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The Effect of SPTLC2 on Promoting Neuronal Apoptosis Is Alleviated By Neurochemical Research (2019) 44:2113–2122 https://doi.org/10.1007/s11064-019-02849-7 ORIGINAL PAPER The Efect of SPTLC2 on Promoting Neuronal Apoptosis is Alleviated by MiR‑124‑3p Through TLR4 Signalling Pathway Xinhong Su1 · Yuqin Ye2 · Yongxiang Yang1 · Kailiang Zhang3 · Wei Bai1 · Huijun Chen1 · Enming Kang1 · Chuiguang Kong1 · Xiaosheng He1 Received: 12 March 2019 / Revised: 6 July 2019 / Accepted: 27 July 2019 / Published online: 1 August 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract To investigate the role and mechanism of microRNA-124-3p (miR-124-3p) and serine palmitoyltransferase long chain base subunit 2 (SPTLC2) in neuronal apoptosis induced by mechanical injury. Transient transfection was used to modify the expression of miR-124-3p and SPTLC2. After transfection, neuronal apoptosis was evaluated in an in vitro injury model of primary neurons using TUNEL staining and western blot. The correlation between miR-124-3p and SPTLC2 was identifed through a dual luciferase reporter assay in HEK293 cells. A rescue experiment in primary neurons was performed to further confrm the result. To explore the downstream mechanisms, co-immunoprecipitation was performed to identify proteins that interact with SPTLC2 in toll-like receptor 4 (TLR4) signalling pathway. Subsequently, the relative expression levels of TLR4 pathway molecules were measured by western blot. Our results showed that increased miR-124-3p can inhibit neuronal apoptosis, which is opposite to the efect of SPTLC2. In addition, miR-124-3p was proved to negatively regulate SPTLC2 expression and suppress the apoptosis-promoting efect of SPTLC2 via the TLR4 signalling pathway. Keywords miR-124-3p · SPTLC2 · Apoptosis · TLR4 Introduction MicroRNA-124 (miR-124) is one of the most abundant brain-specifc microRNAs (miRNAs) [3], and miR-124-3p Traumatic brain injury (TBI) is a leading culprit of morbid- is a broadly conserved subtype of the miR-124 family. MiR- ity, disability, and mortality around the world. It has been 124-3p has been reported to play a protective role in neurons established that neuron loss is the main cause of secondary after TBI [4] and to be involved in proliferation, diferentia- brain injuries that result in disability after TBI [1]. Apopto- tion, invasion, and apoptosis [5–7]. It has also been observed sis occurs minutes to weeks following TBI, ofering a poten- to target proviral integration site 1 to suppress astrocytoma tial window for therapeutic interventions [2]. Thus, fnding [8], target signal transducerand activator of transcription 3 to efective ways to repress apoptosis during this period may attenuate neuronal injury [9], and target annexin a5 to inhibit improve the prognosis of TBI. the mitochondrial pathway of apoptosis [10]. Suppression of other target genes by miR-124-3p may have synergistic efects on neuronal apoptosis [11]. However, the mecha- Xinhong Su and Yuqin Ye have contributed equally to this work. nisms through which these efects would occur are still not clear and thus merit further investigation. * Xiaosheng He Serine palmitoyltransferases (SPTs) can initiate de novo [email protected] biosynthesis of sphingolipids, which are important constitu- 1 Department of Neurosurgery, Xijing Hospital, Air Force ents of cell membranes. Sphingolipids are also involved in Military Medical University, No. 169 Changle Western modulating apoptosis, cell proliferation and diferentiation Road, Xi’an 710032, Shanxi, China as second messengers [12]. Mammalian SPTs are composed 2 Department of Neurosurgery, PLA 921rd Hospital, of three diferent subunits, serine palmitoyltransferase long Changsha 410000, Hunan, China chain base subunit 1 (SPTLC1), SPTLC2 and SPTLC3 [13]. 3 Department of Orthopedic Surgery, Orthopedic Oncology It has been found that the tissue distribution of SPTLC2 Institute of Chinese PLA, Tangdu Hospital, Air Force mRNA corresponds with the activity distribution of SPTs Military Medical University, Xi’an 710032, Shanxi, China Vol.:(0123456789)1 3 2114 Neurochemical Research (2019) 44:2113–2122 [14]. The amino acid sequence of SPTCL2 is similar to that 3.1-SPTLC2 mutant type (miR-NC + SPTLC2-MT) and of aminolevulinate synthase; both have a domain that forms miR-mimic + SPTLC2-MT (n = 3 in each group). a Schif base with pyridoxal phosphate [15, 16]. Overexpres- The primary cortical neurons were prepared from embry- sion of SPTLC2 has been shown to decrease the viability onic day 18–19 C57BL/6 mice (Laboratory Animal Center of HEK293 and HepG2 cells and to induce apoptosis via of the Air Force Military Medical University, Xi’an, China) elevation of cellular ceramide, sphingoid bases, and dihy- as described [23, 24]. All relevant experiments on animals droceramide [17]. However, the regulatory elements that in this study were approved by the Ethics Committee for control endogenous SPTLC2 gene transcription have not Animal Experimentation of the Air Force Military Medi- been identifed. cal University and were conducted under the