 h:12 h circadian cycle, with free access to food and water. All the rats were randomly assigned to four groups (n = 6 per group): Sham group, SCI group, ShNC + SCI group, and ShTERC + SCI group. For SCI induction [15], following anesthetization using 35 mg/kg pentobarbital sodium (P-010, Sigma-Aldrich, USA), the rats in the SCI, ShNC + SCI, and ShTERC + SCI groups were incised at the back posterior to the lower thoracic region. Back muscles were separated to expose the dorsal surface of the spinal cord at T10, after which the lower thoracic cord was transected by using sterile scissors. Lentiviral vectors carrying ShTERC or ShNC (lentivirus-ShTERC/ShNC) were constructed by Genepharma (Shanghai, China). For TERC knockdown, 8 µL of normal saline containing lentivirus-ShTERC/ShNC (10⁷ TU/mL) was injected to each rat in the ShTERC + SCI and ShNC + SCI groups at two sites, 2 mm away from the margin of the head or back ends of the incision and 1 mm away from the central vein of the back. The rats in the Sham group were given anesthetization using 35 mg/kg pentobarbital sodium, without surgery and lentivirus injection. Twelve hours after SCI induction, all the rats were sacrificed by decollation under anesthetization and their spinal cords were harvested.

2.2 Hematoxylin–eosin staining

Rat spinal cords were fixed in 4% paraformaldehyde (16005, Sigma-Aldrich, St. Louis, MO, USA) for 24 h, followed by transparentization using xylene (95682, Sigma-Aldrich, USA), dehydration with gradient ethanol, and embedment in paraffin (1496904, Sigma-Aldrich, USA). The paraffin-embedded spinal cords were sliced into 5 μm thick sections by using a slicer (pfm3005E, Dakewe Biotech Co., Ltd, Shenzhen, China), dewaxed by xylene, and rehydrated by gradient ethanol. Then, the sections were stained with hematoxylin (H3136, Sigma-Aldrich, USA) for 7 min, and later differentiated by 1% hydrochloric alcohol (56694, Sigma-Aldrich, USA) for the removal of excessive pigments. After developing blue in weakly alkaline water, the sections were stained with eosin (E4009, Sigma-Aldrich, USA) for 2 min. Following rinse with distilled water, dehydration using gradient ethanol, and transparentization utilizing xylene, the stained sections were observed by an optical microscope (IX71; Olympus, Tokyo, Japan) under ×100 magnification.

2.3 Cell culture and treatment

Rat adrenal pheochromocytoma PC-12 cells were purchased from Procell (CL-0412, Wuhan, China), and were cultured in RPMI-1640 media (A4192301, Thermo Fisher, USA) supplemented with 15% horse serum (164215, Procell, China), 5% fetal bovine serum (FBS, F2442, Sigma-Aldrich, USA), and 1% streptomycin–penicillin (V900929, Sigma-Aldrich, USA) at 37°C with 5% CO₂. LPS ( L7770, Sigma-Aldrich, USA) was used to establish SCI on PC-12 cells [17]. PC-12 cells were treated by LPS at different concentrations (2, 4, 8, and 16 μg/mL) for 12 h.

2.4 Cell transfection

miR-34a-5p mimic/inhibitor, and mimic/inhibitor control were purchased from RIBOBIO (miR10000815-1-5/miR20000815-1-5 and miR1N0000001-1-5/miR2N0000001-1-5, Guangzhou, China). ShTERC was constructed with MISSION pLKO.1-puro eGFP shRNA Control plasmids (SHC005, Sigma-Aldrich, USA) and sequence (Forward Oligo: 5′-CCGGTAGCTGTGGGTTCTGTTCTTTCTCGAGAAAGAACAGAACCCACAGCTATTTTTG-3′; Reverse Oligo: 5′-AATTCAAAAATAGCTGTGGGTTCTGTTCTTTCTCGAGAAAGAACAGAACCCACAGCTA-3′) and added in animal experiment, and the empty plasmid was used as shNC. Lipofectamine 3,000 transfection reagents (L3000015, Thermo Fisher, Waltham, MA, USA) were used to transfect the above plasmids into PC-12 cells alone or in combination. Briefly, PC-12 cells were plated at a density of 1 × 10⁴ cells/well in 96-well plates. When the cells reached 80% confluence, the above plasmids (0.2 µg) and lipofectamine 3,000 transfection reagents (0.15 µL) were diluted in both Opti-MEM media (10 µL) (31985062, Thermo Fisher, USA) and P3000 reagents (0.4 µL), and then incubated together at 37°C for 10 min. After incubation, gene–lipid complexes were obtained and used to incubate the cells at 37°C for 24 or 48 h.

