Over the last few decades, the role of non-coding RNAs in a plethora of diseases has attracted increasing attention. These RNAs are involved in the epigenetic regulation of physiological as well as pathological mechanisms and are present in most biological fluids. For this reason, they represent promising non-invasive biomarkers of diagnosis and prognosis in several diseases. In this work, the role of microRNAs as biomarkers in seminal plasma has been analyzed in the field of male infertility related to testicular germ cell tumors (TGCTs). TGCTs are the most recurrent tumors in young men with the highest incidence between the ages of 20 and 40 years and represent more than 60% of all cancers diagnosed in this age range. TGCTs have considerably increased in the past 50 years, and, although they are generally curable tumors when early diagnosed, some patients may become refractory to chemotherapy or may be subjected to relapse. The most used serum tumor markers for the diagnosis and follow-up lack sensitivity, being raised in less than 60% of TGCT cases often showing false negative and false positive results. Identification of valid biomarkers would allow not only a precise and early diagnosis but also to assess therapy response and early detection of relapse, improving follow-up monitoring. According to many studies, one of the major risk factors include infertility. Thus, since there is most likely a correlation between TGCTs and infertility, not only because infertility could be a consequence of testicular damage due to TGCTs but also because infertility may represent a risk factor for TGCTs, it is important to find valid biomarkers that could be useful in both tumor diagnosis and prognosis and fertility evaluation. In this work, miRNA profiling in seminal plasma from subjects affected by TGCTs and healthy subjects presenting normal or impaired spermiograms, was performed by high-throughput technologies. The results obtained were subsequently validated by single-assay Real-Time and Digital PCR systems. From this analysis we identified 3 differentially expressed miRNAs, in particular miR-221-3p and miR-222-3p were found to be down-regulated in TGCTs patient with respect to healthy subjects, while miR-126-3p resulted up-regulated in the same comparison. The performed ROC curve and correlation analysis and an in-depth bioinformatics analysis based on weighted gene co-expression networks allowed as to identify those miRNAs as biomarkers of infertility related to TGCTs. Furthermore, this work is part of the project “Research and Innovation Staff Exchange (RISE), Call: H2020-MSCA-RISE-2020 diaRNAgnosis” which involves several research centers as well as biotech companies and whose focus is to identify valid biomarkers of testicular and prostate cancer and develop a PCR-free detection system with high sensitivity and specificity. With the results obtained from the expression analysis, during my experience of 6 months at DESTINA Genomica S.L. (Spain), I was able to set up and test the specificity and the sensitivity of complementary probes for the detection of the miR-126-3p, because being overexpressed it is considered more suitable for the PCR-free evaluation.
Negli ultimi decenni, il ruolo degli RNA non codificanti in molte malattie ha attirato una crescente attenzione. Questi RNA sono coinvolti nella regolazione epigenetica di meccanismi fisiologici e patologici e sono presenti nella maggior parte dei fluidi biologici. Per questo motivo, rappresentano promettenti biomarcatori non invasivi per la diagnosi e la prognosi di diverse malattie. In questo lavoro, il ruolo dei microRNA come biomarcatori nel plasma seminale è stato analizzato nel campo dell'infertilità maschile legata ai tumori testicolari a cellule germinali (TGCT). I TGCT sono i tumori più ricorrenti nei giovani uomini, con un'incidenza maggiore tra i 20 e i 40 anni, e rappresentano oltre il 60% di tutti i tumori diagnosticati in questa fascia di età. Sono aumentati notevolmente negli ultimi 50 anni e, sebbene siano generalmente tumori curabili se diagnosticati precocemente, alcuni pazienti possono diventare refrattari alla chemioterapia o andare incontro a ricadute. I marcatori tumorali sierici più utilizzati per la diagnosi e il follow-up mancano di sensibilità, questi infatti sono rilevati in meno del 60% dei casi di TGCT e mostrano spesso risultati falsi negativi e falsi positivi. L'identificazione di biomarcatori validi consentirebbe non solo una diagnosi precisa e precoce, ma anche di valutare la risposta alla terapia e di individuare precocemente la recidiva, migliorando il monitoraggio del follow-up. Secondo molti studi, uno dei principali fattori di rischio è l'infertilità. Pertanto, poiché è molto probabile che esista una correlazione tra i TGCT e l'infertilità, non solo perché l'infertilità potrebbe essere una conseguenza del danno testicolare dovuto ai TGCT, ma anche perché l'infertilità potrebbe rappresentare un fattore di rischio per i TGCT, è importante trovare biomarcatori validi che possano essere utili sia per la diagnosi e la prognosi del