The Influence Of Hypothalamic Mir-505Overexpression On The Puberty Onset In Mice

Objective: MiRNAs (MicroRNAs) is a kind of natural small non-codingRNA, consisting of20to25nucleotides. With the improvements of researchtechniques and the deepening of research contents, accumulating results showed thatmiRNAs play a role in the regulation of most protein-coding genes expression andtake part in the control of a large number of biological processes. They have beenidentified as potential biological targets, disease regulator and also can be used asdrug targets. A variety of human diseases, including cancer are often accompanied byabnormal level of miRNAexpression.Our previous genetic research showed that miR-505, located in the mouse Xchromosome:57647578-57647667, could have participated in the regulation ofpuberty onset timing in mice, yet research on its biological function is in the initialstage. miR-505was proved to inhibit cell proliferation by inducing apoptosis, andthe only validated target ASF/SF2played a role in mTOR signaling regulation. Tostudy the potential function of miR-505in puberty onset regulation, a hypothalamicmiR–505-3p overexpression mouse model was constructed by in suit injection. Wecompare the puberty and fertility-related phenotypes between the model mice and thecontrols, and confirmed the relationship between miR-505and sexual development.Methods: The hypothalamus region was positioned by stereotaxicinstruments and the lentiviral vector was administered by in suit injection. Toillustrate the miR-505expression in mouse hypothalamus tissues, frozen sections ofmouse hypothalamus were made and microRNA FISH (fluorescence in situhybridization) method with LNA(locked nucleic acids) probes and TSA(tyrosinesignal amplification) system, as well as real time-PCR were applied. In order tocompare puberty onset phenotypes between injected model mice and the untreatedcontrolsl, the data of weight gain, vaginal opening time, the number of offspring born,abortion rates were recorded and analyzed. Overies were wax-fixed and sliced intosections. HE stained ections were investigated by the ovarian follicles histological morphology. Concentration of luteinizing hormone and gonadotropin-releasinghormone were detected in juvenile mice serum using enzyme linked immunosorbentassay(ELISA). To illustrate the miR-505expression in mouse hypothalamus tissues,frozen sections of mouse hypothalamus were made and microRNA FISH methodwith LNAprobes and TSA ystem was applied.Results: The steady elavation of miR-505expression was observed in theinjected region of hypothalamus in juvenile mice using FISH and RT-PCR method,which implied.A hypothalamic miR-505overexpression mouse model mediated bylentivirus has been established successfully Compared with untreated mice, the modelmice manifested lower growth rates，the average weight light2-3g; Vulva openingtime36.67±0.7993days, was delayed three days; increased abortion rate by12%,infertility8%,fewer offspring number, delayed luteal appearance, reduce LHconcentrations. Moreover,we found that the position of miR–505-3p overexpresseven affected the phenotypesConclusion: A hypothalamic miR-505overexpression mediated bylentivirus mouse model has been established successfully by in situ injection. LNA-fluorescence in situ hybridization(FISH) is a stable and repeatable method in thedetection of microRNA on frozen sections from mouse brain. The significantdifferences between model mice and normal mice in a series of developmentalphenotypes indicated that miR-505gene played a role in the regulation of pubertyonset in mice. Theresults provide a theoretical basis for the further investigation of thebiological function of miR-505.