2.5 Flow cytometry

The apoptosis of PC-12 cells was measured by Annexin V-FITC/PI apoptosis detection kit (40302ES20, Yeasen, Shanghai, China). After transfection and LPS treatment, PC-12 cells were digested in EDTA-free trypsin (T2600000, Sigma-Aldrich, USA), and washed with phosphate buffered saline (PBS; AM9624, Thermo Fisher, USA) thrice. The cells were then resuspended by 1× Binding Buffer to be 1 × 10⁶ cells/mL and added with Annexin V-FITC solution (5 μL) and propidium iodide (PI) solution (10 μL). After 10 min incubation in the dark, apoptotic cells were examined by a flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA).

2.6 Cell counting kit (CCK)-8 assay

After transfection and LPS treatment, PC-12 cells were plated in 96-well plates at a density of 5 × 10³ cells/well. CCK-8 reagent (96992, Sigma-Aldrich, USA) was added to the cells at a ratio of 1:10, following which cell incubation was conducted at 37°C for 1 h. Cell viability was calculated based on cell absorbance recorded by a microplate reader (ELx808, BioTek, Winooski, VT, USA) at 450 nm.

2.7 Wound healing assay

After transfection and LPS treatment, PC-12 cells were plated at a density of 2 × 10⁴ cells/well in 6-well plates. The cells were cultured in serum-free RPMI-1640 until cell monolayers were formed. Subsequently, a gap was scraped by a sterile pipette tip on each monolayer. After scraped cells were removed, the gaps were photographed at 0 and 24 h, and the monolayers were incubated at 37°C for 24 h. Uncovered areas from eight randomly selected fields were observed by an inverted microscope (IX71; Olympus, Tokyo, Japan) under ×100 magnification. The widths of the areas were analyzed using an image analysis system (Wound Healing ACAS, ibidi, Munich, Germany).

2.8 Transwell assay

The invasive ability of transfected PC-12 cells was evaluated by Transwell chambers (3428, Corning, NY, USA). Matrigel (356234, Corning, USA) (dilution 1:3) was laid onto the upper chamber. After transfection and LPS treatment, PC-12 cells were suspended by serum-free RPMI-1640 to be 2 × 10⁵ cells/mL, and 100 µL of this cell solution was poured into the upper chamber. RPMI-1640 (600 µL) containing 10% FBS was added into the lower chamber. After 24 h incubation at 37°C, non-invading cells in the upper chamber were removed, following which remanent cells were washed by PBS twice, fixed in 4% paraformaldehyde (P6148, Sigma-Aldrich, USA) and stained with Giemsa (800 µL) (10092013, Thermo Fisher, USA). Stained cells from eight randomly selected fields were observed by an inverted microscope (IX71; Olympus, Tokyo, Japan) under ×200 magnification.

2.9 Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Rat spinal cord tissues were homogenized by a homogenizer (UH-05, Union-Biotech, Shanghai, China). Total RNA and total miRNA from PC-12 cells and rat spinal cord homogenates were extracted by Trizol reagents (15596026, Thermo Fisher, USA) and PureLink miRNA Isolation Kits (K157001, Thermo Fisher, USA), respectively. cDNAs were synthesized via the reverse transcription of the extracted total RNA and total miRNA by SuperScript IV reverse transcriptases (3531295001, Sigma-Aldrich, USA). cDNA amplification was performed with PowerUp SYBR Green Master Mix (A25742, Thermo Fisher, USA) and analyzed by the Real-Time PCR Detection System (CFX Connect, Bio-Rad, Philadelphia, PA, USA), the primers sequence was showed in Table 1. PCR reaction was initiated at the following conditions: 95°C for 10 min, 40 circles of 95°C for 15 s, and 60°C for 60 s. TERC was normalized to GAPDH and miR-34a-5p was normalized to U6. The relative gene expressions were presented by the 2^−ΔΔCt method [18].

