Preliminary Study Of Autophagic SNARE Proteins On Regulating Pluripotent Genes Expression Of Mouse Embryonic Stem Cells

Autophagy is a highly conserved metabolic pathway in eukaryotes,which plays a crucial role in degrading long-lived proteins,removing damaged organelles and maintaining homeostasis of intracellular environment.Autophagy is broadly divided into three types:macroautophagy,microautophagy and chaperone-mediated autophagy.And macroautophagy(hereafter referred to as autophagy)is the focus of our work and involves the formation of autophagosomes,the maturation of autophagosomes,the degradation of substrate,and the regeneration of lysosome.It is note worthy that the maturation of autophagosomes is regulated by a large number of proteins.This project focuses on SNARE proteins,including Snap29,Stx17 and Vamp8.Embryonic stem cells(ESCs)have received increasing attention because of their pluripotency and strong self-renewal ability.The maintenance of ESCs pluripotency is regulated by a variety of transcription factors,including Oct4,Nanog and Sox2.The loss of Oct4,Nanog and Sox2 would destroy the stability of ESCs.As for autophagy in ESCs,Atg3,ULK1 and other key factors involved in the formation of autophagosomes have been reported to play an important role in the maintenance of mouse ESCs pluripotency,but the role of autophagic SNARE proteins in ESCs has not been reported,and the specific mechanism of autophagy regulating ESCs pluripotency remains to be studied.For this problem,we preliminarily investigated the role of autophagic SNARE proteins in the maintenance of ESCs pluripotent genes expression.Firstly,we designed specific small interfering RNAs(siRNAs)targeting Vamp8,Stx17 and Snap29,and the siRNAs were transfected into mouse ESCs to produce specific knockdown phenotype in ESCs.Then we'll detect the effect on key polyfunctional genes expression in mouse ESCs.Secondly,the Vamp8,Stx17 and Snap29 genes in ESCs of mice were knocked out by CRISPR/Cas9.At the early stage of the experiment,we designed and synthesized sgRNA for mouse ESCs,connected sgRNA with the carrier containing Cas gene,and entered ESCs through lentivirus packaging.In the end,the specific gene was knocked out by using the repair mechanisms inside the organism.Finally,we detected the effect of stable knockout cell lines on the expression of ESCs pluripotent genes,cell cycle and apoptosis.Real-time quantitative polymerase chain reaction,western blot and flow cytometry were used for this process.The results of this study showed that:(1)The designed and synthesized siRNA could reduce the expression of Snap29,Stx17 and Vamp8,respectively.However,the expression of Oct4,Nanog and Sox2 did not be changed significantly after the decrease of SNARE expression.(2)In the process of using CRISPR/Cas9 technology to knockout Snap29,Stx17 and Vamp8 respectively,after a large number of selective cloning experiments,only knockout Snap29 cell lines were obtained.(3)The stable cell lines of knock out Snap29 were used to study the expression of mouse ESCs pluripotent genes,cell cycle and apoptosis.The current results showed that the knockout of Snap29 did not significantly change the expression of Oct4,Nanog and Sox2 in mouse ESCs.In addition,cell cycle and apoptosis of mouse ESCs did not be changed significantly after knockout Snap29.In summary,through siRNA interference with Vamp8,Stx17,and Snap29,we found that the expression of key polyfunctional genes in ESCs was not significantly affected.The knockout of Snap29 by CRISPR/Cas9 also did not significantly affect the expression of key polyfunctional genes in ESCs.Cell cycle and apoptosis were not significantly changed in the ESCs of knockout Snap29 mice.However,the effects of SNARE proteins on pluripotency,especially on developmental potential of ESCs,remain to be further studied.