| 高炜,张莉,金培田.长链非编码 RNA脑源性神经营养因子反义 RNA靶向微小 RNA-495-3p调控脂多糖诱导的大鼠肺泡巨噬细胞凋亡和炎症反应[J].安徽医药,2022,26(6):1235-1239. |
| 长链非编码 RNA脑源性神经营养因子反义 RNA靶向微小 RNA-495-3p调控脂多糖诱导的大鼠肺泡巨噬细胞凋亡和炎症反应 |
| LncRNA BDNF-AS targets miR-495-3p to regulate LPS-induced apoptosis and inflammatory response in rat alveolar macrophages |
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| DOI：10.3969/j.issn.1009-6469.2022.06.042 |
| 中文关键词: 巨噬细胞，肺泡 脑源性神经营养因子 RNA，反义 RNA，长链非编码 微小 RNA-495-3p 凋亡 炎症反应 |
| 英文关键词: Macrophages, alveolar Brain-derived neurotrophic factor RNA, antisense RNA, long noncoding MiR-4953p Apoptosis Inflammatory response |
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| 中文摘要: |
| 目的探讨长链非编码 RNA(lncRNA)脑源性神经营养因子反义 RNA(BDNF-AS)对脂多糖( LPS)诱导的大鼠肺泡巨噬细胞凋亡和炎症反应的影响及分子机制。方法该研究起止时间为 2018年 12月至 2019年 12月。用 1 mg/L的 LPS处理大鼠肺泡巨噬细胞 NR8383细胞作为 LPS组；正常培养的细胞作为 Con组。将 BDNF-AS小分子干扰 RNA(si-BDNF-AS)及其阴性对照( si-NC)、微小 RNA(miR)-495-3p及其阴性对照( miR-NC)转染至 NR8383细胞中再用 1 mg/L的 LPS处理，记为 LPS+siBDNF-AS组、 LPS+si-NC组、 LPS+miR-495-3p组、 LPS+miR-NC组；将 si-BDNF-AS分别与 anti-miR-NC、anti-miR-495-3p共转染至 NR8383细胞中再用 1 mg/L的 LPS处理，记为 LPS+si-BDNF-AS+anti-miR-NC组、 LPS+si-BDNF-AS+anti-miR-495-3p组。实时荧光定量 PCR检测 lncRNA BDNF-AS和 miR-495-3p的表达水平；酶联免疫吸附测定(ELISA)法检测白细胞介素 -1β(IL-1β)肿瘤坏死因子 -α(TNF-α)水平；流式细胞术检测细胞凋亡；蛋白质印迹法检测 B细胞淋巴瘤 -2(Bcl-2)、 Bcl-2相关 X蛋白( Bax)表、达；荧光素酶报告实验检测 BDNF-AS对 miR-495-3p的靶向关系。结果 LPS诱导的大鼠肺泡巨噬细胞中 BDNF-AS高表达［( 1.00±0.06)比( 3.41±0.31)］，miR-495-3p低表达［( 1.02±0.07)比( 0.47±0.04)］IL-1β［( 22.14±2.25)ng/L比( 513.20±41.22)ng/ L］、 TNF-α［(184.33±18.65)ng/L比(1 125.65±110.36)ng/L］水平升高，细胞凋亡率［(，8.11±0.81)%比( 36.22±3.21)%］升高， Bax表达水平升高， Bcl-2表达水平降低( P<0.05)。抑制 BDNF-AS表达或过表达 miR-495-3p后， IL-1β［( 526.14±46.87)ng/L比 |
| 英文摘要: |
| Objective To investigate the effect of lncRNA BDNF-AS on lipopolysaccharide (LPS) induced alveolar macrophageapoptosis and inflammatory response in rats and its molecular mechanism.Methods The study started and ended from December2018 to December 2019. Rat alveolar macrophage NR8383 cells were treated with 1 mg/L LPS as the LPS group. Normal cultured cellsserved as the Con group. BDNF-AS small interfering RNA (si-BDNF-AS) and its negative control (si-NC), miR-495-3p oligonucleotide mimics (miR-495-3p mimics) and negative control mimic NC sequences (miR-NC) was transfected into NR8383 cells, which were then treated with 1 mg/L LPS, and recorded as LPS+si-NC group, LPS+si-BDNF-AS group, LPS+miR-NC group and LPS+miR-495-3p group; si-BDNF-AS was co-transfected with anti-miR-NC and anti-miR-495-3p into NR8383 cells which were then treated with 1 mg/L LPS and recorded as LPS+si-BDNF-AS+anti-miR-NC group and LPS+si-BDNF-AS+ anti-miR-495-3p group. Real-time fluorescence quantitative PCR was used to detect the expressions of lncRNA BDNF-AS and miR-495-3p; enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels; flow cytometry was performed to detect apoptosis; Western blotting was employed to detect B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 related X protein (Bax) protein expressions;luciferase reporter assay was used to detect the targeting relationship of BDNF-AS to miR-495-3p.Results LPS-induced rat alveolar macrophages showed high expression of BDNF-AS [(1.00±0.06) vs. (3.41±0.31)], low expression of miR-495-3p [(1.02±0.07) vs. (0.47± 0.04)], and low expression of IL-1β [(22.14±2.25) ng/L vs. (513.20±41.22) ng/L], TNF-α [(184.33±18.65) ng/L vs. (1 125.65±110.36) ng/ L], apoptosis rate [(8.11±0.81)% vs. (36.22±3.21)%], Bax expression were increased, the expression level of Bcl-2 was decreased, and the difference was statistically significant (P<0.05). After inhibiting BDNF-AS expression or overexpressing miR-495-3p, IL-1β [(526.14±46.87) ng/L vs. (132.41±12.58) ng/L, (536.14±51.02) ng/L vs. (175.98±18.41) ng/L], TNF-α [ (1203.33±185.22) ng/L vs. (354.26±21.58) ng/L, (1 248.65±115.28) ng/L vs. (628.47±53.69) ng/L] levels were decreased, and the apoptosis rate [(38.14±3.71)% vs. (12.03±1.25)%, (36.98±3.61)% vs. (14.87±1.42)%], the expression level of Bax was decreased, and the expression level of Bcl-2 was increased, and the difference was statistically significant (P<0.05). BDNF-AS targeted regulation of miR-495-3p, and inhibition of miR495-3p expression reversed the inhibition of lncRNA BDNF-AS expression on LPS-induced rat alveolar macrophage apoptosis and in‐flammation.Conclusion Inhibition of lncRNA BDNF-AS expression may inhibit LPS-induced rat alveolar macrophage apoptosis and inflammatory response by up-regulating miR-495-3p. |
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