2.10 Western blot

Total protein from transfected PC-12 cells was extracted by RIPA Buffer (89900, Thermo Fisher, USA), and quantitated by BCA kit (A53227, Thermo Fisher, USA). Marker (4 μL) (PR1910, Solarbio, Beijing, China) and the extracted protein (40 μg) were separately loaded and subjected to electrophoresis on 10 or 12% SDS-PAGE gel (P0670 or P0672, Beyotime, Shanghai, China), followed by electroblotting onto PVDF membranes (P2438, Sigma-Aldrich, USA). The membranes were blocked by 5% non-fat milk in Tris Buffered Saline with 1% Tween 20 (TBST, TA-125-TT, Thermo Fisher, USA) for 1 h, and incubated with primary antibodies at 4°C overnight, including those against XBP-1 (#40435, 55 kDa, 1:1,000, Danvers, MA), IL-6 (#12912, 24 kDa, 1:1,000), TNF-α (#11948, 25 kDa, 1:1,000), t-p38 (#9212, 40 kDa, 1:1,000), p-p38 (#4511, 43 kDa, 1:1,000), t-Akt (#4691, 60 kDa, 1:1,000), p-Akt (#4060, 60 kDa, 1:2,000), Bcl-2 (#3498, 26 kDa, 1:1,000), Bax (#14796, 20 kDa, 1:1,000), cleaved caspase-9 (#9509, 37 kDa, 1:1,000), cleaved caspase-3 (#9661, 17 kDa, 1:1,000), and GAPDH (#5174, 37 kDa, 1:1,000) purchased from Cell signaling technology in USA. The membranes were then washed with TBST and incubated with secondary antibody Goat anti-Rabbit IgG (A32731, 1:10,000, Thermo Fisher, USA). Immunoreactive bands were visualized by enhanced chemiluminescence reagent kit (WP20005, Thermo Fisher, USA) on an imaging device (iBright CL750, Thermo Fisher, USA) and analyzed quantitatively by ImageJ software (1.52 s version, National Institutes of Health, Bethesda, MA, USA).

2.11 Dual-luciferase reporter assay

The binding sites between TERC and miR-34a-5p and between miR-34a-5p and XBP-1 was performed using TargetScan V7.2 (http://www.targetscan.org/mamm_31/). Sequences of TERC (wild type (wt): 5′-TTCTCCGGAGGCACCCACTGCCA-3′, mutant type (mut): 5′-TTCTCCGGAGGCACCCACGACCA-3′), and XBP-1 (wt: 5′-GCTTTCATC-CAGCCACTGCCC-3′, mut: 5′-GCTTTCATC-CAGCCCGTGCCC-3′) were separately cloned to pmirGLO vectors (E1330, Promega, Madison, WI, USA) for the obtainment of reporter plasmids. PC-12 cells were cultured in 12-well plates at a density of 1 × 10⁷ cells/well. When the cells reached a 70% confluence, the cells were co-transfected with the reporter plasmids (100 ng) and miR-34a-5p mimic/miR-NC (100 ng) by Lipofectamine 3,000 reagents. After 48 h of transfection at 37°C, dual luciferase reporter assay was performed with a dual-luciferase reporter assay system (E1980, Promega, USA). The firefly luciferase activity, which indicated the binding specificity, was normalized to renilla luciferase activity, and measured by a luminometer (GloMax®20/20, Promega, USA).

2.12 RNA immunoprecipitation (RIP) assay

Binding relationship between miR-34a-5p and TERC or XBP-1 was validated by using Magna RIP Kits (17-704, Sigma-Aldrich, USA). Cells were treated with lysis buffer (P0013B, Beyotime, China) on ice, followed by a 10 min centrifugation at 10,000× g at 4°C. After that, supernatant was obtained and was precleared with magnetic beads. Then, the precleared supernatant was re-suspended in RIP Wash Buffer and incubated with protein A/G magnetic beads bound with anti-Argonaute2 (Ago2) antibody (ab32381, 1:50, Abcam, UK) or monoclonal antibody IgG (ab172730, 1:100, Abcam, UK) overnight at 4°C. Afterwards, the precipitate was harvested and digested with Proteinase K and qRT-PCR was used for assessing the enrichment of TERC or XBP-1.

2.13 Statistical analysis

Statistical analyses were performed with Graphpad prism (version 8.0, GraphPad Software Inc., San Diego, CA, USA). All data were obtained from independent experiments performed in triplicate and expressed as mean value ± standard deviation (SD). Differences among multiple groups were analyzed by one-way analysis of variance (ANOVA), and those between two groups were analyzed by independent t-test, followed by Tukey’s post-hoc test. P < 0.05 was regarded statistically significant.

