Int J Stem Cells.  2020 Mar;13(1):55-64. 10.15283/ijsc19139.

Estrogen Promotes cAMP Production in Mesenchymal Stem Cells by Regulating ADCY2

• Zhao G
• Li X
• Miao H
• Chen S
• Hou Y

• KMID: 2500566
• DOI: http://doi.org/10.15283/ijsc19139

Abstract

Background and Objectives
The maternal-fetal interface is an important source of mesenchymal stem cells (MSCs), and it is influenced by high levels of estradiol (E2) during pregnancy. It is highly important to study the role of E2 in MSCs for both clinical application and understanding of the mechanisms underlying pregnancy related diseases.
Methods and Results
In this study, differently expressed genes (DEGs) were found in the MSCs after exposure to E2. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs was performed and the integrated regulatory network of DEGs-miRNA was constructed. A total of 390 DEGs were found in the MSCs exposed to E2, including 164 upregulated DEGs (e.g. ADCY2, VEGFA and PPY) and 226 downregulated DEGs (e.g. KNG1, AGT and NPY). Additionally, 10 miRNAs (such as miR-148A/B, miR-152, miR-182) identified the integrated regulatory network of DEGs-miRNAs. Among them, the expression of ADCY2 was significantly upregulated, and this was associated with multiple changed genes. We confirmed that the expression of ADCY2 is significantly promoted by E2 and subsequently promoted the production of cAMP in MSCs. We also found that E2 promoted ADCY2 expression by inhibiting miR-152 and miR-148a.
Conclusions
E2 promotes the expression of cAMP through miR-148a/152-ADCY2 in MSCs. It is suggested that E2 plays a key role in the growth and function of MSCs.

Keyword

Estradiol; Mesenchymal stem cells; miRNAs; ADCY2; cAMP

Figure

• 

  Fig. 1 Changes in the transcriptome of MSCs exposed to E2. (A) The heatmap showed all significantly differentially expressed genes (DEGs) (p<0.05) identified between the MSC treated with E2 and that without E2. The colors blue, white and red indicate increasing levels of DEGs expression. E2-1, E2-2, E2-3 and E2-4 show E2 samples and Ctl-1, Ctl-2, Ctl-3 and Ctl-4 show four control samples. (B) The enrichment results of GO and KEGG pathway (top five p values). Category: molecular function (MF), biological process (BP), cellular component (CC). Count represents the number of DEGs enriched to the KEGG pathway. Term represents the information of GO function. (C) The protein-protein interaction (PPI) network of DEGs and modules. PPI network, Red nodes represent the up-regulated DEGs; green nodes indicated down-regulated DEGs. The edges between them represent the protein-protein interaction. (D) Modules in PPI, red nodes represent the up-regulated DEGs; green nodes indicate down-regulated DEGs.

• 

  Fig. 2 Integrated regulatory network. Red nodes are up-regulated DEGs, green nodes are down-regulated DEGs, and lines between DEGs represent connections; the purple V-types indicate transcription factors (TFs). The purple rhombuses represent miRNA.

• 

  Fig. 3 E2 promotes cAMP production through upregulating ADCY2 in MSCs. (A) After treatment with 100 nM E2 for 24 h, mRNA level of ADCY2 was assessed using qPCR. (B) After treatment with 100 nM E2 for 24 h, protein level of ADCY2 was detected by western blot analysis. (C) After treatment with 100 nM E2 for 48 h, the concentration of cAMP in the culture medium of MSCs was detected by ELISA. (D) Small interfering fragment of ADCY2 (siADCY2) was transfected into MSCs before E2 treatment. Then the level of cAMP was checked by ELISA. ***p<0.001.

• 

  Fig. 4 E2 promotes ADCY2 expression through inhibiting miR-148a/miR-152. (A) miR-148a and miR-152 have binding sequences on 3’UTR of ADCY2. (B) Co-transfected with luciferase report plasmid including 3’UTR of ADCY2 and miR-148a/152 into MSCs, then the luciferase activity was detected. (C) After transfection with miR-148a and miR-152 mimics for 48 h, protein level of ADCY2 in MSCs was detected by western blot analysis. (D, E) After transfection with miR-148a and miR-152 inhibitors for 48 h, protein levels of ADCY2 and cAMP in MSCs were detected. (F, G) After treatment with 100 nM M E2 for 24 h, the levels of miR-148a and miR-152 were determined using qPCR. (H) miR-148a or miR-152 mimics were transfected into E2-treated MSCs, and then the expression of cAMP was detected determined by ELISA. **p<0.01, ***p<0.001.

Reference

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