The Study Of Roles Of MicroRNAs In Pelvic Organ Prolapse In Postmenopausal Women

Background：Pelvic organ prolapse (POP) is a common disease of women in the aged, whichimpacts their quality of life seriously. Epidemiological research with large samplesrevealed that pregnancy, vaginal delivery, menopause, aging, and chronically increasingabdominal pressure were major risk factors for POP. Now it is generally accepted thatpelvic floor tissue injury and dysfunction in its remodeling, derived from thoseabove-mentioned factors, are the main mechanisms of POP. According to the theoryof genetics and genomics, all human diseases are directly or indirectly associated withgenes and gene mutation is the molecular basis of disease. Large numbers of geneticand epidemiological studies at home and abroad suggested that genetic factors are involvedin the occurrence and development of POP. Etiological research of POP has proventhat pelvic connective tissue, muscle and nerve injuries are the main reasons of POP.These studies only focused on disease itself, morphological changes or other aspects.There is no such kind of molecule mechanism which has been deeply studied and widelyaccepted. Generally speaking, the imbalances of synthesis and degradation ofextracellular matrix (ECM) in pelvic support tissues are the molecular basis of POP.The main component of ECM is collagen which in the form of fascia and ligamentsgives pelvic tissues tensile strength, and plays an important role in maintaining the normalposition and normal functions of the pelvic organs. The content, distribution andarrangement of collagen are important factors for maintaining the tissue function. Thereare28kinds of collagen subtypes，which has a different distribution in different tissues.In fascia and ligaments, collagen type I is the main component while collagen type Ⅲconstitutes mainly in the vaginal wall tissue. Although collagen’s importance in thepathogenesis of POP has been widely confirmed, it still doesn’t reach an agreementabout collagen expression level. Therefore confirming collagen’s expression modeland revealing its regulation mechanism will help to elucidate the pathogenesis of POPand to develop effective prevention or treatment measures.MicroRNA (miRNA) is a type of endogenous non-coding RNA that has about18～25nucleotides. It widely exists in eukaryotic cells. Studies have suggested that the maturemiRNA can degrade its target mRNAs or repress the translation of target mRNAs. miRNAparticipates in a variety of physiological and pathological processes includingorganogenesis, cell proliferation and apoptosis, fat metabolism and so on, by negatively regulating its target gene. Recently more attention had been paid to the role of miRNAin the regulation of ECM. Researches have revealed miR-590，miR-155，miR-7，miR-21，miR-30、miR-29and miR-133may play important role in the regulation ofcollagen, elastin and transforming growth factor β. These studies provided ideas forthe investigation of miRNA in POP.Application of high-throughput chip technology for miRNA expression profiling,bioinformatics prediction and identification of target genes, and biological functionexperimental method have been widely used in this regard.Exploration the function ofmiRNA in gene expression regulation network and revealing its important role in thedevelopment of disease will give laboratory support for diagnosis and treatment of disease.Vaginal tissue and fascia, ligaments and other connective tissues of the pelvic have acommon embryonic origin, and research has shown that changes in vaginal tissue mayreflect changes in the composition of the basin fascia. Vaginal walls are the mostvulnerable parts of pelvic support tissue and are the common injured tissues in POP.Understanding the morphological function of vaginal walls and the underlying molecularmechanisms will greatly help to elucidate the development of POP. Until now, thereis still no research of tissue-specific miRNA expression profiling in POP. Therefore itis worth carrying out this kind of study.Objective：1. Basing on bioinformatics and statistics, this study tried to find out miRNA withdifferent expression model in POP by miRNA expression profiling with an aimingto point out a direction in the research of miRNA in POP.2. With the help of existed bioinformatics platform and with the deep understandingof miRNA’s structural and functional characteristics, this study, with miRNA asthe start point, tried to build a gene expression profile, with an aiming to screenout those genes really meaningful in the pathogenesis of POP.3. This study tried to confirm collagen’s expression model at mRNA and protein level,with an aiming to reveal the molecular basis of collagen in POP.4. This study tried to estimate the expression level of miRNA target gene’s mRNA,with an aiming to reveal the regulatory mechanism of miRNA underlying POP andto provide new ideas for further research in the prevention and treatment of POP.Methods:1. After strict matching of age, menopausal status, body mass index, number of vaginal deliveries and related medical history, five post-menopausal patients with POP andfive without POP were selected. Tissues of anterior vaginal fornix near the middleline were collected for the study.2. Extract total RNA in the samples using mirVana miRNA Isolation Kit, and detectits purity and integrity. Labeling the total RNA by using FlashTag Biotin RNALabeling Kit. After miRNA expression profiling by using Affymetrix GeneChip miRNA Array3.0, the differences of miRNA between the POP and the controlgroups were analyzed using SAM（significance analysis of Microarrays）. Thescreening criteria are: Fold Change≥2for up regulation, Fold Change≤0.5for down regulation, and Q-value≤5%for statistically significant.3. Using TargetScan, PicTar and miRanda with the help of bioinformatics to predicttarget gene for the miRNA with significant difference. In order to decrease the falsepositive rate and improve the accuracy of prediction, only those genes predictedby all these three prediction software were selected as candidate target genes.4. Review literatures of POP related to genomics research and find out genesdifferentially expressed in pathogenesis of POP.5. Those genes shared by step4and step5were left for further study.6. Expand