miR-486调控PTEN影响LPS诱导的肺泡上皮细胞A549凋亡的实验研究

目的:探讨微小RNA-486(miR-486)对脂多糖(LPS)诱导的肺泡上皮细胞A549凋亡的影响和机制。方法:以LPS处理肺泡上皮细胞,RT-qPCR测定miR-486表达变化。在肺泡上皮细胞中转染miR-486模拟物(miR-486 mimics),RT-qPCR检测LPS条件下的转染效果。流式细胞术测定凋亡变化,Western blot检测细胞中cleaved caspase-3(C-caspase-3)和C-caspase-9蛋白表达变化。靶基因预测软件预测第10号染色体同源缺失性磷酸酶-张力蛋白(PTEN)可能是miR-486的靶基因,利用萤光素酶报告载体鉴定靶向关系。在肺泡上皮细胞中共转染pcDNA 3.1-PTEN和miR-486 mimics,检测上调PTEN对miR-486 mimics调控LPS诱导的A549细胞凋亡的作用。结果:LPS处理后的肺泡上皮细胞中miR-486的表达水平显著下降(P<0.05)。miR-486 mimics可以显著上调LPS条件下肺泡上皮细胞中miR-486的表达水平(P<0.05)。LPS处理后的肺泡上皮细胞凋亡率及C-caspase-3和C-caspase-9蛋白表达水平显著升高(P<0.05);上调miR-486可以显著下调LPS诱导的肺泡上皮细胞凋亡(P<0.05)。miR-486靶向负调控PTEN的表达。pcDNA 3.1-PTEN可以显著提高miR-486 mimics转染后LPS诱导的肺泡上皮细胞中PTEN的表达,促进细胞凋亡和细胞中C-caspase-3和C-caspase-9蛋白的表达(P<0.05)。结论:miR-486靶向抑制PTEN的表达,减少LPS诱导的肺泡上皮细胞凋亡。

miR-486 targeted regulation of PTEN on LPS-induced apoptosis of alveolar epithelial cell A549

AIM:To investigate the effect of microRNA-486(miR-486)on lipopolysaccharide(LPS)-induced apoptosis of alveolar epithelial cell A549.METHODS:A549 cells were treated with LPS,and the expression of miR-486 was detected by RT-qPCR.miR-486 mimics were transfected into LPS-induced A549 cells,and RT-qPCR was used to detect the up-regulation effect.The apoptotic rate was analyzed by flow cytometry and the protein levels of cleaved caspase-3(C-caspase-3)and C-caspase-9 were determined by Western blot.The target gene prediction software was used to predict the target gene PTEN of miR-486.Luciferase reporter vector was used to identify the target relationship.pcDNA 3.1-PTEN and miR-486 mimics were co-transfected into A549 cells to detect the effect of PTEN up-regulation on apoptosis of miR-486 mimics transfected A549 cells stimulated with LPS.RESULTS:After LPS treatment,the expression of miR-486 in A549 cells was significantly decreased(P<0.05).Transfection of miR-486 mimics significantly up-regulated the expression of miR-486 in A549 cells stimulated with LPS(P<0.05).The apoptotic rate of A549 cells and the protein levels of C-caspase-3 and C-caspase-9 were significantly increased after LPS treatment(P<0.05).Up-regulation of miR-486 significantly down-regulated LPS-induced apoptosis of A549 cells(P<0.05).The expression of PTEN was negatively regulated by miR-486.Transfection of pcDNA 3.1-PTEN significantly increased the expression of PTEN,promoted the apoptosis and increased the protein levels of C-caspase-3 and C-caspase-9 in A549 cells stimulated with LPS after co-transfection with miR-486 mimics(P<0.05).CONCLUSION:miR-486 inhibits PTEN expression and reduces LPS-induced apoptosis of A549 cells.