Precocious Puberty and Covid-19 Into Perspective: Potential Increased Frequency, Possible Causes, and a Potential Emergency to Be Addressed

A significant increase in precocious puberty, rapidly progressive puberty and precocious menarche has been reported in Italy since the initial lockdown because of the pandemic, and this could represent a new emergency to be addressed during this pandemic. There is a need, therefore, for further understanding and research. Many causes could account for this. Initially, it was thought that the changes in life-style, in screen time, and sleeping habits could be the cause but if considered individually these are insufficient to explain this phenomenon. Likely, changes in central nervous mediators, and an increase in catecholamines could contribute as a trigger, however, these aspects are poorly studied and understood as well as the real perceptions of these children. Finally, staying more indoors has certainly exposed these children to specific contaminants working as endocrine disruptors which could also have had an effect. It would be of utmost importance to compare this phenomenon worldwide with appropriate studies in order to verify what is happening, and gain a new insight into the consequences of the covid-19 pandemic and into precocious puberty and for future prevention

GH and IGF System: The Regulatory Role of miRNAs and lncRNAs in Cancer

Growth hormone (GH) and the insulin-like growth factor (IGF) system are involved in many biological processes and have growth-promoting actions regulating cell proliferation, differentiation, apoptosis and angiogenesis. A recent chapter in epigenetics is represented by microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) which regulate gene expression. Dysregulated miRNAs and lncRNAs have been associated with several diseases including cancer. Herein we report the most recent findings concerning miRNAs and lncRNAs regulating GH and the IGF system in the context of pituitary adenomas, osteosarcoma and colorectal cancer, shedding light on new possible therapeutic targets. Pituitary adenomas are increasingly common intracranial tumors and somatotroph adenomas determine supra-physiological GH secretion and cause acromegaly. Osteosarcoma is the most frequent bone tumor in children and adolescents and was reported in adults who were treated with GH in childhood. Colorectal cancer is the third cancer in the world and has a higher prevalence in acromegalic patients

MicroRNA global profiling in cystic fibrosis cell lines reveals dysregulated pathways related with inflammation, cancer, growth, glucose and lipid metabolism, and fertility: an exploratory study

Cystic fibrosis (CF), is due to CF transmembrane conductance regulator (CFTR) loss of function, and is associated with comorbidities. The increasing longevity of CF patients has been associated with increased cancer risk besides the other known comorbidities. The significant heterogeneity among patients, suggests potential epigenetic regulation. Little attention has been given to how CFTR influences microRNA (miRNA) expression and how this may impact on biological processes and pathways

Specific miRNAs Change After 3 Months of GH treatment and Contribute to Explain the Growth Response After 12 Months

Context: There is growing evidence of the role of epigenetic regulation of growth, and miRNAs potentially play a role. Objective: The aim of this study is to identify changes in circulating miRNAs following GH treatment in subjects with isolated idiopathic GH deficiency (IIGHD) after the first 3 months of treatment, and verify whether these early changes can predict growth response. Design and methods: The expression profiles of 384 miRNAs were analyzed in serum in 10 prepubertal patients with IIGHD (5 M, 5 F) at two time points before starting GH treatment (t-3, t0), and at 3 months on treatment (t+3). MiRNAs with a fold change (FC) >+1.5 or <-1.5 at t+3 were considered as differentially expressed. In silico analysis of target genes and pathways led to a validation step on 8 miRNAs in 25 patients. Clinical and biochemical parameters were collected at baseline, and at 6 and 12 months. Simple linear regression analysis and multiple stepwise linear regression models were used to explain the growth response. Results: Sixteen miRNAs were upregulated and 2 were downregulated at t+3 months. MiR-199a-5p (p = 0.020), miR-335-5p (p = 0.001), and miR-494-3p (p = 0.026) were confirmed to be upregulated at t+3. Changes were independent of GH peak values at testing, and levels stabilized after 12 months. The predicted growth response at 12 months was considerably improved compared with models using the common clinical and biochemical parameters. Conclusions: MiR-199a-5p, miR-335-5p, and miR-494-3p changed after 3 months of GH treatment and likely reflected both the degree of GH deficiency and the sensitivity to treatment. Furthermore, they were of considerable importance to predict growth response.Context: There is growing evidence of the role of epigenetic regulation of growth, and miRNAs potentially play a role. Objective: The aim of this study is to identify changes in circulating miRNAs following GH treatment in subjects with isolated idiopathic GH deficiency (IIGHD) after the first 3 months of treatment, and verify whether these early changes can predict growth response. Design and methods: The expression profiles of 384 miRNAs were analyzed in serum in 10 prepubertal patients with IIGHD (5 M, 5 F) at two time points before starting GH treatment (t-3, t0), and at 3 months on treatment (t+3). MiRNAs with a fold change (FC) >+1.5 or <-1.5 at t+3 were considered as differentially expressed. In silico analysis of target genes and pathways led to a validation step on 8 miRNAs in 25 patients. Clinical and biochemical parameters were collected at baseline, and at 6 and 12 months. Simple linear regression analysis and multiple stepwise linear regression models were used to explain the growth response. Results: Sixteen miRNAs were upregulated and 2 were downregulated at t+3 months. MiR-199a-5p (p = 0.020), miR-335-5p (p = 0.001), and miR-494-3p (p = 0.026) were confirmed to be upregulated at t+3. Changes were independent of GH peak values at testing, and levels stabilized after 12 months. The predicted growth response at 12 months was considerably improved compared with models using the common clinical and biochemical parameters. Conclusions: MiR-199a-5p, miR-335-5p, and miR-494-3p changed after 3 months of GH treatment and likely reflected both the degree of GH deficiency and the sensitivity to treatment. Furthermore, they were of considerable importance to predict growth response