the sample size of POP group and control group to10respectively and usereal-time fluorescence quantitative PCR and Western Blot to verify the accuracy ofmiRNA chip and to analyze the different expression of target gene mRNA and proteinin the vaginal tissues of postmenopausal POP group and control group.7. Obtaining the sequence of miRNA target gene’s3’UTR, cloning it intopsiCHECKTM-2vector to construct a wild type reporter vector; Mutating miRNAtarget gene’s3’UTR, cloning it into psiCHECKTM-2vector to construct a mutationtype reporter vector. After co-transfection of the wild type or mutation type vectorwith miRNA mimics or miRNA negative control into293T cells, Dual Luciferasereport system was used to detect the activity of Luciferase enzymes.Results:1. The total RNA extracted from tissue samples were analyzed using UVSpectrophotometry. The quantity of RNA was9.89ug to21.88ug. A260/280wasbetween1.99and2.03. Gel electrophoresis revealed that the RNA had goodintegrity. All these parameters showed that the RNA samples meet the criterion ofAffymetrix GeneChip miRNA3.0. 2. All the quality control parameters of the10miRNA microarrays including B2Oligo,Spike-in Control Oligos, Eukaryotic Hybridization Controls were assessed to bequalified for further study. This meant that the Affymetrix GeneChip miRNA3.0research model was successfully builded.3. The Affymetrix GeneChip miRNA3.0had screened out44miRNAs which wereup regulated by at least two folds in vaginal tissues in post menopause POP patients.4. After target gene prediction for the differently expressed miRNAs usingbioinformatics and combination analysis with existed gene profiling in POP,hsa-miR-196a was selected for further study and COL3A1was predicted with thegreatest possibility to be its target gene.5. Expand the sample size of POP group and control group to10cases respectively.Real-time fluorescence quantitative PCR revealed that hsa-miR-196a was up regulatedat21.71folds in POP when compared with control group. This further verifiedthe results of miRNA microarray.6. Real-time fluorescence quantitative PCR and western blot confirmed the significantdown regulation of COL3A1at both mRNA and protein level in the vaginal tissuein post menopause POP patients. This gave a clue that COL3A1was the targetgene of hsa-miR-196a.7. After co-transfection of the wild type of expression vector containing COL3A1’s3’UTR with hsa-miR-196a mimics or hsa-miR-196a NC into293T cells, DualLuciferase report system was used to detect the activity of Luciferase enzymes andrevealed that Luciferase enzymes activity in hsa-miR-196a mimics group wassignificantly down regulated when compared with hsa-miR-196a NC group or miRNAblank group. After co-transfection of the mutation type of expression vectorcontaining COL3A1’s3’UTR mutation form with hsa-miR-196a mimics orhsa-miR-196a NC into293T cells, Dual Luciferase report system revealed thatLuciferase enzymes activity in hsa-miR-196a mimics group had no significantdifference with that in hsa-miR-196a NC group or miRNA blank group. All thesedata confirmed that COL3A1really was the target gene of hsa-miR-196a.Conclusions:1. MiRNA microarray is an important tool to study miRNA and makes it easier andconvenient for identifying miRNA in a particular growth stage or related to specificdisease. In this study, Affymetrix GeneChip miRNA3.0analysis revealed44 miRNAs which were up regulated by at least two folds in vaginal tissues in postmenopause POP patients. These miRNAs may play important roles in the occurrenceand development of POP.2. Bioinformatics is a powerful means for studying miRNA. Combined analysis of thosemiRNA target genes predicted simultaneously by different algorithms, with gene chiptechnology can greatly decrease the scope of candidate genes for further study. Thiswill make further study in POP more practical and more scientific. MiRNA predictionalgorithms of different type have different emphases and different filter parameters.The combination of different algorithms to predict miRNA target genes can narrowthe scope of the candidate genes, but may greatly increase the probability of falsenegatives. Many of target genes of hsa-miR-196a which had been verified byexperimental methods did not appear in the predicted gene set in this study. Thereforein biological research, the prediction algorithms should be carefully chosen accordingto objectives of the study.3. Dysregulation of COL3A1is the molecular basis of POP. In POP COL3A1was downregulated significantly at both mRNA and protein level.4. Hsa-miR-196a was up regulated significantly in POP and COL3A1was its target gene.The seed nucleotide sequence of hsa-miR-196a binds to the3’UTR of COL3A1todegrade COL3A1mRNA or repress its translation.5. Hsa-miR-196a plays a role in the occurrence and development of POP.During itspathological process, hsa-miR-196a is significantly up regulated in vaginal tissuesand by binding to its target-COL3A1mRNA, down regulates the expression ofCOL3A1protein significantly. Considering that collagen type Ⅲ is the maincomponent of the ECM in vaginal tissue, this mechanism may be of great importancein POP.Perspective：This study had found out44miRNAs which were up regulated by at least two foldsin vaginal tissues in post menopause POP patients. Considering that each miRNA mayhas tens even hundreds of target genes and each gene may be regulated by many miRNAs,there is a lot research to carry out before the complete understanding of their mechanisms.The study of the role of miRNA in the process of POP has just begun.MiRNAs are small non-coding RNAs with20-25nucleotides. Its mature form isprotected by proteins form RNase. Therefore miRNA can steadily exists in blood, urine and other body fluids and can be easily detected. Great amount of research have revealedthat dysregulation of miRNA may be the cause of disease. Expression of miRNA differsbetween different diseases. miRNAs may be bio markers for specific condition. Thedeep study of miRNA in POP field may help to screen out those people who arepredisposed to POP and to promote early intervention to these individuals. Additionallymany researches had been done to develop target treatment strategy through gene silenceusing miRNA. This provides ideas for the study of intervention measures for POP.