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急性肺损伤(ALI)是一种流行性炎症性肺疾病,是急性呼吸窘迫综合征(ARDS)的重要原因,其发病率和重症病人死亡率均很高。尽管在治疗策略方面已取得重大进展,但ARDS和ALI的年死亡率仍为40%,且医疗费用较高[1]。ALI作为一种进展性疾病,以促炎性因子的过度产生、炎症细胞浸润以及肺泡上皮细胞的凋亡为主要特征[2]。因此,有效地控制肺泡上皮细胞炎症和凋亡对改善病人预后意义重大。长链非编码RNA(lncRNA)通过染色体重塑、转录和转录后加工等多种方式参与调节基因表达,在广泛的生物学领域具有重要功能[3-4]。研究[5]证实,在低氧诱导的心肌细胞损伤中lncRNA嗜酸性粒细胞转录因子(EGOT)低表达,脑钠肽能通过促进lncRNA EGOT表达,抑制促凋亡蛋白表达从而缓解低氧诱导的心肌细胞损伤。lncRNABase网站在线预测显示,EGOT与miR-320a之间存在结合位点。既往研究[6]显示,高氧刺激后肺泡上皮细胞微泡中miR-320a表达增加促进巨噬细胞介导的肺部炎性反应。基于以上研究,本研究探讨lncRNA EGOT、miR-320a在ALI/ARDS中的作用及可能机制,以期为ALI/ARDS提供有效的临床治疗策略。
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与Con组相比,LPS组A549细胞中lncRNA EGOT表达明显降低(P<0.01),miR-320a表达明显升高(P<0.01)(见表 1)。
分组 EGOT miR-320a Con 1.00±0.07 1.00±0.08 LPS 0.39±0.03 2.61±0.23 t 24.03 19.83 P <0.01 <0.01 表 1 lncRNA EGOT和miR-320a在LPS刺激的肺上皮细胞中的表达(ni=9;x±s)
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EGOT过表达对LPS诱导的肺泡上皮细胞炎症因子表达的影响与Con组相比,LPS组A549细胞中EGOT表达降低(P<0.05),培养液中IL-6和IL-1β水平均升高(P<0.05);与LPS+pcDNA组相比,LPS+pcDNA-EGOT组A549细胞中EGOT表达升高(P<0.05),培养液中IL-6和IL-1β水平均降低(P<0.05)(见表 2)。
分组 EGOT IL-6/(pg/mL) IL-1β/(pg/mL) Con 1.00±0.05 106.48±10.42 46.17±4.94 LPS 0.43±0.04* 353.51±25.90* 180.73±12.98* LPS+pcDNA 0.41±0.04* 349.84±19.43* 177.28±14.47* LPS+pcDNA-EGOT 2.31±0.22*#▲ 139.69±12.53*#▲ 64.96±6.26*#▲ F 528.68 482.33 419.16 P <0.01 <0.01 <0.01 MS组内 0.014 328.478 110.363 q检验:与Con组比较*P<0.05;与LPS组比较#P<0.05;与LPS+pcDNA组比较▲P<0.05 表 2 lncRNA EGOT过表达对LPS诱导的肺泡上皮细胞炎症因子表达的影响(ni=9;x±s)
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与Con组相比,LPS组A549细胞凋亡率、Bax蛋白表达均升高(P<0.05),Bcl-2蛋白表达降低(P<0.05);与LPS+pcDNA组相比,LPS+pcDNA-EGOT组A549细胞凋亡率、Bax蛋白表达均降低(P<0.05),Bcl-2蛋白表达升高(P<0.05)(见图 1~2、表 3)。
分组 凋亡率/% Bcl-2蛋白 Bax蛋白 Con 6.52±0.60 0.73±0.07 0.17±0.02 LPS 25.45±2.28* 0.28±0.03* 0.55±0.04* LPS+pcDNA 26.34±2.81* 0.26±0.02* 0.58±0.05* LPS+pcDNA-EGOT 10.59±1.01*#▲ 0.62±0.05*#▲ 0.24±0.02*#▲ F 256.47 234.86 324.49 P <0.01 <0.01 <0.01 MS组内 3.619 0.002 0.001 q检验:与Con组比较*P<0.05;与LPS组比较#P<0.05;与LPS+pcDNA组比较▲P<0.05 表 3 lncRNA EGOT过表达对LPS诱导的肺泡上皮细胞凋亡的影响(ni=9;x±s)
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LncBase Predicted v.2在线预测分析显示,miR-320a与EGOT之间存在结合位点(见图 3)。双荧光素酶报告实验显示,与miR-NC和WT-EGOT共转染组相比,miR-320a mimics和WT-EGOT共转染组A549细胞相对荧光素酶活性降低(P<0.05);与miR-NC和MUT-EGOT共转染组相比,miR-320a mimics和MUT-EGOT共转染组A549细胞相对荧光素酶活性变化差异无统计学意义(P>0.05)(见表 4)。与pcDNA组相比,pcDNA-EGOT组A549细胞miR-320a表达水平降低(P<0.05);与si-NC组相比,si-EGOT组A549细胞miR-320a表达水平升高(P<0.05)(见表 5)。
