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The Expression Of Micrornas And Their Regulatory Role On Target Genes In Rhesus Monkey Endometrium During

Posted on:2012-08-04Degree:DoctorType:Dissertation
Country:ChinaCandidate:J L LiuFull Text:PDF
GTID:1110330338963317Subject:Basic veterinary science
Abstract/Summary:PDF Full Text Request
The establishment of endometrial receptivity is a prerequisite for successful pregnancy and is controled by a complex mechenism in which many factors are involved. MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression. Howerver, the contribution of miRNAs during endometrial receptivity is still unknown.This study was to use rhesus monkey as an animal model and compare both miRNA and mRNA expression profiles in the endometrium during menstrual cycle between days 15 (pre-receptive phase) and 21 (receptive phase) by deep sequencing. A total of 326 known miRNAs are detected. Statistical analysis showed that there are 89 down-regulated miRNAs and 22 up-regulated miRNAs on day 21 compared to day 15. Real-time RT-PCR was performed to verify 8 selected miRNAs. The expression levels measured by real-time RT-PCR were consistent with that from deep sequencing. Additionally, we identified 112 potential novel miRNAs that had not been reported before, 11 of which show a significant homology with known miRNA from other species.Prior to prediction of their target genes, the 3'-UTR boundaries for each gene are identified in the genome within which miRNA binding sites reside using 3'-end tag sequencing of mRNA. Of the 9,943 genes expressed in the rhesus monkey endometrium, about 40% exibit alternative 3'-UTRs. Progesterone receptor (PGR) gene exhibits an ultra long 3'-UTR (~10 kb) along with a short variant (~2.5 kb) in the endometrium of rhesus monkey. These two 3'-UTR variants were confirmed by Northern blot. Evolutionary analysis showed that these 3'-UTR sequences are poorly conserved between primates and rodents, suggesting a species-biased miRNA binding pattern.Using the TargetScan algorithm, we identified the binding sites of miR-96 and miR-375 in the 3'-UTR of the PGR gene. Luciferase assay demonstrated that PGR is a valid target of miR-96 and miR-375. The binding site of miR-96 is conserved in rhesus monkey and human. In the mouse, the binding is defective due to 3-bp mismatches. MiR-375 targeting PGR is rhesus-specific. Due to 2~3 bp mismatches in the binding sites, PGR escapes the regulation by miR-375 in human and mouse.Moreover, we found that miR-219-5p is also a regulator of PGR. It regulates PGR expression through a primate-specific long non-coding RNA (lncRNA) immediately downstream of the PGR locus. In conclusion, we identified a subset of miRNAs associated with rhesus monkey endometrial receptivity. Target gene analysis indicates that some of these miRNAs regulate the expression PGR gene in a species-specific manner. Our study provides new insights into the molecular mechanisms underlying endometrial receptivity.
Keywords/Search Tags:endometrial receptivity, microRNA, rhesus monkey, deep sequencing, PGR, lncRNA
PDF Full Text Request
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