Abstract
MicroRNAs (miRNAs) are known post-transcriptional regulators of various biological processes including ovarian follicle development. We have previously identified miRNAs from human pre-ovulatory ovarian granulosa cells that are expressed from the intronic regions of two key genes in normal follicular development: FSH receptor (FSHR) and CYP19A1, the latter encoding the aromatase enzyme. The present study aims to identify the target genes regulated by these miRNAs: hsa-miR-548ba and hsa-miR-7973, respectively. The miRNAs of interest were transfected into KGN cell line and the gene expression changes were analyzed by Affymetrix microarray. Potential miRNA-regulated genes were further filtered by bioinformatic target prediction algorithms and validated for direct miRNA:mRNA binding by luciferase reporter assay. LIFR, PTEN, NEO1 and SP110 were confirmed as targets for hsa-miR-548ba. Hsa-miR-7973 target genes ADAM19, PXDN and FMNL3 also passed all verification steps. Additionally, the expression pattern of the miRNAs was studied in human primary cumulus granulosa cell culture in relation to the expression of their host genes and FSH stimulation. Based on our findings we propose the involvement of hsa-miR-548ba in the regulation of follicle growth and activation via LIFR and PTEN. Hsa-miR-7973 may be implicated in the modulation of extracellular matrix and cell-cell interactions by regulating the expression of its identified targets.
Highlights
Granulosa cell functions are essential in follicular development, maturation, and atresia
MiRNAs hsa-miR-548ba and hsa-miR-7973 were previously identified by deep sequencing of mural granulosa cells (MGC) and CGC populations isolated from women undergoing controlled ovarian stimulation and in vitro fertilization
Both miRNAs are of intronic origin: hsa-miR-548ba gene resides in the follicle stimulating hormone receptor (FSHR) gene and hsa-miR-7973 is located in the intron of the CYP19A1 gene[11]
Summary
Granulosa cell functions are essential in follicular development, maturation, and atresia. Development of ovarian follicles is a complex process including oocyte maturation, granulosa cell proliferation and differentiation. MiRNAs hsa-miR-548ba and hsa-miR-7973 were previously identified by deep sequencing of MGC and CGC populations isolated from women undergoing controlled ovarian stimulation and in vitro fertilization Both miRNAs are of intronic origin: hsa-miR-548ba gene resides in the follicle stimulating hormone receptor (FSHR) gene and hsa-miR-7973 is located in the intron of the CYP19A1 gene[11]. Follicle stimulating hormone (FSH) activates time-related changes in granulosa cell gene expression by binding to FSHR promoting proliferation, differentiation, antrum formation, and oocyte maturation. Estrogens are produced by aromatization of androgens by aromatase enzyme encoded from CYP19A1 gene[14] Both FSHR and aromatase are crucial for follicle development and maturation[13]
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