Early-life origin of prostate cancer through deregulation of miR-206 networks in maternally malnourished offspring rats

doi:10.21203/rs.3.rs-1546693/v1

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Early-life origin of prostate cancer through deregulation of miR-206 networks in maternally malnourished offspring rats

https://doi.org/10.21203/rs.3.rs-1546693/v1

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The Developmental Origins of Health and Disease (DOHaD) has provided the framework to assess how early life experiences can shape health and disease throughout the life course. While maternal malnutrition has been proposed as a risk factor for the developmental programming of prostate cancer (PCa), the molecular mechanisms remain poorly understood. Here, we found an association between deregulation of steroidogenesis and impairment of the ventral prostate (VP) growth in young offspring rats exposed to maternal low protein diet (LPD) during gestation and lactation. Reanalysis of RNA-seq data demonstrated that miR-206 was upregulated in the VP of young maternally malnourished offspring. Target prediction and in vitro studies identified Plasminogen (PLG) as a direct target of miR-206. To give further insights into the participation of the miR-206-PLG network in prostate carcinogenesis in the progeny submitted to maternal LPD. RT-qPCR analysis revealed deregulation of the miR-206-PLG network in the VP of older rats that developed prostate carcinoma in situ. Furthermore, mimic studies revealed a negative correlation between miR-206 and estrogen receptor α (ESR1) expression in PNT2 cells. Together, we demonstrate that early life estrogenization associated with deregulation of miR-206-networks can contribute to the developmental origins of PCa in maternally malnourished offspring. Understanding the molecular mechanisms by which early life malnutrition affects offspring health can encourage the adoption of a governmental policy for the prevention of non-communicable chronic diseases related to the DOHaD concept.

DOHaD

prostate

maternal low protein diet

steroidogenesis

miRNAs

Non-communicable chronic diseases (NCDs) are reaching epidemic proportions worldwide, with 41 million deaths each year. The most deadly NCDs are related to cardiovascular diseases, followed by several types of cancers, respiratory tract diseases, and diabetes. Almost 80% of these deaths occur in low- and middle-income countries [1]. Although NCDs are diagnosed mainly in adults, a growing body of evidence strongly supports that exposure to adverse conditions during the formative periods disrupts normal developmental biology and predisposes individuals to high-risk NCDs across the life span. The reprogramming of physiologic intrauterine/early postnatal development forms the basis of the Developmental Origins of Health and Disease (DOHaD) concept [2, 3].

The DOHaD concept has emerged over the past 50 years from the outstanding epidemiologic observations of Barker and Osmond (1986) that poor nutrition early in life was associated with increased infant mortality and ischaemic heart disease in adults in England and Wales [4]. Nowadays, a broad range of epidemiological and experimental studies have confirmed and extended Barker's observations, consolidating gene-environment interactions during early life as a critical risk factor for the increased incidence of NCDs later in life. Although the initial focus of DOHaD studies has been directed to obesity, diabetes, and cardiovascular disease, exposures to environmental stressors have been considered a important risk factor for the developmental origins of several malignancies, including breast and prostate cancers [5–8]. The first author to propose the prenatal origin of prostate cancer (PCa) was William Gardner in 1995, who states that "The origins of prostatic diseases, including carcinoma, are to be found in the in utero influences upon the developing prostate" [6]. Later, Keinan-Boker et al. (2009) demonstrated an increased risk of PCa in Jewish men exposed early in life to famine and stress during the Holocaust. Although tragic, this event allowed researchers to explore the potential association between early-life exposure to stress conditions and increased risk for PCa with aging [7].

In the last decade, clinical and experimental studies have provided significant insights into the molecular mechanisms involved in the developmental origins of PCa. In this regard, intrauterine and early postnatal exposure to estrogens or estrogenic compounds, including endocrine-disrupting chemicals (such as bisphenols and phthalates), have been proven to interfere with prostate developmental biology and the susceptibility of prostatic diseases with aging in both humans [5, 9, 10] and rodent models [11–14]. As such, steroidogenesis deregulation induced by maternal malnutrition has been associated with impairment of prostate growth in young rats [15–20] and higher risk of PCa with aging [21–23].

Accumulating data have suggested modifications in epigenetic markers as the main mechanistic framework explaining how malnutrition during early life impacts offspring health [3, 24]. However, the role of maternal malnutrition on the deregulation of microRNAs-mRNAs networks during prostate development with long-lasting consequences for carcinogenesis has not yet been evaluated. We hypothesize that maternal malnutrition during pregnancy and lactation changes the offspring's steroidogenic profiles parallel with the deregulation of miRNA-mRNA networks during prostate development, thereby creating fertile soil for slow-growing PCa with aging. To address this hypothesis, we used a rat model of maternal malnutrition to evaluate changes in the steroidogenic pathway and the deregulation of key mRNA-miRNA networks potentially involved in the developmental origin of prostate cancer in older offspring rats.

