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Cited by 27

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Target prediction and validation of microRNAs expressed from FSHR and aromatase genes in human ovarian granulosa cells

Rooda, Ilmatar, Hensen, Kati, Kaselt, Birgitta, Kasvandik, Sergo, Pook, Martin, Kurg, Ants, Salumets, Andres, Velthut-Meikas, Agne·2020·Scientific Reports
DOI 10.1038/s41598-020-59186-xPMC PMC7010774
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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.

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Figure 1 The rationale and methods used to identify and validate miRNA targets. Each arrow represents a filtering step, the conditions of which are specified in the text.
Figure 1 The rationale and methods used to identify and validate miRNA targets. Each arrow represents a filtering step, the conditions of which are specified in the text.CC BY · PubMed Central
Figure 2 Cluster analysis of gene expression changes upon transfection of KGN cells with cel-miR-39p, hsa-miR-548ba or hsa-miR-7973 miRNA mimic. Gene expression changes were analyzed 72 h after transfection on Affymetrix microarray. Only statistically significant results are presented (adjusted p-value < 0.01, n = 4).
Figure 2 Cluster analysis of gene expression changes upon transfection of KGN cells with cel-miR-39p, hsa-miR-548ba or hsa-miR-7973 miRNA mimic. Gene expression changes were analyzed 72 h after transfection on Affymetrix microarray. Only statistically significant results are presented (adjusted p-value < 0.01, n = 4).CC BY · PubMed Central
Figure 3 Validation of microarray results by RT-qPCR. (a) Potential target genes of hsa-miR-548ba and (b) Potential target genes of hsa-miR-7973. Gene expression change is calculated by comparing expression levels to samples transfected with control miRNA cel-miR-39-3p. Results are displayed as average fold change ±SD on log2 scale (*p < 0.05; **p < 0.01; Student t-test, n = 3). All presented microarray results were statistically significant (adjusted p < 0.01).
Figure 3 Validation of microarray results by RT-qPCR. (a) Potential target genes of hsa-miR-548ba and (b) Potential target genes of hsa-miR-7973. Gene expression change is calculated by comparing expression levels to samples transfected with control miRNA cel-miR-39-3p. Results are displayed as average fold change ±SD on log2 scale (*p < 0.05; **p < 0.01; Student t-test, n = 3). All presented microarray results were statistically significant (adjusted p < 0.01).CC BY · PubMed Central
Figure 4 Validation of miRNA binding on the 3′UTR sequences of their potential target mRNAs by luciferase reporter assay. (a) hsa-miR-548ba potential target genes. (b) hsa-miR-7973 potential target genes. Results are shown as average normalized luciferase signal ± SEM, (*p < 0.05; **p < 0.01; Student one-tailed t-test, n = 3). RLU – relative light unit.
Figure 4 Validation of miRNA binding on the 3′UTR sequences of their potential target mRNAs by luciferase reporter assay. (a) hsa-miR-548ba potential target genes. (b) hsa-miR-7973 potential target genes. Results are shown as average normalized luciferase signal ± SEM, (*p < 0.05; **p < 0.01; Student one-tailed t-test, n = 3). RLU – relative light unit.CC BY · PubMed Central
Figure 5 miRNA and host gene expression levels in primary human cumulus granulosa cells. mRNA and miRNA expression levels were normalized to SDHA or hsa-mir-132-3p, respectively. (a) hsa-miR-548ba and FSHR expression and (b) hsa-miR-7973 and CYP19A1 expression. Results are displayed as average fold change ± SD on log2 scale (*p < 0.05; **p < 0.01; Student t-test between cells exposed to 1 IU/ml FSH vs non-treated cells, n = 3).
Figure 5 miRNA and host gene expression levels in primary human cumulus granulosa cells. mRNA and miRNA expression levels were normalized to SDHA or hsa-mir-132-3p, respectively. (a) hsa-miR-548ba and FSHR expression and (b) hsa-miR-7973 and CYP19A1 expression. Results are displayed as average fold change ± SD on log2 scale (*p < 0.05; **p < 0.01; Student t-test between cells exposed to 1 IU/ml FSH vs non-treated cells, n = 3).CC BY · PubMed Central

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Metadata

Authors
Rooda, I., Hensen, K., Kaselt, B., Kasvandik, S., Pook, M., Kurg, A., Salumets, A., & Velthut-Meikas, A.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-02-10
Volume
10
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-020-59186-x
PMC
PMC7010774
Cited by
27
References
69
Language
en
Licence
Funders
  • FP7 People: Marie-Curie Actions (EU324509)
  • Haridus- ja Teadusministeerium (IUT34-16)
  • Ettevõtluse Arendamise Sihtasutus (EU48695)
  • Horizon 2020 (692065)
  • MSCA-RISE-2015 program MOMENDO (691058)

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CoChat. (2026). Citation check: Target prediction and validation of microRNAs expressed from FSHR and aromatase genes in human ovarian granulosa cells. https://app.cochat.ai/papers/10.1038/s41598-020-59186-x
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Rooda, I., Hensen, K., Kaselt, B., Kasvandik, S., Pook, M., Kurg, A., Salumets, A., & Velthut-Meikas, A. (2020). Target prediction and validation of microRNAs expressed from FSHR and aromatase genes in human ovarian granulosa cells. Scientific Reports. https://doi.org/10.1038/s41598-020-59186-x

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