Research Of The Function Of S100A10 In Cell Proliferation And Autophagy

S100A10 is a special member of the S100 family,a family of small acid Ca²⁺-binding proteins.S100A10 is Ca²⁺insensitive because of primary amino acid replacements in its Ca²⁺-binding loops that lock the protein in a permanently active state.Usually S100A10 functions as a tight heterotetrameric complex with Annexin A2,mainly locates at the cell membrane,especially at the plasma membrane and the membrane of early endosomes.Besides it can also be secreted outside the cell to extracellular matrix(ECM).S100A10 has been proposed to play a key role in many processes such as trafficing and location of ion channels,recruitment of receptors,endocytosis,exocytosis,membrane organization,cytoskeleton rearrangement and vascular fibrinolysis.In the human and animal models,dysregulation of S100A10 has been reported to the pathogenesis of inflammatory,autoimmune and malignant disease,especially oncogenesis,as well as depression.At the present,the mechanism of S100A10 in cell proliferation is lack of research,especially in autophagy.CRISPR/Cas9 Systerm is a powerful tool in genome editing.CRISPR/Cas9Systerm has been paid broad attention ever since it was first reported on January 3,2013,and CRISPR/Cas9 Systerm has experienced rapid progress in its technology and applicability in the past two years,becoming a simple and efficient tool for genome editing.The purpose of this paper is aimed to study the effect of S100A10 on cell proliferation and the molecular mechanisms of cell proliferation influenced by S100A10,as well as the function of S100A10 in autophagy.The CRISPR/Cas9system is used in the preparation of S100A10 knock out cell lines and knock out mice.To study the function of S100A10 in cell proliferation,two classical methods of studying gene function were used,namely gain of function and loss of function.Firstly,S100A10 was knocked out in MEF cell lines by using CRISPR/Cas9system,CCK8,cell counting,RTCA(real time cell analysis)and Brdu embedded analysis showed that S100A10 knockout cell lins grew slowly.In order to further verify the results,exogenous S100A10 was introduced to restore the expression of S100A10 in S100A10 knockout MEF Cell lines,which showed a recovered cell proliferation.Moreover,using labeled actin by phalloidin ring peptide,I observed that S100A10 knockout affected the cytoskeleton,which made the cells more spreading.The wounding assay showed that cells migrated slowly after S100A10 knockout,indicating that S100A10 can promote cell migration.In order to comprehensively confirm that S100A10 can promote cell proliferation,the S100A10 knockout mice were made using CRISPR/Cas9 system and the MEF cell from the S100A10 knockout homozygous mice were used to observe the effect of S100A10 on cell proliferation.It was also found that the growth of S100A10 knockout cell was inhibited.To explore the function of S100A10 in autophagy by S100A10 knockout MEF cells and knockout mice,the endogenous LC3 protein was detected in S100A10^+/+and S100A10^-/-.LC3B protein levels when starvation induction at different time periods(0.5,1,1.5,2 h)were ignificantly reduced,which indicated that S100A10 can promote MEF autophagy.Further statistics analysis of autophagy puncta using LC3B immunofluorescence showed that the autophagy puncta were decreased after S100A10 knockout.When recovery of the S100A10 expression,the autophagy was also increased.At the same time,the autophagy was also decreased in the S100A10 knockout primary MEF cells.Co-immunoprecipitation showed that the S100A10 and Annexin A2 can interact with LC3.In the S100A10 knockout MEF cell line and primary MEF cells,I observed that S100A10 can stablize Annexin A2 at the protein level,and in the wild-type MEF cell line,both S100A10 and Annexin A2 protein levels were increased with the starvation time prolonged,which suggested that S100A10 may regulate autophagy process through Annexin A2.