klotho通过IGF-1/PI3K调控滋养层细胞胰岛素抵抗影响妊娠期糖尿病发生的机制研究

Mechanism of klotho gene regulation on insulin resistance in trophoblast cells through IGF-1/PI3K signaling pathway to affect development of gestational diabetes mellitus

摘要

目的 本研究旨在探讨klotho通过IGF-1/PI3K信号通路调控滋养层细胞的胰岛素抵抗(IR)影响妊娠期糖尿病(GDM)发生的作用机制。方法 利用The Human Protein Atlas数据库预测klotho在胎盘中的表达情况,使用人胎盘滋养层细胞HTR-8-Svneo细胞系建立IR模型。使用shRNA技术降低klotho基因在细胞中的表达。实验分为4组:对照组(Control组)、IR组、IR+shRNA-klotho组和IR+shRNA-klotho-NC组(IR+shRNA组阴性对照)。利用RT-qPCR检测klotho mRNA表达,CCK-8法评估细胞增殖,TUNEL法检测细胞凋亡,Western印记检测相关蛋白表达,并通过葡萄糖摄取实验评估细胞的葡萄糖摄取能力。结果 数据库研究显示,klotho在胎盘中有较高水平的表达。研究发现,与Control组相比,IR组细胞的klotho mRNA表达水平显著升高(t=6.063,P<0.001),24 h与48 h细胞增殖能力减弱(t=5.312、8.647,P<0.001),凋亡率上升(t=6.258,P<0.001)。而通过shRNA转染降低klotho表达后,相较于IR+shRNA-klotho-NC组,IR+shRNA-klotho组24 h与48 h细胞增殖能力得到改善(t=5.082、11.092,P<0.001),凋亡率降低(t=4.640,P<0.001)。Western印记检测结果显示,与对照组相比,IR组细胞中klotho蛋白表达量显著升高(t=7.842,P<0.001),胰岛素样生长因子1(IGF-1)蛋白表达量显著下降(t=6.950,P<0.001),且IGF-1R、磷脂酰肌醇-3-激酶(PI3K)和IRS-1蛋白磷酸化水平也显著下降(t=3.813、3.075、8.733,P<0.05)。降低klotho表达后,相较于IR+shRNA-klotho-NC组,IR+shRNA-klotho组细胞klotho蛋白表达量则明显下降(t=6.558,P<0.001),而IGF-1明显上升(t=8.206,P<0.001),IGF-1R、PI3K和IRS-1蛋白磷酸化水平也显著上升(t=4.601、3.296、12.326,P<0.05)。此外,相较于Control组,IR组细胞的葡萄糖摄取量显著降低(t=5.788,P<0.001),而降低klotho表达后,相较于IR+shRNA-klotho-NC组,IR+shRNA-klotho组细胞葡萄糖摄取量显著升高(t=4.173,P<0.001)。结论 降低klotho表达可以改善滋养层细胞的IR,促进滋养层细胞增殖,降低凋亡,并提高葡萄糖摄取能力,并推测是通过IGF-1/PI3K信号通路调控产生效应。这些发现为理解和治疗GDM提供了新的分子靶点。

Abstract

Objective To explore mechanism by which klotho regulates insulin resistance (IR) in trophoblast cells through insulin like growth factor 1 (IGF-1)/phosphatidylinositol 3-kinase (PI3K) signaling pathway to affect development of gestational diabetes mellitus (GDM). Methods The Human Protein Atlas (HPA) database was used to predict expression of klotho in human placenta. The human placental trophoblast HTR-8-Svneo cell line was used to establish an IR model by treating cells with different concentrations of insulin. The expression of klotho gene in the cells was reduced using shRNA technology. The human placental trophoblast cells in the experiment were divided into four groups: control group, IR group, IR + shRNA-klotho group, and IR + shRNA-klotho-NC group (negative control for IR + shRNA). Real time quantitative polymerase chain reaction (RT-qPCR) was used to detect klotho mRNA expression; CCK-8 reagent assay was used to assess proliferation of the cells; terminal deoxynucleotidyl tranferase-mediated dUTP-biotin nick end labeling assay (TUNEL) was used to detect apoptosis of the cells; Western blotting was used to detect expression of related protein in the cells and glucose uptake test was used to assess glucose uptake capacity of the cells. Results The studies from the HPA database show that klotho is expressed at relatively high level in the placenta. In our study, it was found that compared to the control group, expression of klotho mRNA in the trophoblast cells in the IR group was significantly higher (t=6.063, P<0.001), while cell proliferation ability weakened at both 24 h and 48 h (t=5.312 and 8.647 respectively, both P<0.001), with an increased apoptosis rate (t=6.258, P<0.001). After reducing klotho expression through shRNA transfection, compared to the IR+shRNA-klotho-NC group, the proliferation ability of the trophoblast cells in the IR+shRNA-klotho group improved at both 24 h and 48 h (t=5.082 and 11.092 respectively, both P<0.001), and the apoptosis rate decreased (t=4.640, P<0.001). Western blot analysis showed that expression of klotho protein in the cells in the IR group was significantly higher than that in the control group (t=7.842, P<0.001), while expression of IGF-1 protein was significantly lower than that in the control group (t=6.950, P<0.001), and phosphorylation levels of IGF-1 receptor (IGF-1R), PI3K, and insulin receptor substrate 1 (IRS-1) were also significantly lower (t=3.813, P<0.001; t=3.075, P=0.003; t=8.733, P<0.001). After the expression of klotho was reduced, the expression amount of IGF-1 protein in the trophoblast cells in the IR+shRNA-klotho group was significantly lower than that in the trophoblast cells in the IR+shRNA-klotho-NC group (t=6.558, P<0.001), while the level of IGF-1 and the phosphorylation levels of IGF-1R, PI3K and IRS-1 protein also significantly higher (t=8.206, P<0.001; t=4.601, P<0.001; t=3.296, P=0.002; t=12.326, P<0.001). Furthermore, compared to the control group, the glucose uptake of the trophoblast cells in the IR group was significantly lower (t=5.788, P<0.001). After the expression of klotho gene was reduced, compared to the IR+shRNA-klotho-NC group, the glucose uptake of the trophoblast cells in the IR+shRNA-klotho group was significantly higher (t=4.173, P<0.001). Conclusion Reducing expression of klotho gene can improve IR in trophoblast cells, promote cell proliferation, reduce cell apoptosis, and enhance glucose uptake. It is hypothesized that these effects exert through the IGF-1/PI3K signaling pathway. These findings provide new molecular targets for understanding and treating GDM.

关键词

klotho / 胰岛素抵抗 / 妊娠期糖尿病 / IGF-1/PI3K信号通路 / 滋养层细胞

Key words

klotho gene / insulin resistance / gestational diabetes mellitus / IGF-1/PI3K signaling pathway / trophoblast cell

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