Study On Expression And Function Of Microrna During Proliferation And Differentiation Of Cochlear Progenitor Cells

Cochlea development and hair cell regeneration are two challenging research subjects in otology. After birth, cochlear progenitor cells could still be isolated from the cochlear sensory epithelia of neonatal murine. These progenitor cells are multipotential, proliferating, and sphere-forming; they can give rise to all cell types in the cochlea including hair cells and cochlear supporting cells after 14 days of differentiation culture in vitro. Thus, progenitor cells could be used for cell replacement therapy in hearing loss in the future. Furthermore, inducting cochlear progenitor cell differentiation in vitro provides a research model to study the mechanisms of cochlea development and hair cell regeneration.miRNAs are small (approximately 22 nucleotides) noncoding endogenous RNA molecules that regulate gene expression by directly cleaving targeted 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. miRNAs play important roles in regulating the cell fate determination of stem cells, as well as in cellular proliferation, differentiation and maturation. Previous studies have shown that miRNAs are essential for the development and function of the cochlea in vertebrates. The conditional knock-out of Dicer, which is required for the maturation of miRNAs in mice inner ear, could induce severe developmental defects in hair cells. It was recently reported that mutations in the miR-96 gene could result in autosomal dominant, progressive hearing loss in both humans and mice.No data is available on miRNAs on the differentiation or proliferation of cochlear progenitor cells. Thus, in this study, we aimed to profile dynamic changes in miRNA expression during the differentiation process of cochlear progenitor cells. This will help us understand their complex roles during the differentiation and proliferation of cochlear progenitor cells.1. Isolation, culture and differentiation of cochlear progenitor cells and neural stem cellsCochlear progenitor cells and neural stem cells were respectively isolated from cochlear sensory epithelia of postnatal day 0 to postnatal day 3 SD rats and from the olfactory bulb of rat embryos at embryonic day 15. The two kinds of cells were incubated under standard conditions in a CO2 incubator. To ensure that cell spheres were formed from single cells, Cochlear progenitor cells and neural stem cells were plated at low density. The proliferation medium consisted of DMEM/F12, supplemented with B27 and N2, EGF and bFGF, penicillin. For the induction of cell differentiation, Cochlear progenitor cell and neural stem cell spheres were transferred into six-well dishes with poly-l-lysine treated coverslips. Cochlear progenitor cell and neural stem cell differentiation was induced by filling with 10% fetal calf serum. After 16 h, the differentiation medium was replaced with serum-free DMEM/F12 supplemented with N2 and B27. Media was half-changed every two days. The cochlear progenitor cell and differentiated cells cultured for 14 days were identified in culture by immunocytochemistry.We found sphere-forming capacity of the two kinds of cells.The proliferating cells from cochlea were nestin positive and BrdU positive.The differentiated cells expressed myosinⅦA which is a hair cell marker. The reproductiveactivity of cochlear progenitor cells is much weaker than that of neural stem cells. Cochlear progenitor cells should not be considered as stem cells.2. Dynamic changes in miRNA expression during differentiation of cochlear progenitor cells by microarrayThe floating cell spheres of CPCs and CPCs-derived adherent differentiated cells cultured for 6 and 14 days, were collected and were washed in PBS. Total RNA was harvested using TRIzol according to manufacturer instructions. Puri?ed RNAs were prepared for miRNA expression analysis. miRNA microarray tests include labeling, hybridization, scanning, normalization, and data analysis. The samples were labeled and were hybridized on the miRCURY? LNA Array after passing RNA quantity measurement using NanoDrop 1000. The slides were scanned using the Axon GenePix 4000B microarray scanner. Scanned images were imported into GenePix Pro 6.0 software for grid alignment and data extraction. Triplicate arrays were performed under each differentiation time point for three parallel cultures. The resulting signal intensity values were normalized using the median normalization, and were then used to obtain means and