Mechanism Of Fascin In Regulating Of Mitochondrial Function And Gluconeogenesis Key Enzyme PCK2

Fascin,a member of actin-bundling family proteins,is expressed in a spatially and temporally restricted manner during development.Fascin is the most frequently upregulated actin regulatory protein in essentially all the carcinoma.Fascin plays a key role in tumor invasion and metastasis.Fascin is a potential therapeutic target for malignant tumors,which will provide new opportunities for cancer prevention and treatment.However,the underlying mechanism and normal cellular function of Fascin are largely unknown.The studies of this thesis were based on inhibition of the Fascin disrupts the stability of mt F-actin,leading to loss of mt DNA and reduction of biosynthesis of respiratory complexes,energy stress and cell migration.To elucidate how Fascin works in normal tissue,we designed in vivo and in vitro experiments.This study found that the loss of Fascin gene,the survival rate of mice will be reduced,further studies found that Fascin gene deletion affects the gluconeogenesis.We first established a Fascin knockout mouse model,using the CRISPR/Cas9 technology.Futher Fascin knockdown and overexpression cell models were also established,using CRISPR/Cas9,RNAi and point mutation technology in NIH3T3 cells.Real-time PCR and Western blot were used to detect changes gene expression.Electron microscopy and mitochondrial respiraton were utilized to observe mitochondrial morphology and mitochondrial oxygen consumption rate.The experimental results obtained are as follows:1.Fascin knockout mice were established by deletion of 583 bp of Fascin genomic DNA,which spans exons 2and 3 of Fascin m RNA.Homozygous Fascin knockout mice Fascin does not express in protein levels.This study obtained the complete Fascin gene knockout mice.2.Fascin knockout mice lost weight and developed leukoplakia in the abdomen,toes and tail.A portion of the mice died at fetal stage suggesting that Fascin is involved in mouse development.3.Mitochondrial expansion was observed in liver of Fascin knockout mice.We also observed the reduction ofthe mitochondrial oxygen consumption rate and the mitochondrial oxidative phosphorylation level in Fascin knockout mice.In addition,the electron transportchain complex assembly was decreased in these mice.Our findings indicate a pivotal role of Fascin in mitochondrial function.4.Phosphorylation level of the energy receptor protein AMPK was elevated in Fascin knockout mice and Fascin knockdown cells,indicating that depletion of Fascin activates AMPK.5 Kyoto Encyclopedia of Genes and Genomes(KEGG)database analysis revealed that AMPK activation will phosphorylate transcription factor(HNF4?).The phosphorylated HNF4? inhibits the transcriptional reduction of gluconeogenesis key enzyme PEPCK,and ultimately reduces gluconeogenesis.AICAR is an AMPK activator,treatment reduced PEPCK expression in NIH3T3 cells,whereas Dorsomorphin inhibitor administration induced PEPCK expression in NIH3T3 cells,implying that Fascin relies on AMPK to regulate gluconeogenesis.6 In normal mouse tissues,when Fascin was low expressed in the liver,the expression of PEPCK was also reduced,while the expression of Fascin gene was gradually increased in liver tissue with age.7 The protein level of PEPCK was reduced after depletion of Fascin gene in NIH3T3 cells,whereas PEPCK was increased following sodium pyruvate treatment.However,the expression levels of G6 P and FBP were essentially no change.These data suggest that Fascin specifically regulates PEPCK to modulate gluconeogenesis.Fascin expression was increased in sodium pyruvate treatment of NIH3T3 cells and fasting mouse.These observations further suggest that the occurrence of gluconeogenesis depends on Fascin.8 Knockdown of HNF4? reduced PEPCK expression,suggesting that Fascin regulates gluconeogenesis through HNF4?.The role of Fascin in promoting invasive metastasis of cancer cells has been extensively studied,but the role of Fascin in regulating mitochondrial function to affect gluconeogenesis has not been reported.Loss of Fascin in mice causes perinatal death,probably because Fascin affects mitochondrial function,activates AMPK,phosphorylates transcription factors HNF4? ? PGC1?,and reduces the transcription of the key gluconeogenesis enzyme PEPCK,ultimately can cause the inhibition of gluconeogenesis.To investigate the mechanism of Fascin mediated mitochondrial function in regulating gluconeogenesis,and to elucidate the role of Fascin in gluconeogenesis,which is providing new treatments for perinatal mortality in infants.Fascin can also be used one of the indicators for prenatal diagnosis.