2.1 Animals and diets

Naive adult (90 days age) females (n = 30) and males (n = 10) Sprague Dawley rats were used. The animals were kept under a controlled temperature (22–25°C), relative humidity (55%), and a photoperiod (12h/12h), with free access to water and food. Breeding proceeded overnight in a harem configuration (1 male to 3 females). After determination of pregnancy through detection of spermatozoa in the vaginal smear (considered gestational Day 1 - GD1), pregnant rats were distributed in Control group (CTR, n = 15): Pregnants rats fed with a normal protein diet (17% protein) during the gestation and lactation; and Gestational and Lactational Low Protein group (GLLP, n = 15): Pregnants rats fed with a low protein diet (6% protein) during the same periods. The diets followed the AIN-93 standards described by Reeves et al. (1993) and were provided by PragSoluções (PragSoluções, SP, Brazil) [25]. The diets were previously used in other studies [15, 21–23].

Litters were reduced to eight pups (four males and four females) on a postnatal day (PND) 1 to maximize lactation performance [26]. Maternal body weight and food intake were recorded every 3rd day until GD 21. Dam and offspring biometric parameters were measured until the end of the experiment. One male offspring (21 days old) from each litter was euthanized by an overdose of ketamine and xylazine, followed by decapitation. Blood and ventral prostate (VP) were collected and processed as described below. The procedures were approved by the Biosciences Institute/UNESP Ethics Committee for Animal Experimentation (Protocol #1178).

2.2 Serum blood hormonal analysis

Blood samples from male offspring (n = 8/group) were centrifuged (2400 g for 20 min), and sera were used to quantify total cholesterol (Labtest®, R76-2/100, Brazil, sensitivity: 0.06 mg/dL), pregnenolone (LifeSpanBioSciences®, LS-F39295, USA, sensitivity: 9.375 pg/mL); dehydroepiandrosterone (DHEA) (Monobind®, CA 7425 − 300, USA, sensitivity: 0.10 ng/mL), estrogen (17βestradiol, Monobind®, CA 4925 − 300, USA, sensitivity: 8.2 pg/mL) and testosterone (17β-hydroxy-4-androstene-3-one, Monobind®, CA 3725-300A, USA, sensitivity: 0.038 ng/mL). The hormonal quantifications were made in 96-well plates using the ELISA plate reader (Epoch™, Biotek Instruments, VT, USA) following the manufacturer's protocol.

2.3 Morphology analysis and gelatin-zymography analyses

Samples of VP lobes from CTR and GLLP groups (n = 6/group) on PND 21 were processed for histological analysis as described by Santos et al. (2019). The slides (5µm) were stained with Hematoxylin/Eosin or with picro sirius red. The slides were analyzed using using the image analyzer Leica Q-win software (Version 3 for Windows) coupled a Leica DMLB 80 microscope. The collagen fiber volume was determined by a red color automatic detection in 10 different microscopic fields (200X) from 6 different VP lobe sections. The collagen volume was shown as a percentage of red-stained areas per total prostatic area.

The gelatin zymography was performed as described in Justulin et al., (2010) [27]. Briefly, frozen VP lobes (n = 6/group) were homogenized in extraction buffer (50 mM of Tris buffer pH 7.5 plus 0.25% Triton-X 100), centrifuged, and the total proteins were quantified by the Bradford method [28]. Aliquots (28 µg protein) were subjected to electrophoresis in 0.1% gelatin-containing polyacrylamide gels (8% acrylamide) in a Bio-Rad MiniProtean II system (Bio-Rad Laboratories Inc., Richmond, CA, USA). Next, the gels were shaken with 2.5% Triton-X100 and incubated overnight in 50 mM Tris-HCl (pH 8.4) containing 5 mM CaCl₂ and 1µM ZnCl₂ at 37°C. Then, the gels were stained with Coomassie Blue, and areas of proteolysis were measured using ImageJ software (National Institutes of Health). The results were expressed in fold change as the mean ± SD.