1. Ethics statement: All animal experiments were performed in accordance with the guidelines of the China Council on Animal Care and Use. This study was approved by the Committee of Experimental Animals of Zhejiang Academy of Traditional Chinese Medicine (approval number: ZATCM Animal Ethic Review no. [2019]048). Every effort was made to minimize pain and discomfort to the animals. The animal experiments were performed in Zhejiang Academy of Traditional Chinese Medicine.

3.1 TERC was overexpressed but miR-34a-5p was lowly expressed in the spinal cord of SCI rats, and knockdown of TERC promoted the miR-34a-5p expression and alleviated histopathological abnormalities

TERC has been found to promote cellular inflammation [13]. A significantly upregulated TERC was detected in the spinal cord tissues of SCI rats through qRT-PCR, compared to the TERC level in the spinal cord tissues of the sham rats (Figure 1a). miR-34a-5p has been found to be downregulated in SCI [16]. Consistently, qRT-PCR analysis showed that miR-34a-5p level was downregulated in the spinal cord tissues of SCI rats, compared with that in the spinal cord tissues of the sham rats (Figure 1b). To explore the role of TERC on SCI-associated miR-34a-5p downregulation and histopathological changes, lentivirus-shTERC was employed. Injection of lentivirus-shTERC successfully downregulated TERC, and led to an increased level of miR-34a-5p (Figure 1a and b). Histopathological examination by hematoxylin–eosin staining illustrated that the sham rats had structurally dense and regular spinal cord tissues, which were enriched with neuronal cells showing large cell bodies, delicate nuclear staining and good morphological differentiation and presented dense and uniform interstitium with no inflammatory cell infiltration, whereas structurally relatively disordered spinal cord tissues, which harbored neuronal cells with eosinophilic change, cell body shrinkage, nucleus pyknosis, and degeneration and showed obviously loose and edematous interstitium with local congestion and bleeding and more inflammatory cell infiltration, were observed in the SCI rats; notably, injection of lentivirus-shTERC pronouncedly alleviated the above pathological conditions in the spinal cord tissues of the SCI rats, as following TERC knockdown, the SCI rats presented complete and regular spinal cord tissues with mild loose interstitium, a small number of interstitium-infiltrating inflammatory cells, and abundant intercellular neuronal cells that were mostly well differentiated and hardly showed pyknotic changes (Figure 1c).

Figure 1

Knockdown of TERC, which was upregulated in SCI, revoked miR-34a-5p downregulation, and alleviated histopathological abnormalities in the spinal cord of SCI rats. (a and b) The expressions of TERC and miR-34a-5p in the spinal cords of SCI rats were analyzed by qRT-PCR. (c) Spinal cord histopathological changes were examined via hematoxylin–eosin staining (magnification: ×100; scale: 50 µm; black arrows: a basically complete and regular organizational structure; blue arrows: a relatively disorganized organizational structure; yellow arrows: pyknosis and degeneration of the nucleus; green arrows: obviously loose and edematous interstitium; red arrows: local congestion and bleeding; gray arrows: slightly loose interstitium). ^*** P or ^&&& P < 0.001; ^* vs Sham; ^& vs ShNC + SCI.

3.2 TERC level was upregulated while miR-34a-5p level was downregulated in LPS-induced PC-12 cells

Next, SCI models in vitro were established with LPS-induced PC12 cells. As shown in CCk-8 assay, treatment with LPS concentration-dependently decreased the viability of PC-12 cells (Figure 2a). Given that the viability of PC-12 cells was decreased by half after the PC-12 cells were treated with LPS of 4 μg/mL, 4 μg/mL was selected as the optimal concentration for the establishment of SCI models in vitro. QRT-PCR analysis showed that TERC level was upregulated, but miR-34a-5p level was downregulated in LPS-induced PC-12 cells, compared to those in the control cells (Figure 2b and c).

Figure 2

TERC level was upregulated while miR-34a-5p level was downregulated in LPS-induced PC-12 cells. (a) The viability of PC-12 cells after treatment with different concentrations of LPS was measured by CCK-8 assay. (b and c) The expressions of TERC and miR-34a-5p in LPS-induced PC-12 cells were analyzed by qRT-PCR. ^{^} P < 0.05, ⁺⁺ P < 0.01, ^{^^^} P < 0.001; ^{^} vs 0; ⁺ vs Control.

