Effects and mechanisms of LINC00657 on oxidative glucose deprivation-induced injury in mouse hippocampal neurons
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摘要:目的
探讨LINC00657对氧糖剥夺(OGD)诱导的小鼠海马神经元细胞损伤的影响及作用机制。
方法对小鼠海马神经元细胞HT22进行OGD处理，建立损伤模型，将正常培养的HT22细胞作为对照; 将si-NC、si-LINC00657、微小RNA(miR)-NC、miR-224-3p mimics分别转染至HT22细胞，然后进行OGD处理; 向HT22细胞共转染si-LINC00657和anti-miR-NC, 或共转染si-LINC00657和anti-miR-224-3p, 然后进行OGD处理。采用实时荧光定量聚合酶链反应(qRT-PCR)检测LINC00657、miR-224-3p相对表达量; 采用CCK-8法、流式细胞术分别检测细胞存活率、细胞凋亡率; 采用试剂盒检测乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)水平; 采用双荧光素酶报告基因实验检测miR-224-3p过表达对野生型LINC00657载体(WT-LINC00657)、突变型LINC00657载体(MUT-LINC00657)荧光素酶活性的影响。
结果与对照细胞相比, OGD诱导的HT22细胞中LINC00657表达上调, miR-224-3p表达下调，差异有统计学意义(P < 0.05); 分别与转染si-NC或转染miR-NC相比，转染si-LINC00657或转染miR-224-3p mimics后，细胞存活率、SOD活性和GSH-Px活性升高，细胞凋亡率、LDH活性和MDA水平降低，差异有统计学意义(P < 0.05); miR-224-3p过表达降低了WT-LINC00657的荧光素酶活性，差异有统计学意义(P < 0.05); 与共转染si-LINC00657和anti-miR-NC的细胞相比，共转染si-LINC00657和anti-miR-224-3p的细胞存活率降低，细胞凋亡率升高, LDH活性、MDA水平升高, SOD、GSH-Px活性降低，差异有统计学意义(P < 0.05)。
结论干扰LINC00657可通过上调miR-224-3p而促进细胞增殖，并抑制细胞凋亡和氧化应激反应，从而减轻OGD诱导的小鼠海马神经元细胞损伤。
Abstract:ObjectiveTo investigate the effects and mechanisms of LINC00657 on oxidative glucose deprivation (OGD)-induced injury in mouse hippocampal neurons.
MethodsMouse hippocampal neuron cell line HT22 was given OGD treatment to establish an injury model, with normally cultured HT22 cells as controls. The si-NC, si-LINC00657, microRNA(miR)-NC, and miR-224-3p mimics were transfected into HT22 cells, followed by OGD treatment. Co-transfection of si-LINC00657 and anti-miR-NC, or co-transfection of si-LINC00657 and anti-miR-224-3p, was performed in HT22 cells before OGD treatment. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the relative expression levels of LINC00657 and miR-224-3p. CCK-8 assay and flow cytometry were used to detect cell viability and apoptosis rate, respectively. Kits were used to detect the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the level of malondialdehyde (MDA). Dual-luciferase reporter gene assay was used to detect the effect of miR-224-3p overexpression on the luciferase activity of wild-type LINC00657 vector (WT-LINC00657) and mutant LINC00657 vector (MUT-LINC00657).
ResultsCompared with controls, the expression of LINC00657 was upregulated and the expression of miR-224-3p was downregulated in OGD-induced HT22 cells (P < 0.05). Compared with transfection of si-NC or miR-NC, transfection of si-LINC00657 or miR-224-3p mimics resulted in increased cell viability, SOD activity, and GSH-Px activity, as well as decreased apoptosis rate, LDH activity, and MDA level(P < 0.05). Overexpression of miR-224-3p reduced the luciferase activity of WT-LINC00657 (P < 0.05). Compared with cells co-transfected with si-LINC00657 and anti-miR-NC, cells co-transfected with si-LINC00657 and anti-miR-224-3p showed decreased cell viability, increased apoptosis rate, increased LDH activity and MDA level, and decreased SOD and GSH-Px activities (P < 0.05).
ConclusionInterference with LINC00657 can promote cell proliferation, inhibit apoptosis and oxidative stress response by upregulating miR-224-3p, thereby alleviating OGD-induced injury in mouse hippocampal neurons.