guidelines for Based on the biological prediction (TargetScan.org), the the care and use of laboratory animals. Neuronal cells were sequence “CAC GGA A” of miR-124-3p could bind to the cultured in poly-L-lysine-coated 6-well plates at a density sequence “GUG CCU U” of SPTLC2. SPTLC2 is abundant of 700,000/well in DMEM (Gibco) containing 10% FBS in all cells [18] and miR-124-3p enriches in neurons [19], (ScienCell). After 6 h incubation, the medium was replaced and both of them involve in cell apoptosis mentioned above. with serum-free Neurobasal™ Medium (Gibco) supple- Meanwhile, miR-124-3p can regulate toll-like receptor 4 mented with 2% B27 (Gibco), 0.5 mM glutamine (Gibco) (TLR4) signalling pathway [20], which involves in pro- and penicillin–streptomycin (Gibco). Half of the medium cess of apoptosis too [21]. Myeloid diferentiation factor was changed every 3 days. Cells were maintained at 37 °C 88 (MYD88) and nuclear factor kappa B (NF-κB) are two in an atmosphere of 5% CO2. The cells were randomly key moleculars of TLR4 signalling pathway. According to divided into six groups: Sham, Injury, Injury + SPTLC2- GENEMANIA database, SPTLC2 may bond to MYD88 NC, Injury + pcDNA 3.1-SPTLC2 (Injury + SPTLC2), directly to regulate TLR4 pathway. A prior study showed Injury + miR-NC and Injury + miR-mimic (n = 3 in each that NF-κB binding sites were located in SPTLC2 promoter group). region [22]. So, the current study was aimed at examining their relationship in neuronal apoptosis. Cell Transfection In this study, we identify SPTLC2 as a direct target of miR-124-3p, fnding that overexpression of miR-124-3p dra- After being cultured for 7 days, the primary neurons were matically inhibits the expression of SPTLC2, attenuating transfected using the Lipofectamine 2000 reagent (Invitro- neuronal apoptosis after injury. SPTLC2 is further observed gen, Carlsbad, CA, USA) according to the manufacturer’s to bind to MYD88. As the relative expression levels of miR- instructions. MiR-124-3p mimics (100 nM) (Genomeditech, 124-3p and SPTLC2 are changed, TLR4 signalling pathway Shanghai, China) and Lipofectamine 2000 reagent (10 μl) related molecules expression levels also show corresponding (Invitrogen) were each diluted into 125 μl Neurobasal™ changes. These results suggest that a miR-124-3p/SPTLC2/ Medium (Gibco) for 5 min and then mixed for 20 min at TLR4 pathway is involved in the regulation of neuronal room temperature. The neurons were treated with the trans- apoptosis after injury. fection solutions following three washes with phosphate bufered saline (PBS). After 6 h of transfection, the trans- fection solutions were replaced by Neurobasal™ Medium (Gibco) supplemented with 2% B27 (Gibco) and 0.5 mM Materials and Methods glutamine (Gibco). Analogously, a scrambled sequence con- trol (100 nM), pcDNA3.1-SPTLC2 (2 μg) and pcDNA3.1 Cell Culture empty vector (2 μg) (Genomeditech) were individually trans- fected into neurons with Lipofectamine 2000 (Invitrogen) HEK293 cells (Shanghai Institute of Cell Biology, Chinese using the same method. Academy of Sciences) were cultured in Dulbecco’s modifed Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) Mechanical Injury Model supplemented with 10% foetal bovine serum (FBS, Scien- Cell, San Diego, CA, USA). The cells were maintained at After 24 h transfection, the primary neurons were subjected 37 °C in an atmosphere of humidifed air with 5% CO 2. The to mechanical injury. Traumatic axonal injury is a consist- cells were randomly divided into six groups: miR-124-3p ent component of TBI and has been recognized as a major mimic negative control + pcDNA 3.1-SPTLC2 empty vec- pathology of TBI [25]. In the present study, we employed tor negative control (miR-NC + SPTLC2-NC), miR-124-3p a plastic stylet to scrape the cells [26, 27]. Briefy, 35 mm mimic + SPTLC2-NC (miR-mimic + SPTLC2-NC), miR- Petri dishes were manually scratched with a 10 μl plastic NC + pcDNA 3.1-SPTLC2 wild type (miR-NC + SPTLC2- stylet needle following a 9 × 9 square grid template (with WT), miR-mimic + SPTLC2-WT, miR-NC + pcDNA 4 mm of space between each line). This scratch injury has 1 3 Neurochemical Research (2019) 44:2113–2122 2115 been used to model TBI in cortical neurons in vitro, as the The membranes were blocked by 5% low-fat milk and then scratch injury activates the damage of neurons at the primary incubated with anti-SPTLC2 antibody (1:200, #sc-398704, site, which then extends to the entire population of neurons. Santa Cruz, Cambridge, USA), anti-MYD88 (1:100, #sc- The injured neurons were used for subsequent experiments. 74532, Santa Cruz), anti-interleukin 1 receptor associated kinase 1 (IRAK1) (1:200, #sc-5288, Santa Cruz), anti-tumor Quantitative Real‑Time PCR necrosis factor receptor associated factor 6 (TRAF6) (1:200, #sc-8409, Santa Cruz), anti-NF-κB (1:200, #sc-71675, At scheduled timepoints, total RNA was extracted using Santa Cruz), anti-cleaved caspase 3 (1:500, #WL02117, Trizol (Wanleibio) according to the manufacturer’s instruc- Wanleibio), anti-B cell lymphoma 2 associated X protein tions.
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