分组 WT-EGOT MUT-EGOT miR-NC 0.99±0.05 0.97±0.06 miR-320a 0.38±0.04 1.00±0.07 t 28.58 0.98 P <0.01 >0.05 表 4 双荧光素酶报告实验结果(ni=9;x±s)
分组 miR-320a F P MS组内 pcDNA 1.00±0.05 579.59 <0.01 0.018 pcDNA-EGOT si-NC 0.50±0.05* 1.03±0.08# si-EGOT 2.97±0.25*#▲ q检验:与pcDNA组比较*P<0.05;与pcDNA-EGOT组比较#P<0.05;与si-NC组比较▲P<0.05 表 5 lncRNA EGOT靶向调控miR-320a的表达(ni=9;x±s)
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与LPS+anti-miR-NC组比较,LPS+anti-miR-320a组A549细胞miR-320a表达、Bax蛋白表达、细胞凋亡率均明显降低(P<0.01),Bcl-2蛋白表达明显升高(P<0.01),培养液中IL-6和IL-1β水平均明显降低(P<0.01)(见图 4~5、表 6)。
分组 miR-320a IL-6/(pg/mL) IL-1β/(pg/mL) 凋亡率/% Bcl-2蛋白 Bax蛋白 LPS+anti-miR-NC 1.00±0.06 348.12±16.39 181.36±14.88 27.92±2.51 0.27±0.03 0.60±0.04 LPS+anti-miR-320a 0.54±0.05 152.01±16.50 78.06±7.03 12.56±1.18 0.58±0.04 0.30±0.03 t 17.67 25.30 188.83 16.61 18.60 14.40 P <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 表 6 抑制miR-320a表达对LPS诱导的肺泡上皮细胞炎症因子和细胞凋亡的影响(ni=9;x±s)
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EGOT过表达对LPS诱导的肺泡上皮细胞炎症因子和细胞凋亡的作用与LPS+pcDNA-EGOT+miR-NC组比较,LPS+pcDNA-EGOT+miR-320a组A549细胞miR-320a表达、Bax蛋白表达、细胞凋亡率均明显升高(P<0.01),Bcl-2蛋白表达明显降低(P<0.01),培养液中IL-6和IL-1β水平均明显升高(P<0.01)(见图 6~7、表 7)。
分组 miR-320a IL-6/(pg/mL) IL-1β/(pg/mL) 凋亡率/% Bcl-2蛋白 Bax蛋白 LPS+pcDNA-EGOT+miR-NC 1.00±0.06 137.82±11.14 63.34±6.47 11.79±1.02 0.64±0.06 0.22±0.02 LPS+pcDNA-EGOT+miR-320a 2.53±0.23 311.34±28.45 145.64±12.94 20.51±1.84 0.32±0.03 0.45±0.03 t 19.31 17.04 17.07 12.44 14.31 19.14 P <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 表 7 miR-320a过表达逆转lncRNA EGOT过表达对LPS诱导的肺泡上皮细胞炎症因子和细胞凋亡的作用(ni=9;x±s)
lncRNA EGOT靶向miR-320a对LPS诱导肺泡上皮细胞炎症反应和细胞凋亡的影响
Effect of lncRNA EGOT on LPS-induced inflammation and apoptosis of alveolar epithelial cells by targeting miR-320a
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摘要:
目的探讨长链非编码RNA (lncRNA)嗜酸性粒细胞转录因子(EGOT)对脂多糖(LPS)诱导的肺泡上皮细胞A549凋亡、炎症反应的影响及可能机制。 方法将A549细胞分为对照组(Con)、LPS组、LPS+pcDNA组、LPS+pcDNA-EGOT组、LPS+anti-miR-NC组、LPS+anti-miR-320a组、LPS+pcDNA-EGOT+miR-NC组、LPS+pcDNA-EGOT+miR-320a组。实时荧光定量PCR (RT-qPCR)检测EGOT和miR-320a表达水平;流式细胞术检测细胞凋亡;试剂盒检测白细胞介素(IL)-6、IL-1β水平。双荧光素酶报告基因实验和RT-qPCR确定EGOT和miR-320a之间靶向作用。 