LncPrep + 96kb 2.2 kb Inhibits Estradiol Secretion From Granulosa Cells by Inducing EDF1 Translocation

Abstract

LncPrep + 96kb is a novel long non-coding RNA expressed in murine granulosa cells with two transcripts that are 2.2 and 2.8 kb in length. However, the potential roles of lncPrep + 96kb in granulosa cells remain poorly understood. In this study, we investigated the effect of the lncPrep + 96kb 2.2 kb transcript on granulosa cells through the overexpression and knockdown of lncPrep + 96kb 2.2 kb. We found that lncPrep + 96kb 2.2 kb inhibited aromatase expression and estradiol production. Endothelial differentiation-related factor 1 (EDF1) is an evolutionarily conserved transcriptional coactivator. We found that EDF1 knockdown inhibited aromatase expression and estradiol production. The RNA immunoprecipitation results also showed that lncPrep + 96kb 2.2 kb can bind to EDF1 and that overexpression of lncPrep + 96kb 2.2 kb induced the translocation of EDF1 from the nucleus to the cytoplasm. The CatRAPID signature revealed that the 1,979-2,077 and 603-690 nucleotide positions in lncPrep + 96kb 2.2 kb were potential binding sites for EDF1. We found that mutating the 1,979-2,077 site rescued the effects of lncPrep + 96kb 2.2 kb on aromatase expression and estradiol production. In conclusion, we are the first to report that specific expression of lncPrep + 96kb 2.2 kb in granulosa cells inhibits the production of estradiol by influencing the localization of EDF1 in granulosa cells. The 1,979-2,077 site of lncPrep + 96kb 2.2 kb contributes to the ability to bind to EDF1.

Keywords: EDF1; aromatase; estradiol; granulosa cell; lncPrep + 96kb.

FIGURE 1

The expression of lncPrep + 96kb in murine ovaries. The negative control is shown in (A). (B,C) Show the positive signal of lncPrep + 96kb. Scale bar = 100 μm.

FIGURE 2

Estradiol production and aromatase expression are regulated by lncPrep + 96kb 2.2 kb. (A) Shows the effect of lncPrep + 96kb 2.2 kb on cell proliferation. (B,C) Show estradiol production after the overexpression and knockdown of lncPrep + 96kb 2.2 kb. (D,E) Show the mRNA expression of aromatase after overexpression and knockdown of lncPrep + 96kb 2.2 kb. (F) Shows the protein expression of aromatase after overexpression and knockdown of lncPrep + 96kb 2.2 kb. The experiment was independently repeated for three times. *p < 0.05, which was a specific two-group comparison.

FIGURE 3

The effect of lncPrep + 96kb 2.2 kb on EDF1. (A) Western blot was used to determine the specific interaction of lncPrep + 96kb 2.2 kb with EDF1. (B) Immunofluorescence was used to examine the location of EDF1 in granulosa cells. Scale bar = 100 μm.

FIGURE 4

Estradiol production and aromatase expression are regulated by EDF1. (A) Shows the effect of EDF1-specific shRNA on estradiol production. (B–D) Shows the effect of EDF1-specific shRNA on aromatase expression at the mRNA and protein levels. The experiment was independently repeated for three times. *p < 0.05, which was a specific two-group comparison.

FIGURE 5

Prediction of the interaction between lncPrep + 96kb 2.2 kb and EDF1. (A) The RNA-binding capability of the EDF1 protein was predicted by the CatRAPID signature module. Overall interaction scores greater than 50% indicate binding capability. (B) CatRAPID fragment module prediction of the interaction profile and matrix for lncPrep + 96kb 2.2 kb and EDF1.

FIGURE 6

The effects of the mutating the lncPrep + 96kb 2.2 kb binding sites on estradiol production and aromatase expression. (A) Shows estradiol production after mutating the lncPrep + 96kb 2.2 kb binding sites. (B) Shows the expression of aromatase after mutating the lncPrep + 96kb 2.2 kb binding sites. (C,D) Show the protein expression of aromatase after mutating the lncPrep + 96kb 2.2 kb binding sites. *p < 0.05. The experiment was independently repeated for three times.