Microarray Analysis Of Differentially Expressed Genes In Vaginal Wall And Related Gene Polymorphism From Women With Stress Urinary Incontinence

BackgroundStress urinary incontinence(SUI) is defined as uncontrolled intraurethra incontinence when abdominal pressure increased by international continence society,SCI.The incidence rate of female SUI is about 40%,it has been regarded as a international disease for further research.SUI and POP(pelvic organ prolapse) are called pelvic floor dysfunction(PFD).The mechanism and etiology of SUI are complex,multiple factors,including anatomic and functional theory.When organism is affected by some factors and perform special biofunctions,its related genes begin to express,this process is called biologic or pathologic molecular figure. So gene map can be used to explore gene function and relationship between regulations,which can help us to know the physiologic and biochemical process of organism.With the development of gene chip,especially the accomplishment of human genome plan,we can get molecular figure by gene chip,to reach the gene level.Objective1.To identify the differentially expressed genes in paraurethra vaginal anterior wall tissues between post-menopause patients with stress urinary incontinence and those without SUI by Human Genome Expression Chip.2.To further explore the possible molecular mechanism associated with stress urinary incontinence(SUI) based on the differential expression profile.3.To compare the different expressed gene on RNA level and protein level between post-menopause SUI patients and pre-memopause SUI patients,find the relationship between ovarian hormone and gene express profile.4.To explore the relationship between significant gene polymorphism and SUI predisposition,to find the genetics evidence for SUI.Methods1.Anterior vaginal wall samples were obtained from three postmenopausal patients with SUI and three age/BMI/parity matched postmenopausal subjects without SUI.2.Total RNA from samples were used to synthesize biotin-labeled cRNA by One-Cycle cDNA Synthesis method and hybridized with Human Genome Chip.3.The differentially expressed genes were identified between SUI and normal controls with the threshold:Fold Change(FC)＞2.0 or＜0.5.The data was further interrogated with Gene Ontology(GO) and Pathway Analysis.4.Four significantly different expressed genes were confirmed by quantitative reverse transcription-polymerase chain reaction and Western Blot,immunochemistry.5.The same different expressed genes were confirmed by quantitative reverse transcription-polymerase chain reaction and Western Blot immunochemistry in post-menopause SUI patients and pre- menopause SUI patients.6.The relationship between ApoE genotype polymorphism and SUI was analyzed by PCR-RFLP.Results1.The quality of total RNA was ensured by quantification and electrophoresis.The hybridization module of Human Genome expression chip was successfully established.2.75 differentially expressed genes were screened between SUI and normal vaginal wall tissue,including 3 function-unknown genes.31 genes were upregulated in SUI group,while 44 downregulated.This differential expression profile was related to multiple functional proteins and pathways by biological analysis.Among these,SNARE signaling pathway and neurodegenerative disorder pathway were the most predominant.3.Quantitative PCR and W.B validated the significant different expression genes.4.ApoE,GRB2 are upregulated in post-menopause SUI patients than in pre-menopause SUI patients,while GOSRI,GBA are downregulated in post-menopause SUI patients than in pre-menopause SUI patients,the differences are no significant.5.ApoE genotype and alleles frequency were no significant difference between SUI and controls,but e4 allele frequency was high in SUI while e3 allele frequency was low in SUI patients.Conclusions1.Genome Expression microarray is an effective approach for the high-throughput analysis of gene expression profile and aids to further explore the underlying molecular mechanism in SUI.75 differentially expressed genes were screened between SUI and normal vaginal wall tissue, including 3 function-unknown genes.31 genes were upregulated in SUI group,while 44 downregulated.2.The pathophysiology of SUI is complex covering multiple functional proteins and pathways.Among these,SNARE signaling pathway and neurodegenerative disorders pathway were the most predominant and APOE,GRB2,GOSR1,GBA may contribute a neurodegenerative role to SUI development.3.Quantitative PCR and Western Blot validated the significant different expression genes from microarray data.4.Neurodegenerative disorder related genes were also differently expressed between post-menopause SUI patients and pre-menopause SUI patients,so the ovarian hormone may coordinate with these genes functional proteins to interfere the SUI developments.5.ApoE genotype and alleles frequency have no relationship with SUI,but e4 seems to be the potential risk factors for SUI.