2.4 Identification of deregulated miRNAs-mRNAs networks in offspring VP

To identify the potential deregulation in miRNAs-mRNAs networks in the VP of young rats, we reanalyzed data of next-generation sequencing (NGS) from the CTR (n = 4) and GLLP (n = 3) groups. These data were generated by our research group and were available for download at the Gene Expression Omnibus (GEO) database under accession numbers GSE180674 (miRNA) and GSE180673 (mRNA). After, the differentially expressed (DE) miRNAs and mRNAs were identified using the DESeq2 package (https://bioconductor.org/packages/DESeq2/)[29]. The cut-off significance for miRNA and mRNA was p-value < 0.05 and Log₂ Fold Change ≥ |+0.66≤|-0.66|. After identifying deregulated miRNAs in the rat VP, the predicted target mRNAs were identified using miRWalk 3.0 platform (http://mirwalk.umm.uni-heidelberg.de/) for Rattus norvegicus [30]. The cut-off criterion for significance was p-value < 0.05. To further identify the deregulated miRNAs-mRNAs network potentially involved with prostatic disorders, the list of predicted mRNAs was integrated with the DE mRNAs identified in our transcriptome dataset, considering miRNAs upregulated and mRNA downregulated and vice versa.

2.5 Identification of miRNAs commonly deregulated in the offspring VP and human PCa

To identify DE miRNAs in patients with PCa, we explored the Prostate Adenocarcinoma (PRAD-TCGA, https://portal.gdc.cancer.gov/projects/TCGA-PRAD) dataset, with data from 550 samples (52 normal samples and 498 tumor samples). The differentially expressed (DE) miRNAs were identified using the DESeq package[29]. The cut-off significance for both miRNA and mRNA were p-value < 0.05 and Log₂ Fold Change ≥ |+0.66≤|-0.66|. Then, we compared the common DE miRNAs in the VP of maternally malnourished offspring with those from patients with PCa.

2.6 Criteria for selection of miRNA commonly deregulated in the offspring VP and human PCa

To select a miRNA commonly deregulated between VP and human PCa for further analysis, we take into account the higher fold change observed in our RNAseq dataset, the miRNA conservation sequences between Rattus norvegicus and human (checked through the miRBase database, http://www.mirbase.org/) [31, 32], and its role on prostate biology [33, 34].

2.7 Identification of selected miRNA-mRNAs networks on human PCa: in silico approach

To further explore the role of selected miRNA-mRNAs networks on PCa, the mRNAs predicted to be regulated by the selected miRNA were subjected to enrichment analysis of molecular pathways, biological processes, and cellular components using the Enrichr tool (https://maayanlab.cloud/Enrichr/) [35, 36]. Subsequently, these mRNAs were applied in overall survival analysis and risk assessment in PCa patients using the SurvExpress platform (http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp) [37]. The Human Protein Atlas (HPA) (https://www.proteinatlas.org/) database was used to demonstrate the immunolocalization of deregulated mRNAs in human prostatic samples [38–40].

2.8 Functional validation of selected miRNA in transfected prostatic cell line

The benign prostate cell line (PNT-2, Cell Bank of Rio de Janeiro, Brazil) was cultured using RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% FBS, 50 µg/mL penicillin-streptomycin, and 0.5 µg/mL amphotericin B (GIBCO/Invitrogen). The cells were grown at 37ºC with 5% CO₂ in a humid atmosphere and used up to passage 20. The culture medium was changed twice a week, and throughout the experimental period, the cells were monitored with an inverted microscope (Zeiss Axiovert). For passage, the cells were released with 0.25% trypsin (GIBCO/Invitrogen) for 5 min at 37°C, resuspended in a fresh medium, and plated again. Based on the significance of enriched terms (p < 0.05) and its relevance for prostate biology, mRNA was selected for validation through RT-qPCR.

2.8.1 miRNA transfection and Cell Viability Assays

Before transfecting, the specific miRNA mimic (mirVanaTM miRNA Mimic, code: 4464066; MC10409, Thermo Fisher, USA) and its control group formed a complex with the Opti-MEM reduced serum medium (Thermo Fisher Scientific, USA). The PNT-2 cells (8 × 10⁴) were plated in 12-well plates in 800µl of complete RPMI medium per well. Once cells became 80% confluent, transfections were performed using RNAiMAX lipofectamine (Thermo Fisher Scientific, USA) with or without 10 nM of specific miRNA mimic for 16 h at 37^oC and 5% CO₂. After 24, 48, and 72 h, cell viability was determined by the MTT reduction method according to the manufacturer's instructions (Sigma-Aldrich)[41, 42]. As absorbance is proportional to cell viability, the percentage of cell viability relative to control cells was quantified in a spectrophotometer (ASYS HITECH GmbH, AUT) using a 96-well plate at 550 nm absorbance.