3.3 TERC regulated the viability, migration, invasion, and apoptosis of LPS-induced PC-12 cells

ShTERC and TERC overexpression plasmids were employed to investigate the effect of TERC on the biological behaviors in SCI in vitro. Transfection of ShTERC or TERC overexpression plasmids successfully led to knockdown or overexpression of TERC, and ShTERC or TERC overexpression promoted or inhibited the expression of miR-34a-5p (Figure 3a and b). As displayed in cellular behavior assessment based on CCK-8, wound healing, and Transwell assays, TERC knockdown increased the viability of LPS-induced PC-12 cells, as well as the migration and invasion by LPS-induced PC-12 cells, whereas TERC overexpression exerts effects opposite to those of TERC knockdown (Figure 3c–e). Following the detection of flow cytometry, the apoptosis of LPS-induced PC-12 cells was found to be inhibited by TERC knockdown, yet enhanced with TERC overexpression (Figure 3f).

Figure 3

TERC regulated the viability, migration, invasion, and apoptosis of LPS-induced PC-12 cells. (a and b) The expressions of TERC and miR-34a-5p in shTERC/TERC overexpression plasmid-transfected PC-12 cells after LPS treatment were analyzed by qRT-PCR. (c) The viability of shTERC/TERC overexpression plasmid-transfected PC-12 cells after LPS treatment was measured by CCK-8 assay. (d) The migratory ability of shTERC/TERC overexpression plasmid-transfected PC-12 cells after LPS treatment was evaluated by wound healing assay (magnification: ×100; scale: 100 µm). (e) The invasive ability of shTERC/TERC overexpression plasmid-transfected PC-12 cells after LPS treatment was assessed by Transwell assay (magnification: ×200; scale: 50 µm). (f) The apoptosis of shTERC/TERC overexpression plasmid-transfected PC-12 cells after LPS treatment was detected by flow cytometry. ⁺ P < 0.05, ^ΔΔ P or ⁺⁺ P < 0.01, ^### P or ⁺⁺⁺ P < 0.001; ^# vs shNC; ^Δ vs NC; ⁺ vs shNC + NC.

3.4 Downregulation of miR-34a-5p, a target of TERC, offset TERC knockdown-induced effects on the viability, migration, and invasion of LPS-induced PC-12 cells

The role of miR-34a-5p in TERC-regulated progression of SCI in vitro was investigated. A successful upregulation or downregulation of miR-34a-5p level was caused by transfection of miR-34a-5p mimic or inhibitor (Figure 4a). Dual-luciferase reporter assay revealed that the luciferase activity of LPS-induced PC-12 cells containing sequences of TERC-wt was decreased by upregulation of miR-34a-5p (Figure 4b). Analysis based on bioinformatics tools showed that there were sites that can bind miR-34a-5p to TERC (Figure 4c). Moreover, RIP assay was conducted to verify the targeting relationship between TERC and miR-34a-5p. It was found that the addition of Ago2 antibody caused a co-enrichment of miR-34a-5p and TERC in PC-12 cells (Figure 4d). TERC knockdown was demonstrated to upregulate miR-34a-5p level and reverse miR-34a-5p inhibitor-induced downregulation of miR-34a-5p. In turn, miR-34a-5p inhibitor offset TERC knockdown-induced upregulation of miR-34a-5p in LPS-induced PC-12 cells (Figure 4e). Through CCK-8, wound healing, and Transwell assays, we observed that miR-34a-5p downregulation decreased the viability, migration and invasion of LPS-induced PC-12 cells. Moreover, miR-34a-5p downregulation offset TERC knockdown-induced promotion on the cell viability, migration and invasion (Figure 4f–j). In addition, those effects induced by miR-34a-5p inhibitor on these biological behaviors were reversed following the knockdown of TERC (Figure 4f–j).