结果与Con组比较,LPS组A549细胞凋亡率、IL-6和IL-1β水平均升高(P < 0.05),EGOT表达水平降低(P < 0.01),miR-320a表达水平明显升高(P < 0.01)。与LPS+pcDNA组比较,LPS+pcDNA-EGOT组A549细胞凋亡率、Bax蛋白表达、IL-6和IL-1β水平均降低(P < 0.05),Bcl-2蛋白表达升高(P < 0.05)。与LPS+anti-miR-NC组比较,LPS+anti-miR-320a组A549细胞凋亡率、Bax蛋白表达、IL-6和IL-1β水平均明显降低(P < 0.01),Bcl-2蛋白表达明显升高(P < 0.01)。与LPS+pcDNA-EGOT+miR-NC组比较,LPS+pcDNA-EGOT+miR-320a组A549细胞凋亡率、Bax蛋白表达、IL-6和IL-1β水平均明显升高(P < 0.01),Bcl-2蛋白表达明显降低(P < 0.01)。双荧光素酶报告实验显示,EGOT靶向负性调控miR-320a表达。 结论lncRNA EGOT通过靶向miR-320a可减轻LPS诱导肺泡上皮细胞炎症反应和细胞凋亡。 Abstract:ObjectiveTo explore the effect of long-chain non-coding RNA eosinophilic transcription factor (EGOT) on lipopolysaccharide (LPS) -induced apoptosis and inflammation of alveolar epithelial A549 cells and its possible mechanism. MethodsA549 cells were divided into control (Con) group, LPS group, LPS+pcDNA group, LPS+pcDNA-EGOT group, LPS+anti-miR-NC group, LPS+anti-miR-320a group, LPS+pcDNA-EGOT+miR-NC group, and LPS+pcDNA-EGOT+miR-320a group.Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of EGOT and miR-320a, flow cytometry was applied to detect apoptosis, and the kit was used to detect the levels of interleukin (IL) -6 and IL-1β.Dual luciferase reporter gene assay and RT-qPCR was employed to confirm the targeting relationship between EGOT and miR-320a. ResultsCompared with the Con group, the apoptosis rate, IL-6 and IL-1β levels of A549 cells in the LPS group were increased(P < 0.01), the EGOT expression level was reduced(P < 0.05), and the miR-320a expression level was significantly increased (P < 0.01).Compared with the LPS+pcDNA group, the apoptosis rate, Bax protein expression, IL-6 and IL-1β levels of A549 cells in the LPS+pcDNA-EGOT group were reduced(P < 0.05), and the Bcl-2 protein expression were increased(P < 0.05).Compared with the LPS+anti-miR-NC group, the apoptosis rate, Bax protein expression, IL-6 and IL-1β levels of A549 cells in the LPS+anti-miR-320a group were significantly reduced(P < 0.01), and the Bcl-2 protein expression were significantly increased(P < 0.01).Compared with the LPS+pcDNA-EGOT+miR-NC group, the apoptosis rate, Bax protein expression, IL-6 and IL-1β levels of A549 cells in the LPS+pcDNA-EGOT+miR-320a group were significantly increased (P < 0.01), and the Bcl-2 protein expression were significantly reduced (P < 0.01).The results of dual luciferase reporter gene assay showed that EGOT targeted and negatively regulated the miR-320a expression. ConclusionsLncRNA EGOT can alleviate LPS-induced inflammation and apoptosis of alveolar epithelial cells by targeting miR-320a. -