2.8.2 Wound healing assay

PNT2 cells were cultured in a 6 well plate using RPMI 1640 medium at 37°C and 5% CO2 until they reached 80–100% confluence. After, a 200-µl plastic tip was used to perform a single-line scratch at the highest diameter in all replicates. Cell debris was removed using two PBS washes, and 1,2 mL RPMI supplemented with 10% FBS was added to each well. The analysis of the wound open area was performed at 0, 24, 48, and 72 h, and the results were expressed as a percentage of wound healing, following the equation: % wound healing = [100 − (wound area at Tnh/wound area at T0h)] × 100, where T0h is the time point immediately after the scratch [43].

2.8.3 In vitro validation of selected miRNA and its predicted targets

Total mRNA of PNT2 cells in all treatments was extracted with TRIzol (Thermo Fisher Scientific, USA) as recommended by the manufacturer. Aliquots of 2 µg of total RNA were reverse transcribed using the High-Capacity Kit RNA-to-cDNA (Life Technologies) in a 10-µl reaction according to the manufacturer's instructions. Aliquots of cDNA from each sample were added to a mix of reagents containing primers "sense" and "anti-sense," and the volume was completed to 10 µl with ultrapure water. Primers were designed specifically for target genes after in silico filtering. The values obtained for all samples were normalized by the ratio between the target genes and the reference genes (ACTIN and GAPDH).

For miRNA reactions, the High-Capacity RNA-to-cDNA Kit (Life Technologies) was used for cDNA synthesis from 100 ng of total RNA according to the manufacturer's recommendations. Stem-loop RT primers were synthesized according to Chen et al. (2005) [44]. The cDNA was amplified with universal and forward primers, and the quantifications were normalized using the reference miRNA (U6). All reactions were used using the SYBR Green PCR Master Mix system (Thermo Fisher, USA). The expression level of the selected miRNA and mRNAs was determined using the QuantStudio™ฏ 12K Flex real-time PCR system (Thermo Fisher Scientific) on 96 plate wells. The values were calculated using the expression ratio of the GLLP/CTR groups. The relative quantification of each gene was performed using the 2^−∆∆CT method normalized using [45]. For these experiments, the cells that received the lipofectamine vehicle were used as the control group. The sequences of the primers were designed on the Primer Express 3.0 software (Supplementary Table 2).

2.9 In vivo validation: selected miRNA, its predicted target, and hormones receptors in offspring VP

To evaluate the expression profile of these miRNA, the target mRNA and hormones receptors in young (PND21) and older (PND540) offspring rats, the total RNA was extracted from samples (n = 6/group) in all experimental groups and both ages using TRIzol®ฏ Reagent (ThermoFisher Scientific) according to the manufacturer's recommendations. The samples of old rats we used were generated in a previous study of our research group [22]. Details of the RT-qPCR reactions were described in item 2.8.3. For the mRNA data, the normalization was conducted using Gapdh and Gusb as reference genes; for the miRNA data, the normalization was conducted using the reference miRNA (U6). Primer sequences were described in Supplementary Table 2.

2.10 Statistical analysis

Statistical analyses were performed using the GraphPadPrism® software (version 5.00, Graph Pad, Inc., San Diego, CA). The results were submitted to normalization analysis (Shapiro-Wilk test). Parametric data were compared using the Student’s t-test. The remaining (dams body weight gain in the lactation, Offspring weight at PND21, DHEA concentration, and Plg expression in VP samples) were submitted to Mann-Whitney. Data were expressed as mean ± SD. The differences were considered statistically significant when p < 0.05. The statistical tests used are described in the figure legends.

2.11 Data representation and analyses

Bar graphs were generated using the GraphPad Prism tool (GraphPad Software). The heatmap with the results of RT-qPCR was created using the Morpheus web tool (https://software.broadinstitute.org/morpheus) [46]. miRNA-mRNAs networks were demonstrated by circus plot graphics generated in the R environment using the "Circlize" package [47]. The UpSet plot generated to represent deregulated miRNAs in the VP of animals submitted to maternal malnutrition and PCa patients was made on the Intervene platform (https://intervene.shinyapps.io/intervene/) [48]. The relationships between the molecular pathways and the predicted targets of miRNA, demonstrated in an alluvium diagram, were performed on the Sankeymatic platform (https://sankeymatic.com/).

3.1 Maternal malnutrition alters biometrical parameters in dams and offspring

Dams from the GLLP group displayed lower body weight gain during pregnancy and lactation, although the relative food and energy intake did not change compared to the control group (Table 1). The offspring from the GLLP group showed a reduced body weight, reduced anogenital distance at PND 1 and 21, and lower VP absolute and relative weight at PND 21 compared to the CTR group (Table 1).

