Regulation Of LncPrep+96kb 2.2kb On Estrogen Secretion In Mouse Ovarian Granulosa Cells

Objectives:LncPrep+96kb is a new long non-coding RNA with two transcripts that are 2.2 kb and 2.8kb in length.LncPrep+96kb is expressed in ovarian granulosa cells.However,its function and mechanism in ovaries are still unclear.In this study,we will investigate the role of lncPrep+96kb 2.2kb in mouse ovarian granulosa cells and possible mechanism.Methods:In situ hybridization was used to investigate the spatiotemporal expression of lncPrep+96kb in mouse ovaries.We cultured the granulosa cells and transfected granulosa cells with lncPrep+96kb 2.2kb overexpression plasmid and knockdown plasmid.CCK-8 assay was employed to observe the effect of lncPrep+96kb 2.2kb on cell viability.We also harvested the culture medium to detect the production of estradiol in granulosa cells.The expression of aromatase were examined by real-time quantitative PCR and Western blot.RNA immunoprecipitation was used to identify the specific binding proteins of lncPrep+96kb 2.2kb.The bioinformatics methods were used to predict the specific binding sites of lncPrep+96kb 2.2kb with EDF1.we also mutated the potential binding sites and observed whether the mutation can rescue the effect of lncPrep+96kb 2.2kb on estradiol synthesis and aromatase expression in granulosa cells.Results:In situ hybridization showed that lncPrep+96kb was mainly expressed in the granulosa cells of primary and secondary follicles in murine ovaries.With the development of the follicles it was decreased.lncPrep+96kb 2.2kb had no effect on granulosa cell viability.Overexpression of lncPrep+96kb 2.2kb inhibited the production of estradiol and aromatase expression.Conversely,knockdown of lncPrep +96kb increased the estradiol secretion and aromatase expression.We identified EDF1,which could specifically bind with lncPrep+96kb 2.2kb through RNA immunoprecipitation and Western blot.Further studies showed that overexpression of lncPrep+96kb 2.2kb induced the translocation of EDF1 from the nucleus to the cytoplasm.After silence of EDF1,the estradiol production and aromatase expression were inhibited.The CatRAPID signature revealed that the 1979-2077 and 603-690 nucleotide positions in lncPrep+96kb 2.2 kb were potential binding sites of EDF1.We found that mutation of the 1979-2077 site rescued the effect of lncPrep+96kb 2.2kb on aromatase expression and estradiol production.Conclusion:(1)Lncprep+96kb 2.2kb inhibits estradiol secretion and aromatase expression in ovarian granulosa cells.(2)Lncprep+96kb 2.2kb specifically binds with EDF1 and promotes EDF1 translocation from the nucleus to the cytoplasm.(3)Silence of EDF1 inhibits estradiol secretion and aromatase expression.(4)LncPrep+96kb 2.2kb 1979-2077 is key site for binding to EDF1,which mediates the effect of lncPrep+96kb 2.2kb on estradiol synthesis and aromatase expression.