Figure 4

Downregulation of miR-34a-5p, a target of TERC, offset TERC knockdown-induced effects on viability, migration, and invasion of LPS-induced PC-12 cells. (a) The expression of miR-34a-5p in miR-34a-5p mimic/inhibitor-transfected PC-12 cells after LPS treatment was analyzed by qRT-PCR. (b–d) The interaction between TERC and miR-34a-5p was checked by (b) dual-luciferase reporter assay, predicted by (c) TargetScan V7.2, and reaffirmed by (d) RIP assay. (e) The expression of miR-34a-5p in miR-34a-5p inhibitor and/or shTERC-transfected PC-12 cells after LPS treatment was determined by qRT-PCR. (f) The viability of miR-34a-5p inhibitor and/or shTERC-transfected PC-12 cells after LPS treatment was measured by CCK-8 assay. (g and h) The migratory ability of miR-34a-5p inhibitor and/or shTERC-transfected PC-12 cells after LPS treatment was evaluated by wound healing assay (magnification: ×100; scale: 100 µm). (i and j). The invasive ability of miR-34a-5p inhibitor/shTERC-transfected PC-12 cells after LPS treatment was assessed by Transwell assay (magnification: ×200; scale: 50 µm). ⁺ P or ^{^} P < 0.05; ^&& P or ^δδ P or ⁺⁺ P or ^{^^} P < 0.01, ^### P or ⁺⁺⁺ P or ^*** P < 0.001, ^△△△ P < 0.001; ^ωωω P < 0.001; ^& vs mimics control; ^δ vs inhibitor control; ^# vs TERC + miR-34a-5p control; ⁺vs shNC + NC; ^* vs shTERC; ^{^} vs miR-34a-5p inhibitor; △ vs IgG in TERC; ^ω vs IgG in miR-34a-5p.

3.5 Knockdown of TERC inhibited apoptosis, inflammation, and p38 activation, yet activated Akt in LPS-induced PC-12 cells via the miR-34a-5p/XBP-1 axis

Finally, western blot analysis revealed that in LPS-induced PC-12 cells, the expressions of proapoptotic cleaved caspase-3, cleaved caspase-9, and Bax, together with the expressions of Akt pathway-related factor p-p38 and p-p38/p38, accompanied with the expressions of inflammation-related IL-6, TNF-α, and XBP-1, were all decreased by TERC knockdown, but increased by miR-34a-5p downregulation (Figure 5a–f). Meanwhile, the expressions of antiapoptotic Bcl-2 and the expressions of Akt pathway-related factor p-Akt and p-Akt/Akt were all increased by TERC knockdown, but decreased by miR-34a-5p downregulation (Figure 5a–f). Moreover, there existed a mutually antagonistic mechanism between the effects exerted by TERC knockdown and those by miR-34a-5p downregulation on the expressions of abovementioned proteins (Figure 5a–f). Furthermore, dual-luciferase reporter assay revealed that upregulation of miR-34a-5p induced decreases in the luciferase activity of LPS-induced PC-12 cells containing sequences of XBP-1-wt (Figure 5g), which suggested that XBP-1 was directly targeted by miR-34a-5p. The existence of this targeting relation was further reinforced by a bioinformatics tool-based prediction, which displayed miR-34a-5p had binding sites complementary to the sites on XBP-1 (Figure 5h), and RIP assay, which showed that miR-34a-5p and XBP-1 were co-enriched after the addition of Ago2 antibody (Figure 5i).

Figure 5

Knockdown of TERC inhibited apoptosis, inflammation, and p38 activation yet activated Akt in LPS-induced PC-12 cells via the miR-34a-5p/XBP-1 axis. (a–f) The expressions of cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, IL-6, TNF-α, XBP-1, t-Akt, p-Akt, t-p38, p-p38, p-Akt/t-Akt, and p-p38/t-p38 in miR-34a-5p inhibitor and/or shTERC-transfected PC-12 cells after LPS treatment were analyzed by Western blot, with GAPDH serving as a control gene. (g–i). The interaction between miR-34a-5p and XBP-1 was checked by (g) dual-luciferase reporter assay, predicted by (h) TargetScan V7.2, and reaffirmed by (i) RIP assay. ⁺ P or ^{^} P < 0.05; ^{^^} P or ⁺⁺ P < 0.01, ⁺⁺⁺ P or ^*** P or ^{^^^} P or ^ΔΔΔ Por < 0.001, ^δδδ P < 0.001; ^ωωω P < 0.001; ⁺vs shNC + NC; ^*vs shTERC^{; ^}vs miR-34a-5p inhibitor; ^Δvs 3′-XBP1 + miR-34a-5p control; ^ωvs IgG in miR-34a-5p; ^δvs IgG in XBP-1.