3.2 Maternal malnutrition affects hormone levels and impairs prostate growth in young male offspring

We observed an increase in total cholesterol and plasma concentrations of testosterone and 17β-estradiol in the GLLP group compared to the CTR group at PND 21. Conversely, plasma concentrations of pregnenolone, progesterone, and DHEA were reduced in the GLLP group (Fig. 1).

The morphological analysis demonstrated an impairment of prostatic growth in the GLLP compared to the CTR group at PND 21 (Figs. 2a and b). We observed a reduction in the lumen of prostatic acini associated with increased collagen deposition in the stromal compartment in the GLLP group compared to the CTR group (Fig. 2c and d). The morphometric data confirmed the impairment of prostatic growth (Fig. 2e). Consistent with the increase in collagen in the stromal compartment, the gelatinolytic activity of the active form of MMP-2 was reduced in the GLLP group compared to the CTR (Fig. 2f).

3.3 Maternal malnutrition alters the miRNA-mRNA networks in the young offspring VP

The integrative analysis identified 49 DE miRNAs (28 up and 21 downregulated) (Supplementary Table 3) and 707 DE mRNA (525 up and 182 downregulated) in the VP from the GLLP group (Supplementary Table 4, Fig. 3a). We evaluated the interaction of 49 DE miRNAs with predicted mRNAs and identified 51,338 potential target mRNAs (Supplementary Table 5). Then, we compared the list of predicted mRNAs with the DE mRNA identified in the VP, resulting in the match of 268 mRNAs potentially regulated by 47 miRNAs (Fig. 3b and Supplementary Table 6), considering miRNAs upregulated and mRNA downregulated and vice versa.

3.4 Similarities between miRNA-mRNA networks in the VP of maternally malnourished offspring and PCa patients

We identified 245 DE miRNAs (118 downregulated and 127 upregulated) in patients diagnosed with PCa using the PRAD-TCGA dataset (Supplementary Table 7). The integrative analysis demonstrated similarities between eight commonly DE miRNAs in the VP from the GLLP group and patients with PCa (miR-1-3p, miR-141-3p, miR-184, miR-206-3p, miR-410-3p, miR-452-5p, miR-496-3p, miR-96-5p) (Fig. 3c). Of these, 5 miRNAs (miR-141-3p, miR-96-5p, miR-410-3p, miR-184, and miR-206-3p) shared identical sequences in both human and Rattus norvegicus (Supplementary Table 8, Fig. 3d). Based on these results, we selected rno-miR-206-3p, which presented a higher fold change among DE miRNAs (1.79) and their predicted target mRNAs (Zfp366, Plg, Rimbp2, Pbld1, Lhfpl2, Dtna, Rmnd5a, Zmat3, Mob3b, Stxbp6, LOC103692716) for further validations (Supplementary Table 6).

3.5 Molecular pathways enriched by the miR-206/predicted targets

Predicted targets of miR-206 enriched molecular pathways related to the regulation of cell-cell adhesion mediated by cadherin (GO:2000047), regulation of IGF transport and uptake by IGFBPs (R-HSA-381426), activation of matrix metalloproteinases (R-HSA-1592389), regulated exocytosis (GO:0045055), negative regulation of exocytosis (GO:0045920), exocyst localization (GO:0051601) development of primary female sexual characteristics (GO:0046545), negative regulation of intracellular estrogen receptor signaling pathway (GO:0033147), regulation of intracellular estrogen receptor signaling pathway (GO:0033146) estrogen receptor binding (GO:0030331), signal transduction by p53 class mediator (GO:0072331), miRNA regulation of p53 pathway in prostate cancer (WP3982), interleukin 1Î² rat insulinoma cell line (GDS4332 ligand:186). Seven DE mRNAs (ZNF366, PLG, ZMAT3, RIMBP2, PBLD, LHFPL2, and STXBP6) were mostly associated with enriched molecular pathways (Fig. 4a).

To further assess the potential role of these seven DE mRNAs in predicting a worse prognosis for PCa (PRAD-TCGA), the SurvExpress tool was used to explore the prognostic index correlating survival analysis and cancer risk assessment. Our set of genes clustered the PCa patients (n = 497) into two distinct groups (at high-risk, n = 128 and low-risk, n = 369), and was strongly associated with shorter survival of patients, as shown in PRAD (hazard ratio (HR) = 9.31; log-rank p-value = 0.001392) (Fig. 4b). Furthermore, data from the HPA database demonstrated the immunolocalization of three targets (PLG, ZNF366, and ZMAT3) in PCa (Fig. 4c).