表 1 lncRNA EGOT和miR-320a在LPS刺激的肺上皮细胞中的表达(ni=9;x±s)
分组 EGOT miR-320a Con 1.00±0.07 1.00±0.08 LPS 0.39±0.03 2.61±0.23 t 24.03 19.83 P <0.01 <0.01 表 2 lncRNA EGOT过表达对LPS诱导的肺泡上皮细胞炎症因子表达的影响(ni=9;x±s)
分组 EGOT IL-6/(pg/mL) IL-1β/(pg/mL) Con 1.00±0.05 106.48±10.42 46.17±4.94 LPS 0.43±0.04* 353.51±25.90* 180.73±12.98* LPS+pcDNA 0.41±0.04* 349.84±19.43* 177.28±14.47* LPS+pcDNA-EGOT 2.31±0.22*#▲ 139.69±12.53*#▲ 64.96±6.26*#▲ F 528.68 482.33 419.16 P <0.01 <0.01 <0.01 MS组内 0.014 328.478 110.363 q检验:与Con组比较*P<0.05;与LPS组比较#P<0.05;与LPS+pcDNA组比较▲P<0.05 表 3 lncRNA EGOT过表达对LPS诱导的肺泡上皮细胞凋亡的影响(ni=9;x±s)
分组 凋亡率/% Bcl-2蛋白 Bax蛋白 Con 6.52±0.60 0.73±0.07 0.17±0.02 LPS 25.45±2.28* 0.28±0.03* 0.55±0.04* LPS+pcDNA 26.34±2.81* 0.26±0.02* 0.58±0.05* LPS+pcDNA-EGOT 10.59±1.01*#▲ 0.62±0.05*#▲ 0.24±0.02*#▲ F 256.47 234.86 324.49 P <0.01 <0.01 <0.01 MS组内 3.619 0.002 0.001 q检验:与Con组比较*P<0.05;与LPS组比较#P<0.05;与LPS+pcDNA组比较▲P<0.05 表 4 双荧光素酶报告实验结果(ni=9;x±s)
分组 WT-EGOT MUT-EGOT miR-NC 0.99±0.05 0.97±0.06 miR-320a 0.38±0.04 1.00±0.07 t 28.58 0.98 P <0.01 >0.05 表 5 lncRNA EGOT靶向调控miR-320a的表达(ni=9;x±s)
分组 miR-320a F P MS组内 pcDNA 1.00±0.05 579.59 <0.01 0.018 pcDNA-EGOT si-NC 0.50±0.05* 1.03±0.08# si-EGOT 2.97±0.25*#▲ q检验:与pcDNA组比较*P<0.05;与pcDNA-EGOT组比较#P<0.05;与si-NC组比较▲P<0.05 表 6 抑制miR-320a表达对LPS诱导的肺泡上皮细胞炎症因子和细胞凋亡的影响(ni=9;x±s)
分组 miR-320a IL-6/(pg/mL) IL-1β/(pg/mL) 凋亡率/% Bcl-2蛋白 Bax蛋白 LPS+anti-miR-NC 1.00±0.06 348.12±16.39 181.36±14.88 27.92±2.51 0.27±0.03 0.60±0.04 LPS+anti-miR-320a 0.54±0.05 152.01±16.50 78.06±7.03 12.56±1.18 0.58±0.04 0.30±0.03 t 17.67 25.30 188.83 16.61 18.60 14.40 P <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 表 7 miR-320a过表达逆转lncRNA EGOT过表达对LPS诱导的肺泡上皮细胞炎症因子和细胞凋亡的作用(ni=9;x±s)
分组 miR-320a IL-6/(pg/mL) IL-1β/(pg/mL) 凋亡率/% Bcl-2蛋白 Bax蛋白 LPS+pcDNA-EGOT+miR-NC 1.00±0.06 137.82±11.14 63.34±6.47 11.79±1.02 0.64±0.06 0.22±0.02 LPS+pcDNA-EGOT+miR-320a 2.53±0.23 311.34±28.45 145.64±12.94 20.51±1.84 0.32±0.03 0.45±0.03 t 19.31 17.04 17.07 12.44 14.31 19.14 P <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 -
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