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表 1 Con组与OGD组细胞中LINC00657、miR-224-3p相对表达量比较($\mathop x\limits^ - $±s)
组别 n LINC00657 miR-224-3p Con组 9 1.00±0 1.00±0 OGD组 9 3.45±0.24* 0.43±0.04* miR-224-3p: 微小RNA-224-3p。与Con组比较, *P < 0.05。 
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表 2 干扰LINC00657表达对OGD诱导的HT22细胞存活率和凋亡率的影响($\mathop x\limits^ - $±s)
组别 n LINC00657 存活率/% 凋亡率/% Con组 9 1.00±0 94.16±5.04 5.53±0.53 OGD组 9 3.56±0.23* 43.79±4.49* 25.34±2.37* OGD+si-NC组 9 3.59±0.28 41.76±4.31 26.41±2.25 OGD+si-LINC00657组 9 1.69±0.14# 87.42±7.67# 9.16±0.82# 与Con组比较, *P < 0.05; 与OGD+si-NC组比较, #P < 0.05。
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表 3 干扰LINC00657表达对OGD诱导的HT22细胞中氧化应激指标的影响($\mathop x\limits^ - $±s)
组别 n LDH/(U/L) MDA/(mmol/L) SOD/(U/L) GSH-Px/(U/L) Con组 9 43.48±3.47 1.51±0.13 29.02±2.47 17.29±1.65 OGD组 9 171.48±11.48* 5.27±0.49* 6.82±0.53* 4.12±0.42* OGD+si-NC组 9 176.85±13.45 5.37±0.51 6.87±0.63 4.08±0.36 OGD+si-LINC00657组 9 52.15±5.12# 1.87±0.17# 22.07±2.25# 13.14±1.27# LDH: 乳酸脱氢酶; MDA: 丙二醛; SOD: 超氧化物歧化酶; GSH-Px: 谷胱甘肽过氧化物酶。
与Con组比较, *P < 0.05; 与OGD+si-NC组比较, #P < 0.05。
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表 4 双荧光素酶报告基因实验结果($\mathop x\limits^ - $±s)
组别 n WT-LINC00657 MUT-LINC00657 miR-NC组 9 1.02±0.05 1.04±0.06 miR-224-3p组 9 0.63±0.05* 1.01±0.05 与miR-NC组比较, *P<0.05。
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表 5 LINC00657对miR-224-3p表达的调控作用($\mathop x\limits^ - $±s)
组别 n miR-224-3p pcDNA组 9 1.00±0 pcDNA-LINC00657组 9 0.34±0.03* si-NC组 9 0.99±0.05 si-LINC00657组 9 3.12±0.32# 与pcDNA组比较, *P < 0.05; 与si-NC组比较, #P < 0.05。
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表 6 miR-224-3p过表达对OGD诱导的HT22细胞损伤的影响($\mathop x\limits^ - $±s)
组别 n miR-224-3p 存活率/% 凋亡率/% LDH/(U/L) MDA/(mmol/L) SOD/(U/L) GSH-Px/(U/L) OGD+miR-NC组 9 1.00±0 41.93±4.09 27.34±2.33 177.04±12.78 5.76±0.53 6.56±0.45 3.94±0.35 OGD+miR-224-3p组 9 2.92±0.24* 79.57±5.31* 11.93±1.07* 62.82±6.18* 2.46±0.23* 18.43±1.47* 10.09±0.83* 与OGD+miR-NC组比较, *P<0.05。
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表 7 下调miR-224-3p逆转了干扰LINC00657对OGD诱导的HT22细胞损伤的影响($\mathop x\limits^ - $±s)
组别 n miR-224-3p 存活率/% 凋亡率/% LDH/(U/L) MDA/(mmol/L) SOD/(U/L) GSH-Px/(U/L) OGD+si-LINC00657+ anti-miR-NC组 9 1.00±0 88.87±5.97 9.08±0.74 50.42±4.16 1.75±0.16 23.15±2.26 14.79±1.33 OGD+si-LINC00657+ anti-miR-224-3p组 9 0.34±0.03* 50.95±4.14* 19.61±1.53* 152.98±13.51* 4.13±0.39* 11.08±1.08* 5.98±0.55* 与OGD+si-LINC00657+anti-miR-NC组比较, *P<0.05。
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