Current Knowledge on Endocrine Disrupting Chemicals (EDCs) from Animal Biology to Humans, from Pregnancy to Adulthood: Highlights from a National Italian Meeting

Wildlife has often presented and suggested the effects of endocrine disrupting chemicals (EDCs). Animal studies have given us an important opportunity to understand the mechanisms of action of many chemicals on the endocrine system and on neurodevelopment and behaviour, and to evaluate the effects of doses, time and duration of exposure. Although results are sometimes conflicting because of confounding factors, epidemiological studies in humans suggest effects of EDCs on prenatal growth, thyroid function, glucose metabolism and obesity, puberty, fertility, and on carcinogenesis mainly through epigenetic mechanisms. This manuscript reviews the reports of a multidisciplinary national meeting on this topic

Ruolo dei miRNA nel predire la risposta all’Ormone della Crescita (GH) in pazienti pediatrici con Deficit di GH (GHD) e loro relazione con l’oncogenesi: miRNA profiling, studi in silico e in vitro.

L’Ormone della crescita (GH) e il suo principale effettore periferico insulin-like growth factor (IGF)-I, sono fondamentali nella crescita e nel metabolismo. I pazienti pediatrici con deficit di GH (GHD) vengono sottoposti a una terapia sostitutiva a lungo termine con GH a dosi che non sono individualizzate e spesso i tassi di crescita osservati non corrispondono all’atteso. Il ruolo del GH nella regolazione di proliferazione cellulare, differenziamento e apoptosi porta a valutarne un possibile effetto pro-oncogenico. I microRNA (miRNA) sono regolatori post-trascrizionali dell’espressione genica coinvolti in molti processi biologici tra cui crescita e cancro. Inoltre i miRNA sono studiati come biomarcatori di patologia e come marcatori per ottimizzare la somministrazione di farmaci. Questo progetto è volto ad identificare, tramite un approccio di miRNA profiling, i miRNA circolanti che variano in pazienti prepuberi con GHD sottoposti a terapia con GH e a valutare se questi miRNA siano utili per predire la risposta al GH in termini di crescita. Inoltre lo studio si pone di valutare l’impatto dei miRNA identificati su pathways associati al cancro utilizzando un approccio in silico e saggi funzionali in vitro in colture primarie. L’analisi di miRNA profiling in campioni di siero di 10 pazienti con GHD (5 maschi, 5 femmine) a due tempi prima dell’inizio della terapia (i.e. baseline) e a 3 mesi di terapia ha portato ad identificare 16 miRNA up-regolati e 2 miRNA down-regolati. L’analisi in silico ha mostrato che questi miRNA sono coinvolti nella crescita e nel cancro e ha consentito di selezionarne 8 con un ruolo putativo nella crescita e nel metabolismo osseo che sono stati sottoposti a validazione in 25 pazienti con GHD. MiR-199a-5p, miR-335-5p e miR-494-3p si sono confermati up-regolati dopo 3 mesi di terapia con GH. L’analisi dell’espressione dei 3 miRNA sui 12 mesi di terapia ha evidenziato che la loro variabilità tende a diminuire dopo 12 mesi. Non sono emerse differenze nei livelli dei miRNA fra pazienti responder e non-responder. Questi 3 miRNA contribuiscono a spiegare la risposta alla terapia in termini di variazione di altezza (0-12 mesi) (Adj.R2=0.79; R2CV=0.43) e variazione di velocità di crescita (0-6 mesi) (Adj.R2=0.75; R2CV=0.63). L’analisi di predizione dei target ha evidenziato che miR-335-5p e miR-199a-5p sono predetti regolare CRIM1 che lega BMP4 e BMP7 nel Golgi, riduce la produzione e il processamento delle preproteine a BMP mature e ne riduce la secrezione. Inoltre miR-199a-5p, miR-335-5p e miR-494-3p sono predetti regolare CLOCK, coinvolto nel ritmo circadiano associato allo sviluppo dell’osso e ROCK1 che è aumentato in diversi tumori e associato a metastasi. Lo studio in vitro degli effetti di questi miRNA sugli osteoblasti HOB ha mostrato, in seguito a trasfezione, che questi miRNA non hanno effetti sulla vitalità cellulare. L’osteopontina (SPP1), COL1A1 e COL1A2 sono stati analizzati come marker di differenziamento nelle cellule HOB e in seguito a trasfezione, i loro livelli di espressione non variano. Inoltre, i 3 miRNA non hanno mostrato effetti sui livelli di espressione di CLOCK, CRIM1 e ROCK1 né sui livelli proteici di CLOCK e ROCK1. In conclusione, questo