Systematic Characterization Of Autophagy In Gestational Diabetes Mellitus And Trophoblasts Under High Glucose

Objective:Autophagy is a dynamic process that degrades and recycles cellular organelles and proteins,the vesicles of which are known as autophagosomes,which fuse with lysosomes.Alterations in autophagy occur in various diseases,but the role of autophagy in gestational diabetes mellitus(GDM)is unknown.We aimed to characterize the roles and functions of autophagy in this disease.Methods:(1)Analysis of morphologic change and autophagy of placental extravillous trophoblasts(EVTs):Transmission electron microscopy was used to observe the ultrastructure of EVTs,the mRNA and protein level of ATG5,LC3-?,P62 were detected by qRT-PCR and Western blotting respectively.(2)Analysis of Genome-wide 5hmC sequencing data of umbilical cord for GDM and NP.(3)Observation of autophagy and cellular function:Treatment of HTR8/SVneo with different concentration of glucose.The mRNA level of ATG5,and the protein levels of ATG5,LC3-II,P62 were detected by qRT-PCR and Western blotting respectively.The distribution of GFP-LC3 was observed by confocal microscopy.CCK8,flow cytometry and transwell assays were used to detect cell proliferation,early cell apoptosis rate and invasion ability respectively.(4)Cellular functions and the level of autophagy after siATG5:The mRNA level of ATG5,and the protein levels of ATG5,LC3-?,P62 were detected by qRT-PCR and Western blotting respectively.The distribution of GFP-LC3 was observed by confocal microscopy.CCK8,flow cytometry and transwell assays were used to detect cell proliferation,early cell apoptosis rate and invasion ability respectively.(5)Detection of gene expression changes after siATG5 by mRNA sequencing.Results:(1)We detected ultrastructure changes of microvilli as well as increased autophagic vacuoles and enhanced autophagy in EVTs of GDM placenta.(2)The ATG5 promoter was marked with 5hmC in umbilical cord of GDM.5hmC specifically regulated signaling including autophagy,apoptosis and insulin response.(3)HTR8/SVneo treated with high glucose showed enhanced autophagy.Meanwhile,cellular functions such as early cell apoptosis rate,invasion ability and early cell apoptosis rate were changed in the present of high glucose.(4)Knockdown of ATG5 ameliorates the effect of apoptosis and invasion caused by high glucose.RNA-seq revealed that many critical pathways such as Macroautophagy and Apoptotic processes were significant changed.Conclusion:Our study demonstrated the significant functions of autophagy in GDM patients and an EVT cell line exposed to excess glucose.High glucose inhibit cell proliferation,early cell apoptosis rate and invasion ability,which can be reversed by knockdown of ATG5.