GAS5 通过抑制 PTEN 表达促进脓毒症中足细胞损伤。
GAS5 promotes podocyte injury in sepsis by inhibiting PTEN expression.
机构信息
Intensive Care Unit, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China.
出版信息
Eur Rev Med Pharmacol Sci. 2018 Dec;22(23):8423-8430. doi: 10.26355/eurrev_201812_16541.
OBJECTIVE
To investigate the potential role of long noncoding RNA (lncRNA) growth arrest specific transcript 5 (GAS5) in sepsis-induced podocyte injury and its underlying mechanism.
MATERIALS AND METHODS
The sepsis model was established by lipopolysaccharide (LPS) induction in podocytes. The expression levels of Nephrin and GAS5 were detected by quantitative Real-time polymerase chain reaction (qRT-PCR) after LPS induction in podocytes for 12 h, 24 h and 36 h, respectively. Western blot was used to detect the expression level of Nephrin in sepsis-induced podocytes. The mRNA expressions of GAS5 and Nephrin in podocytes were detected after transfection of GAS5 siRNA. Albumin influx in podocytes after GAS5 knockdown was detected by Transwell assay. Western blot was used to detect the protein expression of Snail in sepsis after GAS5 knockdown. The target gene of GAS5 was predicted by bioinformatics analysis. QRT-PCR and Western blot were used to detect the protein and mRNA levels of PTEN (phosphatase and tensin homolog deleted on chromosome ten). Nephrin expression and the albumin inflow after PTEN knockdown were then measured. The expression of PI3K/AKT/GSK3β was also detected after GAS5 was downregulated while PTEN was upregulated.
RESULTS
LPS stimulation downregulated the mRNA expression of Nephrin in podocytes and achieved the lowest level at 24 h. The protein expression change of Nephrin was consistent with its mRNA expression. In the septic state, the albumin influx of podocytes remarkably increased, but the function of podocyte barrier was weakened. Besides, GAS5 expression decreased in a time-dependent manner in LPS-induced podocytes. After GAS5 knockdown by siRNA, Nephrin expression and the function of podocyte barrier were significantly reduced. Snail expression was also upregulated in septic state, and GAS5 knockdown increased the expressions of phosphorylated Snail and PI3K/AKT/GSK3β. After knockdown of GAS5, the mRNA and protein levels of PTEN significantly decreased, which was contract to the expression of Snail. However, overexpression of PTEN could reverse the promotive effect of GAS5 on PI3K/AKT activation.
CONCLUSIONS
GAS5 expression decreased in sepsis-induced podocyte injury, and GAS5 was involved in regulating sepsis-induced podocyte injury by reducing PTEN expression.
目的
探讨长链非编码 RNA（lncRNA）生长停滞特异性转录物 5（GAS5）在脂多糖（LPS）诱导的足细胞损伤中的潜在作用及其机制。
材料与方法
通过 LPS 诱导足细胞建立脓毒症模型。分别在 LPS 诱导足细胞 12 h、24 h 和 36 h 后，采用实时定量聚合酶链反应（qRT-PCR）检测 Nephrin 和 GAS5 的表达水平。采用 Western blot 检测 LPS 诱导的足细胞中 Nephrin 的表达水平。用 GAS5 siRNA 转染后检测足细胞中 GAS5 和 Nephrin 的 mRNA 表达。用 Transwell 实验检测 GAS5 敲低后足细胞中白蛋白的内流。用 Western blot 检测 GAS5 敲低后脓毒症中 Snail 的蛋白表达。通过生物信息学分析预测 GAS5 的靶基因。采用 qRT-PCR 和 Western blot 检测磷酸酶和张力蛋白同源物缺失的染色体 10（PTEN）的蛋白和 mRNA 水平。然后测量 PTEN 敲低后 Nephrin 表达和白蛋白内流的变化。下调 GAS5 的同时上调 PTEN 后，还检测了 PI3K/AKT/GSK3β 的表达。
结果
LPS 刺激使足细胞中 Nephrin 的 mRNA 表达下调，在 24 h 时达到最低水平。Nephrin 的蛋白表达变化与其 mRNA 表达一致。在脓毒症状态下，足细胞的白蛋白内流明显增加，但其足细胞屏障功能减弱。此外，LPS 诱导的足细胞中 GAS5 的表达呈时间依赖性下降。用 siRNA 敲低 GAS5 后，Nephrin 表达和足细胞屏障功能明显降低。Snail 的表达在脓毒症状态下也上调，GAS5 敲低增加了磷酸化 Snail 和 PI3K/AKT/GSK3β 的表达。GAS5 敲低后，PTEN 的 mRNA 和蛋白水平显著降低，与 Snail 的表达一致。然而，过表达 PTEN 可以逆转 GAS5 对 PI3K/AKT 激活的促进作用。
结论
在 LPS 诱导的足细胞损伤中，GAS5 的表达降低，GAS5 通过降低 PTEN 的表达参与调节 LPS 诱导的足细胞损伤。
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