progetto ha portato ad identificare 18 miRNA che variano in risposta al trattamento con GH e fra questi miR-199a-5p, miR-335-5p e miR-494-3p si sono confermati. La variazione precoce di questi miRNA contribuisce a spiegare la risposta al GH in termini di variazione di altezza 0-12 mesi e variazione di velocità di crescita 0-6 mesi e potrebbero essere usati in futuro in modelli di predizione per personalizzare la terapia. I dati attualmente disponibili non indicano un ruolo di questi miRNA nell’aumentare il rischio di cancro.Growth Hormone (GH) and its main peripheral effector, insulin-like growth factor (IGF)-I, play a fundamental role in growth processes and metabolism. Pediatric subjects diagnosed with GH deficiency (GHD) undergo long-term GH replacement therapy at dosages that are currently not individualized and growth rates do not always coincide with the expected. The role of GH in the regulation of cell proliferation, differentiation and apoptosis leads to consider possible oncogenic effects. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression, which are involved in many biological processes such as body growth and cancer. Moreover, miRNAs are studied as biomarkers of diseases and as markers to optimize administration of pharmaceutical agents. This project aimed at identifying circulating miRNAs varying in prepubertal GHD patients undergoing GH treatment by using a miRNA profiling approach and at evaluating whether they could be useful to predict the clinical outcome in terms of growth. Furthermore, it aimed at evaluating the impact of the identified miRNAs on pathways related with cancer by using an in silico approach and functional assays in vitro in primary cells. The miRNA profiling analysis on serum samples from 10 GHD patients (5 males, 5 females) at two time points before starting GH treatment (i.e. the baseline), and at 3 months on treatment allowed to identify 16 miRNAs which were upregulated and 2 miRNAs which were downregulated. The in silico analysis evidenced the involvement of these miRNAs in growth and cancer and allowed to select a subset of 8 miRNAs with a putative role in growth and bone metabolism, which underwent a validation step in 25 GHD patients. MiR-199a-5p, miR-335-5p, and miR-494-3p were confirmed to be upregulated after 3 months on GH treatment. The analysis of miRNA expression over 12 months of GH treatment evidenced that miRNA variability tends to decrease after 12 months on treatment. No differences in miRNA levels between responder and non- responder patients were observed. These 3 miRNAs contributed to explain growth response on GH treatment in terms of height variation (0-12 months) (Adj.R2=0.79; R2CV=0.43) and growth velocity variation (0-6 months) (Adj.R2=0.75; R2CV=0.63). A target prediction analysis of these 3 miRNAs evidenced that miR-335-5p and miR-199a-5p are predicted to target CRIM1 which binds with both BMP4 and BMP7 in the Golgi, and determines a reduction in the production and processing of preproteins to mature BMPs and a reduction of secretion of mature BMPs. Moreover, miR-199a-5p, miR-335-5p, and miR-494-3p are predicted to target CLOCK, which is involved in circadian clock regulation known to be linked to bone development and cancer and ROCK1, which is increased in many tumors and related to metastasis. The in vitro study of the effects of these miRNAs on HOB osteoblasts evidenced that these miRNAs had no effects on cell viability after miRNA transfection. Moreover, osteopontin (SPP1), COL1A1 and COL1A2 were analyzed as markers of cell differentiation in HOB cells after miRNA transfection and their expression levels did not change. Furthermore, the 3 miRNAs showed to have no effects on CLOCK, CRIM1, and ROCK1 gene expression nor CLOCK and ROCK1 protein levels. To conclude, this project led to the identification of 18 miRNAs that change in response to GH treatment and among these miR-199a-5p, miR-335-5p, and miR-494-3p were confirmed to change. The early change in these miRNAs contributes to explain growth response in terms of height variation 0-12 months and growth velocity variation 0-6 months, and they could be used in future for prediction models to customize treatment. Current data do not indicate a role of these miRNAs in increasing the risk for cancer