3.6 Effects of transient transfection of prostatic cells with mimic miR-206

After the identification and in silico selection of potential miRNA-mRNA networks altered by maternal malnutrition in the offspring VP, we performed functional assays in benign prostatic PNT2 cells with miR-206 mimetic molecules attempting to elucidate the impact of increased miR-206 expression in the prostate. Cells transfected with the miR-206 mimic did not show morphological changes compared to cells treated with lipofectamine (Fig. 5a-b). The miR-206 expression increased in PNT-2 cells transiently transfected with mimic miR-206 (Fig. 5c). Interestingly, the expression of PLG and ESR1 transcripts decreased in mimic miR-206-treated cells (Fig. 5d-e), highlighting the potential miR-206-PLG-ERS1 interaction. The expression of ESR2 and AR did not change in this condition (Fig. 5f-g). The MTT assays demonstrated a reduction in the viability after 72 h of exposure to mimic miR-206 compared to the control cells (Fig. 5h). The wound-healing assay showed that miR-206 transfected cells delayed wound healing potential after 48 and 72 h of treatment (Fig. 5i-j).

3.7 Validation of miR-206 network in the VP of young and older offspring rats

Consistent with the integrative analysis of RNAseq, miR-206 was upregulated in the VP of young rats from the GLLP group compared to the CTR group on PND21 (Fig. 6a). In parallel, we observed that Plg transcript levels were downregulated in the GLLP group (Fig. 6b). Moreover, we observed an increase in the Ar expression (Fig. 6c) and a reduction of Esr1 and Esr2 in the VP of young rats from the GLLP group compared to the CTR group (Figs. 6d-e).

To give further insights into the participation of the miR-206 in the prostate carcinogenesis in the progeny submitted to maternal LPD, RT-qPCR analysis revealed a reduction of miR-206 expression was observed in the VP of older rats from the GLLP group compared to the CTR group. It was accomplished by an increase of Plg expression (Fig. 6f and g). The Ar appeared downregulated, while Esr1 was upregulated, and the Esr2 did not change in the VP or older rats from the GLLP group compared to the CTR group (Figs. 6h-j).

Figure 7 summarizes the workflow depicting the effects of maternal exposure to a low protein diet on the deregulation of the molecular mechanisms involved in the developmental origins of PCa. We further highlighted the key role of miR206-network and estrogen signaling pathways (through deregulation of ERα) as a potential prostate cancer driver in older offspring submitted to maternal malnutrition.

Over the past decades, the DOHaD concept has provided causal relationships between early-life exposure to environmental risk factors and the developmental origins of non-communicable chronic diseases, including PCa [7, 21, 22]. Although the molecular mechanisms involved in this process remain elusive, the imbalance of sex hormones in both dams and offspring submitted to maternal malnutrition has been pointed to as a key factor related to prostate carcinogenesis in the progeny [6, 9, 10, 21, 22].

To further elucidate how maternal malnutrition interferes with the balance of sex hormones in male offspring, we evaluated the serum cholesterol profile and their intermediate metabolites involved in the steroidogenesis pathways. An increase in total cholesterol was observed in GLLP animais at PND 21. Sohi et al. (2011) have demonstrated an increase in cholesterol levels in male rat offspring submitted to MPR (8% protein) at the same age and associated this result with permanent epigenetic silencing of the hepatic Cyp7a1 promoter, which metabolizes cholesterol to bile acids [49]. The offspring from the GLLP group also showed an imbalaced in plasma concentrations of steroid hormones; while testosterone and estradial were elevated, pregnenolone, progesterone, and DHEA were reduced, suggesting that these intermediate metabolites could be consumed to sustain the higher levels of testosterone and estradiol.

