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 24h and 48h(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 24h and 48h(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分类
医药卫生
