The Molecular Mechanism Of FoxO1 Regulating The Incidence Of Gestational Diabetes Mellitus Through FGF12

Objective: Gestational diabetes mellitus(GDM)is a special type of diabetes that is identified after pregnancy or first occurs with varying degrees of impaired glucose tolerance or symptoms typical of diabetes.Gestational diabetes is harmful to the fetus,newborn and pregnant women.For the fetus,hyperglycemia can cause macrofetus,fetal intrauterine growth restriction,fetal congenital malformation,fetal death,etc.It can cause hypoglycemia and respiratory distress syndrome in neonates.For pregnant women,it can cause complications such as hypertension during pregnancy,infection in pregnant women,polyamniotic fluid,ketoacidosis,dystocia and increased operation rate.It also leads to an increased incidence of type 2 diabetes in pregnant women after childbirth.Therefore,gestational diabetes not only affects fetal development,but also endangers the health of mothers and newborns.It is of great significance to study the molecular mechanism of this disease for its prevention and treatment.Fox O1 is an important transcription factor involved in the regulation of multiple genes.The function of Fox O1 in diabetes has been widely recognized,but its function in GDM is rarely studied.Previous studies have shown that Fox O1 expression level is increased in the placental tissues of pregnant women with GDM,and Mir-142-3p promotes the survival of placental trophoblast cells by inhibiting Fox O1 expression in patients with gestational diabetes.Although a few studies have demonstrated the expression and function of Fox O1 in GESTational diabetes mellitus,its downstream regulatory genes and related signaling pathways are still unclear.Therefore,finding its downstream regulatory genes is of great significance to reveal the function of Fox O1 in gestational diabetes mellitus.Fibroblast growth factor(FGF),also known as heparin binding growth factor,is a kind of polypeptide substance that can promote fibroblast mitosis and mesodermal cell growth,and can also stimulate blood vessel formation and play a role in wound healing and limb regeneration.At present,there are few studies on the function of FGF12.FGF12 can be used as an oncogene to promote the proliferation and metastasis of tumor cells.At the same time,FGF12 can reduce the cell apoptosis induced by radiotherapy and reduce the tissue damage induced by radiotherapy.FGF12 plays a key role in the occurrence and development of many diseases,but the function of FGF12 in gestational diabetes has not been reported.FGF12 has been screened as a significantly differentially expressed gene by sequencing analysis of placental tissues of patients with gestational diabetes in the preliminary experiment,indicating that its role in the pathogenesis of gestational diabetes cannot be ignored.Studies have shown that Fox O1 plays an important role in the occurrence and development of diabetes by participating in the regulation of insulin resistance,islet β cell proliferation,differentiation and apoptosis,glucolipid metabolism,oxidative stress and inflammatory response.At present,it is generally believed that the pathogenesis of gestational diabetes mellitus is similar to type 2 diabetes mellitus,Fox O1 is highly expressed in the placental tissues of gestational diabetes.Therefore,we speculate whether there is a certain relationship between FGF12 and Fox O1,and whether they are involved in the pathogenesis of gestational diabetes.Therefore,the purpose of this study is to investigate the regulatory relationship between Fox O1 and FGF12 gene at the molecular level,so as to further explore the mechanism of Fox O1 in gestational diabetes.Methods: 1.According to the diagnostic criteria of alternative GDM guidelines recommended by The World Health Organization(WHO)in 2013,18 pregnant women with gestational diabetes who delivered by cesarean section in Dalian Maternal and Child Health Hospital in 2019 and 18 pregnant women with OGTT who delivered by normal cesarean section without obstetric complications during the same period were selected as the control group,and the placental tissues voluntarily abandoned were collected.The placental tissues of 2 GDM and 2 normal was sent to Beijing BMG Company for RNA sequencing analysis and the differentially expressed gene FGF12 was screened out.Subsequently,the expression of FGF12 m RNA level in the placental tissue of GDM pregnant women was verified by fluorescence quantitative PCR,and the expression of FGF12 protein level in the placental tissue of GDM pregnant women was verified by immunohistochemistry,and the RNA