MicroRNAs change and target key regulatory genes involved in longitudinal growth in patients with idiopathic isolated growth hormone deficiency (IGHD) on growth hormone (GH) treatment

The growth response in patients undergoing GH treatment is variable depending both on the patient’s basal conditions and on personal innate sensitivity to therapy. MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and are recognised as important regulators of biological and metabolic processes. It is unknown at present whether miRNAs could be early biomarkers of response to GH treatment in a perspective of individualised medicine, and whether they could disclose new information on the effects and regulation of GH. We aimed at identifying all miRNAs varying on GH treatment using a global profiling approach, and at evaluating the principal pathways and biological processes, within growth, impacted by these miRNAs. Ten prepubertal normal weight patients with IGHD were enrolled (5Males,5Females; CA:8,12±0,73yr). Global miRNA profiles (TaqMan Advanced Human CardA) were evaluated at -3, 0 and at +3 months on treatment. MiRNA expression levels at -3 and 0 months were compared and the miRNAs showing a p-value≤0.05 were excluded allowing to identify those miRNAs changing only in response to treatment (+3 months) by either a factor Log22-DDCt>+1.5 or Log22-DDCt<-1.5 (up- or downregulated, respectively). Single miRNA target genes were evaluated and DIANA-miRPathv3.0 software was used for KEGG pathway and Gene Ontology analyses. Overall 30 miRNAs were regulated by GH, 27 were upregulated, and 3 down-regulated. A subset of 8 showed the most stringent criteria. Interestingly, 13 miRNAs were specifically regulated in females only and other 13 in males only, suggesting gender-specific effects. In the entire population, the miRNAs identified, targeted genes involved in the following pathways: ExtraCellularMatrix-receptor (COL1A1, COL2A1, integrins, laminins, SOX9, NF1 genes,etc.), Thyroid hormone (THReceptor, ATP1B2, MAP2K2, PIK3R2, RAF1, NOTCH2, AKT, CASP9, STAT1, FOXO1, MAPK1, etc.), Steroid biosynthesis, mTOR signaling (BRAF, AKT, PIK3CD, IGF1, PTEN, etc.), MAPK signaling (BRAF, SOS2, FGFR3, RAF1, FGF4, EGFR, TGFB1, FGF11, AKT, FLNB, FGF9, NF1, TGFB2, FGFs, FGFRs, TGFB3, etc.), Prolactin signaling (PRLR, STAT3, SOS2, SHC1, RAF1, AKT2, JAK2, SOCS1, FOXO3, STAT1, SOCS7, etc.), PI3K-Akt signaling (FGFR3, FGFR4, COL4A5, IGF1R, EGFR, COL5A1, COL1A1, INSR, IGF1, VEGFB, FGFR1, FGF7, SOS2, RAF1, FGF8, etc.), Phosphatidylinositol signaling (PIK3R3, PTEN, etc.), and N-Glycan biosynthesis pathways. Mutations of 15 of these genes are well known to cause genetic short stature in humans. Furthermore, analyses of the biological processes identified the following as being regulated by the miRNAs identified: fibroblast growth factor receptor signaling, glycosaminoglycan metabolism, phosphatidylinositol-mediated signaling (FGFR3, IGF1R, PTPN11, IGF1, FGF8, FGFR1, etc.), insulin receptor signaling, transforming growth factor beta receptor signaling, androgen receptor signaling and the JAK-STAT cascade involved in GH signaling as expected. In conclusion, GH regulates miRNAs that in turn regulate genes, pathways and biological processes involved with growth. Novel gender specific effects of GH were found. MiRNAs could be explored as biomarkers of response to treatment. Further, some novel genes implicated in the regulation of growth could be identified using this approach