Study On Expression And Function Of MiR-182in The Proliferation And Differentiation Of Cochlear Progenitor Cells In Vitro

In the cochlea of adult mammalian，hair cells can not regenerate spontaneously afterdamaged. The number of hair cells reduce will gradually reduce caused by age, disease,trauma and other factors., when the hair cells’s damage reduced to a certain extent, inducehearing will loss and sensorineural deafness leading to permanent hearing loss. Recently,Stem cells could be used to replacement hair cells in hearing loss in the future.Cochlear progenitor cells have been isolated from the early postnatal rats and mice inprevious studies. These progenitor cells can differentiate into neurons, astrocytes, hair cellsand supporting cells in vitro.Compared with neural stem cells, the CPCs has a great advantage in the regeneration ofhair cells aspects: Firstly, the cochlear progenitor cells is more easily differentiation into haircells; secondly, the CPCs differentiate into hair cells was higher than NSCs. Therefore, CPCsis the best candidate for cell replacement in the damagement of cochlea hair cells.microRNA (miRNA) are a class of small (approximately22-25nucleotides)noncoding endogenous RNA molecules that regulate gene expression by directly cleavingtargeted mRNA or by repressing translation. A single miRNA might target tens to hundreds of mRNAs, leading to great overall change in the molecular constitution of cells. miRNAsplay important roles in regulating the cell fate determination of stem cells, as well as byinfluencing in cellular proliferation,differentiation and maturation. Recently, in vitro and invivo models miRNAs have were identified that microRNAs (miRNAs), which are theextensively expressed expression in the inner ear, and playing important roles in inner eardevelopment and function of inner ear. Previous studies have shown that miRNA183family are essential for the development and function of the cochlea in vertebrates.No data is was available on about miRNA182on the differentiation or proliferation ofcochlea progenitor cells. Thus, in this study, So we aimeded to profile dynamic changes inthe expression of target genes of effected by miRNA182expression during thedifferentiation process of cochlea progenitor cells,. This which willis useful to helpusexplore understand the complex roles of miRNA182during the differentiation andproliferation of cochlea progenitor cells.1. Isolation, culture and differentiation of CPCs from newborn ratsObjective: To set the method of isolation and culture the cochlear progenitor cells.Methods: The cochlear progenitor cells were isolated from the P0-3rat cochlea tissues andcultured. We identified progenitor cells in postnatal rat cochlea and their potential todifferentiate into multiple lineages using immunocytochemistry. Rusults: The acutelyisolated cells formed floating otospheres in1day of CPCs culture. Acutely dissociated cellsfrom the postnatal rat cochlea were expressed nestin and BrdU after7days of culture. Thisdemonstratesd of that the specific markers of stem cells were expressed in these spheresnot only expressed the specific markers of stem cells but also were as well as activelyundergoing mitosis. The differentiated cells express myosin Ⅶa and phalloidin (hair cellsmarker). SEM was performed in order to determine the shape and surface structure of haircells. Conclusions: The progenitor cells were present in the cochlea of newborn SD rats.They and had the capacity of self-renew and possess potential to differentiation into celltypes of inner ear in vitro. This experiment provided a good model of animal was establishedin vitro for studying the mechanisms of controlling the hair cells replacement. 2. The influence effect of microRNA182on the cochlea progenitor cells proliferationand cell cycle of cochlea progenitor cells in vitroObjective: We established the mimics and inhibitor of microRNA182and successfullydelivered them into the cochlear progenitor cells in vitro. Then, the changes effect on of theproliferative proliferation capacity of cochlea progenitor cells were was observed.Methods：Cochlear progenitor cells were dissociated and cultured in vitro. Then the culturedcells were divided into three groups:(1) the control group;(2) the miRNA182mimics group;(3) the miRNA182inhibitor group. Flow cytometry was used for assessing the influenceeffect of the cell cycle and the capacity of proliferation. The expression of P27kip1、FoxO1、FoxO3were analyzed by RT-PCR、Western-blot, respectively. Results: Our datafrom flow cytometry analysis showed that tThe cell cycle distribution of the Cochlearcochlear progenitor cells was affected significantly affected by the overexpression ofmiR-182. However, overexpression of miR-182had no effect on apoptosis. Conclusions:We found that overexpression of miR-182inhibited the proliferation of cochlear progenitorcells. Which by targeting the genes maybe of P27kip1、 FoxO1、and FoxO3.3. Effect and mechanism of miR-182promotes on differentiation of cochlearprogenitor cells differentiation in vitroObjective: To investigate the effects of miR-182in on the differentiation of cochlearprogenitor cells. Methods：Cochlear progenitor cells were dissociated and cultured invitro. Then the cultured cells were divided into three groups:(1) the control group;(2) themiR-182mimics group;(3) the miRNA182inhibitor group. To analyze the process ofprecursor cells differentiation, Cells cells wew collected at7days after culture and themyosinⅦ+cells were measured by flow cytometry for myosinⅦ+cells.. The expression ofSox2、hes1、p27kip1and math1were analyzed by RT-PCR、 Western-blot, respectively.Results: The current study used an in vitro model of embryonic mouse inner ear in astem/progenitor cell culture to demonstrate that:1) miR-182is was expressed duringdifferentiation of inner ear stem/progenitor cell into a hair cell-like fatecell,,2) ectopic miR-182promotesd inner ear stem/progenitor cell differentiation into a hair cell-like fatecell,and3) the function of miR-182may bewas associated with to its putative targets, Sox2、hes1、p27kip1and math1, transcription factors that have been implicated in inner eardevelopment and hair cells fate. Conclusions: Our findings suggest that miR-182could can regulate the differentiation of cochlear progenitor cell in inner ear. Therefore,progenitor cell differentiation and that miRNAs are important regulators of hair celldifferentiation, which providing provide new targets for hair cells’ repair.