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. 2022 Nov 28;23(23):14855.
doi: 10.3390/ijms232314855.

miR-18a-5p Is Involved in the Developmental Origin of Prostate Cancer in Maternally Malnourished Offspring Rats: A DOHaD Approach

Sergio Alexandre Alcantara Santos  1   2 , Luiz Marcos Frediani Portela  1 , Ana Carolina Lima Camargo  1 , Flavia Bessi Constantino  1 , Ketlin Thassiani Colombelli  1 , Matheus Naia Fioretto  1 , Renato Mattos  1 , Bruno Evaristo de Almeida Fantinatti  1 , Michela Alessandra Denti  3 , Silvano Piazza  3 , Sérgio Luis Felisbino  1 , Elena Zambrano  4 , Luis Antonio Justulin  1
Affiliations

miR-18a-5p Is Involved in the Developmental Origin of Prostate Cancer in Maternally Malnourished Offspring Rats: A DOHaD Approach

Sergio Alexandre Alcantara Santos et al. Int J Mol Sci. .

Abstract

The Developmental Origins of Health and Disease (DOHaD) concept correlates early life exposure to stressor conditions with the increased incidence of non-communicable chronic diseases, including prostate cancer (PCa), throughout the life span. However, the molecular mechanisms involved in this process remain poorly understood. In this study, the deregulation of two miRNAs (rno-miR-18a-5p and rno-miR-345-3p) was described in the ventral prostate VP of old rats born to dams fed with a low protein diet (LPD) (6% protein in the diet) during gestational and lactational periods. Integrative analysis of the (VP) transcriptomic and proteomic data revealed changes in the expression profile of 14 identified predicted _targets of these two DE miRNAs, which enriched terms related to post-translational protein modification, metabolism of proteins, protein processing in endoplasmic reticulum, phosphonate and phosphinate metabolism, the calnexin/calreticulin cycle, metabolic pathways, N-glycan trimming in the ER and the calnexin/calreticulin cycle, hedgehog ligand biogenesis, the ER-phagosome pathway, detoxification of reactive oxygen species, antigenprocessing-cross presentation, RAB geranylgeranylation, collagen formation, glutathione metabolism, the metabolism of xenobiotics by cytochrome P450, and platinum drug resistance. RT-qPCR validated the deregulation of the miR-18a-5p/P4HB (prolyl 4-hydroxylase subunit beta) network in the VP of older offspring as well as in the PNT-2 cells transfected with mimic miR-18a-5p. Functional in vitro studies revealed a potential modulation of estrogen receptor α (ESR1) by miR-18a-5p in PNT-2 cells, which was also confirmed in the VP of older offspring. An imbalance of the testosterone/estrogen ratio was also observed in the offspring rats born to dams fed with an LPD. In conclusion, deregulation of the miR-18a-5p/P4HB network can contribute to the developmental origins of prostate cancer in maternally malnourished offspring, highlighting the need for improving maternal healthcare during critical windows of vulnerability early in life.

Keywords: Developmental Origins of Health and Disease (DOHaD); aging; epigenetics; miR-18a-5p/P4HB network; prostate cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Representative histological sections of the VP lobes from the CTR (A,B) and GLLP (C,D) groups at PND 540 stained with hematoxylin/eosin (HE). In the CTR group, the VP histology resembles the structural characteristic of older VP with normal glandular and stromal compartments. In the GLLP group, the glandular lumen was filled with a dysplastic cell mass with microacinar structures (dashed circle). Scale bar: 200 µm (A,C) and 50 µm (B,D). CTR: Control; GLLP: Gestational and Lactation Low Protein.
Figure 2
Figure 2
(A) Representative image of the differential expression of the multiomic data in the offspring VP from the GLLP group compared to the CTR group at PND 540 (for detailed data, see Supplementary Tables S1–S3). Data of microRNome and transcriptome (n = 5 from the CTR group and n = 4 from the GLLP group. Data of proteome (n = 3 for both the CTR and GLLP groups). (B) Differentially expressed (DE) miRNAs identified between the GLLP and the CTR groups. The criteria used to identify DE miRNAs were Log2 Fold Change ≥ |+0.4| ≤ |−0.4| and p < 0.05 and base mean > 20. The prediction analysis was performed on the miRWalk 3.0 tool. (C) Integrative analyzes using predicted _targets of the DE miRNA identified in the VP transcriptome and proteome data from older offspring rats submitted to maternal malnutrition. D and E: RT-qPCR for experimental validation of the DE miRNA miR-18a-5p (D) and miR-345-3p (E) in the offspring VP. Asterisks mean the statistical difference with p < 0.05. CTR: Control; GLLP: Gestational and Lactation Low Protein.
Figure 3
Figure 3
(A) Ontological enrichment terms by KOBAs 3.0. All data were expressed as −Log10 (p-value). The numbers in the bar graph identify the input number of predicted _targets that enriched each term. (B) Circus plot graphic identifying the association of deregulated _targets involved in each enriched term.
Figure 4
Figure 4
(A) Expression profile of miR-18a-5p in PNT-2 cells from the Control Lipo, Mimic, and Negative CTR groups after 24 h of treatment. (B). Representative wound healing assay images after 24, 48, and 72 h. (C). Cellular wound closure after the transfection of PNT-2 cells with lipofectamine and mimic miR-18a-5p after 0, 24, 48, and 72 h post-wound. Mimic group: cells treated with mimic miR-18a-5p. In vitro studies were conducted in three independent experiments performed in triplicate. Data are expressed as mean ± SD. * means a statistical difference between experimental groups with p < 0.05. Scale bar: 200 µm. CTR: Control.
Figure 5
Figure 5
P4hb gene expression in the ventral prostate (A) of offspring rats from the CTR and GLLP groups at PND 540. (B) P4HB gene expression in the PNT-2 human prostate cells from the CTR and transfected with mimic miR-18a-5p groups. (C) P4HB immunostaining in normal and PRAD tissues obtained from The Human Protein Atlas database (https://proteinatlas.org/ (accessed on 4 May 2021)). Data are expressed as mean ± SD. * means a statistical difference between experimental groups with p < 0.05. CTR: Control group; GLLP: Gestational and Lactational Low Protein diet; VP: ventral prostate; PRAD: prostate adenocarcinoma.
Figure 6
Figure 6
Esr1 (A) and Esr2 (C) gene expression profiles in the offspring VP from the CTR and GLLP groups at PND 540. Esr1 (B) and Esr2 (D) gene expression profiles in the PNT-2 human prostate cells from CTR and transfected with mimic miR-18a-5p. Data are expressed as mean ± SD. * means a statistical difference between experimental groups with p < 0.05.
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