sequencing results were confirmed.Western blot was used to verify the expression of Fox O1 in the placenta of GDM pregnant women,and the correlation between Fox O1 and FGF12 expression in the placenta of GDM pregnant women.The effect of Fox O1 on FGF12 gene promoter activity was detected by luciferase reporter assay,and the potential binding sites of Fox O1 on FGF12 gene promoter were analyzed and searched by bioinformatics.2.Taking trophoblast cell HTR8/SVneo as the research object,construct FGF12 knockdown or overexpression plasmid,and establish trophoblast cell HTR8/SVneo cell line with stable knockdown or overexpression of FGF12 through lentivirus system.The knockdown or overexpression of FGF12 was detected by fluorescence quantitative PCR and Western blot.The effects of FGF12 modification on the migration and proliferation of Trophoblast cells were detected by Transwell and clonogenesis assay and CCK8 assay.3.Fox O1 was knocked down or overexpressed in HTR8/SVneo cells,and the expression changes of endogenous FGF12 were detected by fluorescence quantitative PCR and Western blot.The effect of Fox O1 on FGF12 promoter activity in HTR8/SVneo cells was detected by luciferase reporter assay.Chromosomal immunoprecipitation assay confirmed that Fox O1 directly binds to the potential binding site of FGF12 gene promoter and plays a regulatory role.Effects of changing Fox O1 expression on cell biological behavior in FGF12 overexpressed HTR8/SVneo cells and control cells.4.The effects of FGF12 knockdown or overexpression on HTR8/SVneo cell apoptosis induced by high glucose were detected by flow cytometry in simulated high glucose environment in gestational diabetes patients.The regulation of Fox O1 on FGF12 gene in GDM placental tissue was confirmed.Results: 1.By RNA sequencing,91 down-regulated and 145 up-regulated genes were screened out.Fluorescence quantitative PCR confirmed that the m RNA level of FGF12 decreased significantly in the placental tissues of GDM pregnant women with normal glucose tolerance as control.Immunohistochemistry further confirmed that the level of FGF12 protein decreased significantly in GDM placental tissues.The expression of Fox O1 in placenta of GDM pregnant women was increased by Western blot,and the expression of Fox O1 and FGF12 in placenta was negatively correlated.The luciferase reporter gene knockdown overexpression of Fox O1 inhibited FGF12 promoter activity;The transcription factor Fox O1 has a potential binding site(tgttgttgact)upstream of the promoter of FGF12 gene from-1199 bp to-1188 bp.2.The number of migration and proliferation of HTR8/SVneo cells decreased significantly after FGF12 knockdown;After overexpression of FGF12,the number of migration and proliferation of HTR8/SVneo cells increased significantly,and the difference was statistically significant.3.FGF12 m RNA and protein levels were significantly increased after Fox O1knockdown;The m RNA and protein levels of FGF12 were significantly decreased after overexpression of Fox O1.FGF12 promoter activity increased after Fox O1 knockdown in trophoblast cells by luciferase reporter gene assay.FGF12 promoter activity decreased after overexpression of Fox O1.Chromosome immunoprecipitation assay confirmed that Fox O1 can directly bind to FGF12 promoter and regulate its activity.Fox O1 transcriptionally regulates FGF12,reducing the migration and proliferation of HTR8/SVneo cells overexpressing FGF12.4.FGF12 knockdown increased the number of HTR8/SVneo cell apoptosis induced by high glucose.On the contrary,overexpression of FGF12 reduced the number of HTR8/SVneo cell apoptosis induced by high glucose,and the difference was statistically significant.Conclusion: 1.The expression of FGF12 protein and m RNA in GDM placental tissues decreased.The expression of Fox O1 protein was increased in the placental tissues of GDM pregnant women,and the expression of Fox O1 was negatively correlated with FGF12.Transcription factor Fox O1 inhibits FGF12 promoter activity.2.Knockdown FGF12 in trophoblast cells HTR8/SVneo inhibited cell migration and proliferation;Conversely,overexpression of FGF12 promoted cell migration and proliferation.3.Fox O1 expression was negatively correlated with FGF12 expression in trophoblast cells HTR8/SVneo;The transcription factor Fox O1 inhibits FGF12 promoter activity.Transcription factor Fox O1 inhibits proliferation and migration of trophoblast cells HTR8/SVneo by inhibiting FGF12 gene expression.4.Knockdown FGF12 promoted HTR8/SVneo cell apoptosis induced by high glucose;Conversely,overexpression of FGF12 inhibited HTR8/SVneo cell apoptosis induced by high glucose.