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BACKGROUND MicroRNAs (miRNAs) are small non-coding RNAs with important regulatory functions. While well-studied in cancer, less is known about the role of miRNAs in premalignant disease. Complete hydatidiform moles are benign forms of gestational trophoblastic disease that progress to gestational trophoblastic neoplasia in up to 20% of cases; however, there is no well-established biomarker that can predict the development of gestational trophoblastic neoplasia. OBJECTIVE To investigate possible differences in miRNA expression between complete moles progressing to gestational trophoblastic neoplasia compared to those regressing after surgical evacuation. STUDY DESIGN Total RNA was extracted from fresh frozen tissues from 39 complete moles collected at the time of uterine evacuation in Brazil. Thirty cases achieved hCG normalization without further therapy and 9 cases developed gestational trophoblastic neoplasia requiring chemotherapy. Total RNA was also 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 was quantified using miRNA-sequencing. Hits from the sequencing data were validated using a quantitative probe-based assay. Significantly altered miRNAs were then subjected to target prediction and gene ontology analyses to search for alterations in key signaling pathways. Expression of potential miRNA targets was assessed by qRT-PCR and Western blot. Finally, potential prognostic protein biomarkers were validated in an independent set of formalin-fixed paraffin embedded patient samples from the United States (15 complete moles progressing to gestational trophoblastic neoplasia and 12 that spontaneously regressed), using quantitative immunohistochemistry. RESULTS In total, 462 miRNAs were identified in all samples at a threshold of least 1 tag per million. miRNA-sequencing revealed a distinct set of miRNAs associated with gestational trophoblastic neoplasia. Gene ontology analysis of the most altered transcripts showed that the leading pathway was related to response to ischemia (p<0.001). Two of the top three most significantly altered miRNAs were mir-181b-5p (1.65-fold; adj. p=0.014) and mir-181d-5p (1.85-fold; adj. p=0.014), both of which have been shown to regulate expression of BCL2. By RT-PCR, BCL2 mRNA expression was significantly lower in the complete moles progressing to gestational trophoblastic neoplasia than the regressing complete moles (-4.69-fold; p=0.018). Reduced expression of BCL2 was confirmed in tissue samples by Western blot. Immunohistochemistry in the independent patient samples revealed significantly lower cytoplasmic expression of BCL2 in the villous trophoblasts from cases destined for progression to gestational trophoblastic neoplasia compared to those that regressed, both with respect to staining intensity (optic density 0.110 ± 0.102 versus 0.212 ± 0.036; p<0.001) and to the percentage of positive cells (16 ± 28% versus 49.4± 28.05%; p=0.003). CONCLUSIONS Complete moles progressing to gestational trophoblastic neoplasia are associated with a distinct miRNA profile. miR-181 family members and BCL2 may be prognostic biomarkers in predicting gestational trophoblastic neoplasia risk.