MicroRNA Expression Profile In Periurethral Vaginal Wall From Postmenopausal Women With Stress Urinary Incontinence And Analysis Of Their Target Genes

●BACKGROUNDSMicroRNAs (miRNAs) are small, noncoding RNAs molecules of about 22nt, which is the research focus in life science in recent years. The known function of miRNAs is the post-transcriptional negative regulation to target mRNA expression. They are involved in the regulation of a variety of biological processes including developmental timing, signal transduction, cell proliferation and apoptosis, etc. In one word, miRNAs play an important role in both physiological and pathological process. At present, widely used methods in laboratory as follows, setting up the miRNAs expression profiles through the technique of miRNA chip, and then, verifying or identifying the target genes, further, analyzing the miRNA biological function. And the purpose of miRNA study is to reveal their important role in the development of disease, and to be adopted as diagnostic and gene therapeutic index for disease.Stress urinary incontinence (SUI) is one kind of pelvic floor dysfunction (PFD), which is a common health problem in older women, particularly, in post-menopausal women. The mechanism and etiology of SUI are complex, multi-factor, and not yet clear. So far, there is no one molecular mechanisms are widely accepted. Based on epidemiologic data, there appears to be a genetic predisposition for the development of SUI. Our research group obtained some significant differentially expressed genes in SUI from Gene Chip and further confirmation. Until now, there is no study of SUI on the level of the miRNA.The vaginal wall is the main supporting tissue for urethra and changes in components of it are known to be involved in the pathophysiology of SUI. So, periurethral vaginal wall at the level of urethrovesical junction was excised as our experimental samples.●OBJECTIVESThe objective of this study was to assess the expression profile of miRNAs and their predicted target genes in periurethral vaginal wall from post-menopausal women with SUI, to initially establish the "microRNA/target gene/protein" interaction molecular network, and to further explore the pathogenesis of SUI in the respect of the microRNA.●METHODS1. Anterior vaginal wall samples were obtained from 3 postmenopausal patients with SUI and 3 age/BMI/parity/delivery mode/menopausal state matched postmenopausal women without SUI.2. Total RNA from samples were used to synthesize cDNA and analyze with miRNA chip.3. Candidate target genes were obtained through cross-matching select combined the miRNA bioinformatic database with our research group preliminary results from Gene Chip. Then the target genes were subjected to gene ontology and pathway analysis.4. Different expressed miRNAs, target genes and proteins were confirmed by quantitative reverse transcription-polymerase chain reaction, Western Blot and immunohistochemistry methods. Finally, we analyzed the role of differentially expressed microRNAs and target genes in the pathogenesis of SUI.●RESULTS1. The quality of total RNA was ensured by quantification and electrophoresis. The study model of Human MicroRNA expression chip was successfully established.2.12 differentially expressed miRNAs were identified in SUI compared to controlled patients, and 5 of them were up-regulated, and 7 down-regulated. Candidate target genes were obtained through cross-matching selecting which combined the miRNA bioinformatic database with our research group preliminary results from Gene Chip. According to the inverse correlation,4 differentially expressed miRNAs (hsa-miR-124, hsa-let-7a, hsa-mir-330-3p, hsa-miR-93) and 4 corresponding target genes (GRB2、RAPTOR、BICD2、STAT3) were obtained. Further functional analysis showed they may play a neuro-degenerative role in SUI development. 3. RT-PCR and W.B. or immunohistochemistry validated the significant differentially expressed miRNAs, target genes, and proteins.●CONCLUSIONS1.12 differentially expressed miRNAs were identified in SUI compared to controlled patients.5of them were up-regulated, and 7 down-regulated.2. Combined gene expression profile chip with miRNA expression profile chip can greatly reduce the selecting range of the candidate mRNAs and miRNAs, and make further confirmation more accurate and scientific.3. Quantitative PCR and Western Blot validated the significant differentially expressed miRNAs and target genes from chips.4. This study further enriched the pathogenesis hypothesis of nerve injury or degeneration in SUI development from the theoretical view.