Although largely driven by androgens, the developing prostate is sensitive to estrogens. Epidemiological and experimental data have demonstrated that abnormal exposure to an excessive estrogenization early in life may disrupt molecular mechanisms governing prostate developmental biology, with long-lasting consequences for prostate diseases with aging [9, 21, 22, 50]. As such, African-American men are at higher risk of prostatic carcinoma than their Caucasian counterparts, and it has been postulated that this higher risk is related, in part, to the higher estrogens levels observed during pregnancy in the former population [9, 10]. These conditions, termed "developmental estrogenization" or "estrogen imprinting", states that inappropriate exposure to estrogen (due to ethnicity, exposure to the endocrine disruptor, environmental chemicals, maternal malnutrition, among other individual and environmental factors) is memorized ("imprinted") by cells and tissues [12, 13, 51–56]. Understanding the relationship between exposure to an estrogenic environment with long-lasting consequences for prostate diseases is essential for the DOHaD concept. It has been demonstrated in rodent models that exposure to high levels of estrogens early in life blocks epithelial cell differentiation and increases the number of basal epithelial cells [15, 57]. Using proteomic approaches, Santos et al., 2020 identified estrogenic signaling pathways, endoplasmic reticulum functions, oxidative stress, and deregulation of insulin/IGF signaling pathways as the main deregulated mechanisms involved in the VP response to maternal malnutrition in both young and older male offspring [23]. We further demonstrated that the impairment of the glandular compartment in young rats was associated with an increase in collagen fiber deposition in the stroma. The increase in collagen content can be explained, at least in part, by the reduction of the gelatinolytic activity of the active form of the MMP-2, a class of enzyme responsible for extracellular matrix remodeling in the prostate gland [27, 58, 59].

To give further translational insights into the relationship between deregulated miRNAs networks early in life and the developmental origins of PCa, we identified 5 miRNAs with similar nucleotide sequences commonly deregulated in the VP of maternally malnourished offspring and patients with PCa. Of these, miR-206 was selected for further functional validation. RT-qPCR confirmed the upregulation of this miRNA in the VP of young offspring. Accordingly, experimental upregulation of miR-206 led to retardation of mammary gland development through modulation of Wnt and transcription factors Tbx3 and Lef1 [60]. The upregulation of miR-206 in the GLLP group can be associated with stromal collagen accumulation in the impaired VP of young rats since the miR-206/Plg network, which modulates the extracellular matrix remodeling and angiogenesis processes [61, 62], was downregulated in these animals.

It is noteworthy that the prostate is particularly more sensitive to estrogens during critical windows of development [12]. Estrogen actions on the prostate are mainly mediated through major canonical estrogen receptors (ER), including ERα and ERβ. Studies involving knockout mice and developmental exposure to estrogenic compounds indicate that paracrine signaling involving stromal ERα is the dominant form of estrogen-mediated imprinting in the developing uterus [63], ovary [63], mammary gland [64], lung [65], and prostate [51, 66, 67]. Consistently, the deletion of the ERα gene in the stromal fibroblast leads to a reduction in prostate branching morphogenesis [66]. Additionally, Lee et al., (2013) demonstrated that miR-206 epigenetically regulates downregulated ESR1 expression during normal mammary gland development [60]. Although there is no data regarding the Esr1 modulation by miR-206 in the developing prostate, our data demonstrated the potential relationship of miR-206 in modulating ER signaling since miR-206-ERs networks have opposite expression profiles in the VP of young offspring in the GLLP group.

In addition to the miR-206 potential role as an important modulator of gene expression during the developmental process, a body of evidence highlighted its key role as a tumor suppressor miRNA in PCa [35, 36, 64]. However, there is no data regarding the epigenetic modulation of ERs by miR-206 in PCa. Studies with knockout mice for estrogen receptors indicate that Esr1 and Esr2 can have opposite actions on prostate carcinogenesis since Esr1 is recognized to promote cell proliferation, while Esr2 has been described as anti-proliferative and proapoptotic. Altogether, these results suggest that the overall proliferative response to estrogen results from a balance between ERα and ERβ signaling [68–70]. Our functional validation experiments demonstrated downregulation of ESR1 expression in PNT2 cells transfected with miR-206. These results are consistent with deregulation of the miR-206-ESR1 network observed in the VP of young and older offspring rats from the GLLP group. Moreover, it has been demonstrated that in vitro inhibition of miR-206 in PC-3 cells increases cell invasion through upregulated ANXA2 and E-cadherin, and downregulation of N-cadherin and vimentin [35]. Wang et al., (2018) also described the tumor suppressor effect of miR-206 in PCa by negatively regulating cell proliferation and migration by targeting CXCL11 [64]. These authors also demonstrated experimentally that up-regulation of miR-206 inhibits proliferation, migration, invasion, and induced G1/G0 arrest of PCa cells. The expression profile of miRNA extracted from PRAD-TCGA demonstrated the downregulation of miR-206 in patients with PCa. Interestingly, we observed a reduction in the expression profile of miR-206 and an increase in Plg gene expression in the VP of older offspring rats from the GLLP groups, which developed prostate carcinoma in situ [22–24]. Functional validation confirmed the modulation negative correlation of the miR-206/PLG expression, as well as their participation in modulating cell migration and invasion in PNT2 cells. Furthermore, we provide new evidence on the participation of miR206-ESR1 network mediating prostate carcinogenesis in maternally malnourished offspring rats. Overall, our data provide new insights into the participation of an estrogenized environment associated with deregulation of miR-206-PLG-ESR1 in the developmental origin of prostate diseases in maternally malnourished offspring rats. Understanding these mechanisms can encourage the adoption of a governmental policy for the early life prevention of non-communicable chronic diseases, as proposed by the DOHaD concept.