standard deviations for each miRNA.Nearly 100 miRNAs were detected in the microarray. These miRNAs have a wide range of abundances. Of the miRNAs on the microarray, miR-125b-5p, miR-494, and miR-22 were the most abundant during the differentiation of CPCs. There were changes in the expression levels of many miRNAs upon the induction of differentiation. For example, miR-125b-5p, miR-181a, miR-200c, miR-182, miR-96, miR-24, and others, were expressed at higher levels in undifferentiated time points. Their expression decreased and even became undetectable following the induction of differentiation. miR-22, miR-503, miR-351, miR-32*, and others, were expressed at higher levels on day 14 after induction of differentiation (D14). A group of miRNAs peaked on day 6 after induction of differentiation (D6), including miR-743b, miR-98, let-7a, let-7f, miR-340-5p, miR-21, and others. This group was expressed lowly and was undetectable in undifferentiated time points and at D14 in microarray. The temporal regulation of these miRNAs indicated that they might play different roles in differentiating cochlear progenitor cells.3. miRNA expression in CPC and NSC by qRT-PCRExpressions of U6, miR-21, miR-503, let-7a, let-7f, miR-96, miR-182, and miR-183 were assayed using Poly-A tailing-based reverse transcriptase followed by SYBR Green based quantitative PCR with an All-in-One miRNA qRT-PCR Detection Kitaccording to manufacturer instructions. Briefly, 2μg of total RNA from each sample was converted to cDNA in a 25μL reaction containing 1μl Poly-A polymerase, 1μl RTase mix, and 5μl 5×reaction buffer. A total of 2μL of a 1/5 dilution of cDNA in water was added into 10μL of the 2×All-in-One TM Q-PCR mix, with 2μL of each Forward Primer, and 2μL Universal Adaptor PCR Reverse Primer for a total volume of 20μL. The reactions were amplified with the following cycle conditions: 95°C for 10 seconds, 60°C for 20 seconds and 72°C for 10 seconds for 40 cycles. The relative amount of miRNAs was normalized against U6 snRNA and calculated using the 2?ΔΔCT method.Most qRT-PCR results were in accordance with those obtained by microarray analysis. The expression levels of these miRNAs significantly changed during CPC differentiation. These miRNAs have different expression patterns in this study. miR-96 and miR-182, which were undetectable in D6 and D14, were identified by qRT-PCR. The failure to detect them in the microarray may be due to their relatively low levels. miR-96 and miR-183 were not detected in any differentiation time point of NSCs. Only miR-182 was identified in undifferentiated time points of NSCs. However, the relative miR-182 expression level in NSCs was low, at about 5% of CPCs. These result suggest that specific miRNAs such as miR-183 family might influence the cell fate determination of cochlear progenitor cells.4. Methods for transfecting of target gene into CPCsWe compared the transfection efficiencies of three most commonly used transfection methods, lipofectin transfection, electroporation transfection and adenovirus transduction. First, DNA/ lipofectamine compounds mixed in varying proportions (1:3, 1:4, 1:5) were used for transfection. And then different incubation times (6 hours or 12 hours) were tried. After culture for 24h, fluorescencev inverted microscope was used to observe the proportion of EGFP-positive cell among total cells. Second, the important electroporation parameter, electric field intensity, was set to observe the transfection efficiency. We tried four different electric field intensities (2000V/cm, 2500V/cm, 2700V/cm and 3000V/cm) in electric pulse time of 40μs. Third, rAd-EGFP was added into cell culture fluid in different MOIs (100, 150, 200 and 250). After incubation for 2h, fresh and new cell culture fluid was added to take place of fluid containing adenovirus. After culture for 24h, fluorescencev inverted microscope was used to evaluate the transfection efficiency.Trypan blue test was used to analyze the cell survival rate in these groups.Lipofectin transfection has the lowest transfection efficiency (< 2%) and highest cell survival rate. In electroporation transfection group, the electric field intensity of 2500V/cm is a point of balance for transfection efficiency(24.7%) and cell survival rate(51%). Adenovirus transduction is the optimal method for transfecting of target gene into CPCs. It has the highest transfection efficiency (52%) and acceptable cell survival rate (85%). However, we need more data about parameters optimization to support our conclusion.