tumore che per la valutazione della fertilità. In questo lavoro è stato eseguito il profiling dei miRNA nel plasma seminale di soggetti affetti da TGCT e di soggetti sani che presentavano spermiogrammi normali o alterati, mediante tecnologie high-throughput. I risultati ottenuti sono stati successivamente validati mediante sistemi di Real-Time e Digital PCR single assay. Da questa analisi abbiamo identificato 3 miRNA differenzialmente espressi, in particolare miR-221-3p e miR-222-3p sono risultati down-regolati nei pazienti con TGCT rispetto ai soggetti sani, mentre miR-126-3p è risultato up-regolato nello stesso confronto.La curva ROC e l'analisi di correlazione eseguite insieme ad un'approfondita analisi bioinformatica basata sull’analisi delle gene co-expression network pesate, hanno permesso di identificare questi miRNA come biomarcatori dell'infertilità correlata ai TGCT.Inoltre, questo lavoro fa parte del progetto "Research and Innovation Staff Exchange (RISE), Call: H2020-MSCA-RISE-2020 diaRNAgnosis" che coinvolge diversi centri di ricerca e aziende biotecnologiche e il cui obiettivo è identificare validi biomarcatori del cancro ai testicoli e alla prostata e sviluppare un sistema di rilevamento che non necessiti dell’uso della PCR con elevata sensibilità e specificità. Con i risultati ottenuti dall'analisi dell'espressione, durante la mia esperienza di 6 mesi presso l’azienda DESTINA Genomica S.L. (Spagna), ho potuto sviluppare e testare la specificità e la sensibilità di sonde complementari per il rilevamento del miR-126-3p, il quale, essendo sovraespresso è considerato più adatto per la valutazione senza uso della tecnica di PCR.
MicroRNAs come potenziali biomarcatori per i Tumori Testicolari a Cellule Germinali in uomini infertili
FERRARA, CARMEN GAIA ROSSELLA
2024
Abstract
Over the last few decades, the role of non-coding RNAs in a plethora of diseases has attracted increasing attention. These RNAs are involved in the epigenetic regulation of physiological as well as pathological mechanisms and are present in most biological fluids. For this reason, they represent promising non-invasive biomarkers of diagnosis and prognosis in several diseases. In this work, the role of microRNAs as biomarkers in seminal plasma has been analyzed in the field of male infertility related to testicular germ cell tumors (TGCTs). TGCTs are the most recurrent tumors in young men with the highest incidence between the ages of 20 and 40 years and represent more than 60% of all cancers diagnosed in this age range. TGCTs have considerably increased in the past 50 years, and, although they are generally curable tumors when early diagnosed, some patients may become refractory to chemotherapy or may be subjected to relapse. The most used serum tumor markers for the diagnosis and follow-up lack sensitivity, being raised in less than 60% of TGCT cases often showing false negative and false positive results. Identification of valid biomarkers would allow not only a precise and early diagnosis but also to assess therapy response and early detection of relapse, improving follow-up monitoring. According to many studies, one of the major risk factors include infertility. Thus, since there is most likely a correlation between TGCTs and infertility, not only because infertility could be a consequence of testicular damage due to TGCTs but also because infertility may represent a risk factor for TGCTs, it is important to find valid biomarkers that could be useful in both tumor diagnosis and prognosis and fertility evaluation. In this work, miRNA profiling in seminal plasma from subjects affected by TGCTs and healthy subjects presenting normal or impaired spermiograms, was performed by high-throughput technologies. The results obtained were subsequently validated by single-assay Real-Time and Digital PCR systems. From this analysis we identified 3 differentially expressed miRNAs, in particular miR-221-3p and miR-222-3p were found to be down-regulated in TGCTs patient with respect to healthy subjects, while miR-126-3p resulted up-regulated in the same comparison. The performed ROC curve and correlation analysis and an in-depth bioinformatics analysis based on weighted gene co-expression networks allowed as to identify those miRNAs as biomarkers of infertility related to TGCTs. Furthermore, this work is part of the project “Research and Innovation Staff Exchange (RISE), Call: H2020-MSCA-RISE-2020 diaRNAgnosis” which involves several research centers as well as biotech companies and whose focus is to identify valid biomarkers of testicular and prostate cancer and develop a PCR-free detection system with high sensitivity and specificity. With the results obtained from the expression analysis, during my experience of 6 months at DESTINA Genomica S.L. (Spain), I was able to set up and test the specificity and the sensitivity of complementary probes for the detection of the miR-126-3p, because being overexpressed it is considered more suitable for the PCR-free evaluation.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/165721
URN:NBN:IT:UNICT-165721