Funding: Sao Paulo State Research Foundation FAPESP (2018/26120-6; 2017/01063-7; 2021/03405-8) and National Council for Scientific and Technological Development (CNPq grant # 310,663/2018–0).

Author contributions: All authors contributed to the study conception and design. LMFP and LAJ designed the project, performed the conceptualization, animal experimentation, methodology, analyses, interpretation of data, and wrote the manuscript. Material preparation, data collection, and analyses were performed by LMFP, FBC, ACLC, SAAS, LAB, MNF, WRS, and KTC. The first draft of the manuscript was written by LMFP, LAJ, EJRS, and CSM. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Conflict of interest: The authors declare no competing financial interests.

Availability of data and material: The datasets presented in this study can be found in online repositories on The Cancer Genome Atlas (TCGA) projects; The Gene Expression Omnibus (GEO) on accession number(s) GSE180673 and GSE180674.

Ethics approval: This study was performed in line with the principles ethics. Approval was granted by the Biosciences Institute/UNESP Ethics Committee for Animal Experimentation (Protocol #1178).

Code availability: Not applicable.

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Table 1. Biometric parameters of dams and offspring submitted to low protein diet during gestation and lactation.

Parameters	CTR group	GLLP group
Dams body weight gain during pregnancy (g)	47.25 ± 5.99 ^a	37.38 ± 5.02 ^b
Dams body weight gain in the lactation (g)	-0.28 ± 6.70 ^a	-30.44 ± 7.74 ^b
Dams energy intake during pregnancy and lactation (KJ/day)	310.30 ± 22.01	326.20 ± 61.38
Offspring weight at PND1 (g)	7.12 ± 0.55 ^a	5.91 ± 0.57 ^b
AGD at PND1 (mm)	3.30 ± 0.40 ^a	2.89 ± 0.48 ^b
Offspring weight at PND21 (g)	37.23 ± 6.57 ^a	20.03 ± 3.88 ^b
Absolute VP weight at PND21 (mg)	3.24 ± 0.83 ^a	1.440 ± 0.39 ^b
Relative VP weight at PV 21	0.92 ± 0.19 ^a	0.7893 ± 0.16 ^b
AGD at PND21 (mm)	10.69 ± 2.55 ^a	7.64 ± 1.62 ^b

The different letters mean statistical difference between experimental groups with p < 0.05. AGD: anogenital distance; VP: ventral prostate; PND: postnatal day; g: grams; KJ: kilojoule; mm: millimeters; mg: milligram; pg: picogram; ng: nanogram.

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Early-life origin of prostate cancer through deregulation of miR-206 networks in maternally malnourished offspring rats

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Animals and diets

2.2 Serum blood hormonal analysis

2.3 Morphology analysis and gelatin-zymography analyses

2.4 Identification of deregulated miRNAs-mRNAs networks in offspring VP

2.5 Identification of miRNAs commonly deregulated in the offspring VP and human PCa

2.6 Criteria for selection of miRNA commonly deregulated in the offspring VP and human PCa

2.7 Identification of selected miRNA-mRNAs networks on human PCa: in silico approach

2.8 Functional validation of selected miRNA in transfected prostatic cell line

2.8.1 miRNA transfection and Cell Viability Assays

2.8.2 Wound healing assay

2.8.3 In vitro validation of selected miRNA and its predicted targets

2.9 In vivo validation: selected miRNA, its predicted target, and hormones receptors in offspring VP

2.10 Statistical analysis

2.11 Data representation and analyses

3. Results

3.1 Maternal malnutrition alters biometrical parameters in dams and offspring

3.2 Maternal malnutrition affects hormone levels and impairs prostate growth in young male offspring

3.3 Maternal malnutrition alters the miRNA-mRNA networks in the young offspring VP

3.4 Similarities between miRNA-mRNA networks in the VP of maternally malnourished offspring and PCa patients

3.5 Molecular pathways enriched by the miR-206/predicted targets

3.6 Effects of transient transfection of prostatic cells with mimic miR-206

3.7 Validation of miR-206 network in the VP of young and older offspring rats

4. Discussion

Declarations

References

Table

Supplementary Files

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