Elucidating the effect of epigenetic modifications of complete hydatidiform mole microRNA signalling and risk of malignization

• Grant number: 20/08830-6
• Support Opportunities: Regular Research Grants
• Duration: April 01, 2021 - March 31, 2023
• Field of knowledge: Health Sciences - Medicine - Maternal and Child Health
• Principal Investigator: Izildinha Maestá
• Grantee: Izildinha Maestá
• Host Institution: Faculdade de Medicina (FMB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated researchers: Lawrence Hsu Lin ; Ross Berkowitz

Abstract

Subproject 1: MicroRNAs (miRNAs) are small non-coding single stranded RNAs with important regulatory functions. While well-studied in cancer, less is known about the role of miRNAs in premalignant disease. Complete hydatidiform moles (CHM) are benign forms of gestational trophoblastic disease (GTD) that progress to gestational trophoblastic neoplasia (GTN) in up to 20% of cases; however, there is no well-established biomarker that can predict the development of postmolar GTN. Objective: To investigate possible differences in miRNA expression between CHM progressing to GTN compared to those resolving after surgical evacuation. Methods: Total RNA will be extracted from fresh frozen tissues from 49 complete molar pregnancies collected at the time of uterine evacuation. Total RNA will also be extracted from two choriocarcinoma cell lines, JEG-3 and JAR, and an immortalized normal placenta cell line, 3A-subE. miRNA expression in all the samples will be quantified using miRNA-sequencing. Hits from the sequencing data will be validated using a quantitative probe-based assay. Significantly altered miRNAs will then be subjected to target prediction and gene ontology analyses to look for alterations in key signaling pathways. Expression of potential miRNA targets will be assessed by qRT-PCR and Western blotting. Finally, potential prognostic protein biomarkers will be validated in an independent set of formalin-fixed paraffin embedded patient samples (15 CHM progressing to gestational trophoblastic neoplasia and 12 that spontaneously regressed), using quantitative immunohistochemistry. Subproject 2: The mechanisms of complete hydatidiform mole (CHM) transition to gestational trophoblastic neoplasia (GTN) are poorly understood. A subset of microRNAs, known as "Tropho-miRs", is expressed by the trophoblast and plays a role in placentation. CHM has a high rate of abnormal DNA methylation, which results in the aberrant expression of genes that flank microRNA clusters. Few studies have investigated CHM epigenetic changes, including DNA methylation and microRNA expression. Objective: To determine patterns of microRNA expression and DNA methylation in CHM, and to ascertain their influence on the risk of malignant transformation. Methods: Genomic DNA from benign CHM (spontaneous resolution), pre-GTN CHM (development to GTN) and normal placentas will be isolated from fresh tissue samples stored at - 80ºC. Differences in microRNA expression will be based on previously obtained microRNA sequencing (subproject 1). Validation of microRNA expression will be performed by qRT-PCR. Methylation analysis of microRNA promoters will be performed by the Illumina Infinium Methylation EPIC Kit and validated by PCR with bisulfite-converted DNA. qRT-PCR and Western blotting will be used to evaluate the expression of messenger RNA and proteins, regulated by microRNAs. Changes in protein expression will be validated by quantitative immunohistochemical analysis in an independent set of samples of CHM placentas fixed in paraffin blocks. Findings of DNA methylation patterns and microRNA expression in fresh tissue from normal placentas will be compared to those seen in fresh tissue from benign CHM (spontaneous resolution) and pre-GTN CHM (development to GTN). (AU)

Scientific publications

(4)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

ST LAURENT, JESSICA D.; LIN, LAWRENCE H.; OWEN, DAVID M.; MAESTA, IZILDINHA; CASTANEDA, ARNOLD; HASSELBLATT, KATHLEEN T.; GOLDSTEIN, DONALD P.; HOROWITZ, NEIL S.; BERKOWITZ, ROSS S.; ELIAS, KEVIN M.. Loss of Selenoprotein Iodothyronine Deiodinase 3 Expression Correlates with Progression of Complete Hydatidiform Mole to Gestational Trophoblastic Neoplasia. REPRODUCTIVE SCIENCES, JUN 2021. (20/08830-6)

LIN, LAWRENCE H.; MAESTA, IZILDINHA; ST LAURENT, JESSICA D.; HASSELBLATT, KATHLEEN T.; HOROWITZ, NEIL S.; GOLDSTEIN, DONALD P.; QUADE, BRADLEY J.; SUN, SUE Y.; BRAGA, ANTONIO; FISHER, ROSEMARY A.; et al. Distinct microRNA profiles for complete hydatidiform moles at risk of malignant progression. American Journal of Obstetrics and Gynecology, v. 224, n. 4, APR 2021. (20/08830-6)

ST LAURENT, JESSICA D.; LIN, LAWRENCE H.; OWEN, DAVID M.; MAESTA, IZILDINHA; CASTANEDA, ARNOLD; HASSELBLATT, KATHLEEN T.; GOLDSTEIN, DONALD P.; HOROWITZ, NEIL S.; BERKOWITZ, ROSS S.; ELIAS, KEVIN M.. Loss of Selenoprotein Iodothyronine Deiodinase 3 Expression Correlates with Progression of Complete Hydatidiform Mole to Gestational Trophoblastic Neoplasia. REPRODUCTIVE SCIENCES, v. 28, n. 11, p. 3200-3211, NOV 2021. (20/08830-6)

RIBEIRO DA SILVA, VALDETE APARECIDA; MAESTA, IZILDINHA; DE ARAUJO COSTA, ROBERTO ANTONIO; CAMPOS, ALINEDE AVILA; BRAGA, ANTONIO; HOROWITZ, NEIL; ELIAS, KEVIN M.; BERKOWITZ, ROSS. Geographical Health District and Distance Traveled Influence on Clinical Status at Admission of Patients with Gestational Trophoblastic Disease. Revista Brasileira de Ginecologia e Obstetrícia, v. 45, n. 07, p. 9-pg., 2023-07-01. (20/08830-6, 18/15591-8)