Identification of complete hydatidiform mole pregnancy-associated microRnas in plasma.
The presence of pregnancy-associated, placenta-specific microRNAs (miRNAs)  in the plasma of pregnant women has recently been reported (1). Such miRNAs have potential as molecular markers for complete hydatidiform mole (CHM), which is usually diploid and androgenetic in origin. We aimed to identify CHM pregnancy-associated miRNAs in plasma. All participants gave written informed consent, and the Research Ethics Committee of Nagasaki University approved the study.
First, we obtained a set of CHM tissues and blood samples from a CHM pregnant woman at 10 weeks' gestation and normal villous tissue from a woman with an uncomplicated singleton pregnancy who underwent artificial abortion at 10 weeks' gestation. Both the androgenetic origin of the CHM (46,XX) and the biparental origin of normal villous tissue (46,XX) were confirmed by DNA genotyping analysis.
Isolation of total RNA (including small RNAs), assessment of their quality, concentration measurements, construction of a small-RNA library, next-generation sequencing (NGS), miRNA mapping, and NGS analysis of the differential expression of miRNA-encoding genes were performed as described previously (2). NGS analysis of the sample set yielded 16 920 412 reads from CHM tissue, 17 462 519 reads from the patient's blood cells, and 30 447 835 reads from normal villous tissue. All of these sequence data were deposited in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive (http://www.ddbj.nig.ac.jp/index-e. html). The accession ID is DRA001009 (http://trace.ddbj.nig. ac.jp/DRASearch/).
To compare miRNA concentrations across the data set, we corrected the sequencing bias of GC content for each miRNA (3) and normalized the sequencing read count as reads per kilobase of exon model per million mapped reads (RPKM) (4). NGS analysis of the sample set detected 503 miRNAs in CHM tissue, 497 miRNAs in the patient's blood cells, and 715 miRNAs in normal villous tissue. Homo sapiens miRNA 520b (hsamiR-520b), -520f, and -520c-3p were detected in CHM tissue at concentrations > 50 times higher than in normal villous tissue. Conversely, hsa-miR-136-3p, -127-5p, -370, -127-3p, -493-3p, -184, and -493-5p were detected in CHM tissue at concentrations > 50 times lower than in normal villous tissue. We selected hsa-miR-520b, -520f, and -520c-3p (which were undetectable in blood cells) as candidate plasma CHM pregnancy-associated miRNAs. These candidate miRNAs were located on chromosome 19q13.42, which is commonly referred to as the chromosome 19 miRNA cluster (C19MC). The -fold changes (i.e., the RPKM of CHM miRNA divided by the RPKM of normal villous miRNA) for hsa-miR-520b, -520f, and -520c-3p were 62.38 (i.e., 13 760.74 - 220.59), 78.71 (6711.02 / 85.26), and 115.41 (310 662.75 / 2691.72), respectively. Compared with normal villous tissue, the expression of miRNA-encoding genes on C19MC were upregulated, whereas those on C14MC were downregulated in CHM tissue (raw data deposited in the DDBJ).
Subsequently, we analyzed the 3 candidate plasma CHM pregnancy-associated miRNAs by absolute reverse-transcription quantitative real-time PCR (RT-qPCR) with the TaqMan[R] MicroRNA Assay (Applied Biosystems) in villous tissues and plasma samples from 14 CHM pregnancies (CHM group) and 20 uncomplicated pregnancies (control group). There were no significant differences between the CHM and control groups in maternal age or gestational age (data not shown). Preparation and extraction of total RNA containing small RNA molecules from tissue samples or from 1.2-mL samples of cell-free plasma, as well as absolute RT-qPCR analysis, were performed as described previously (1, 2). The intraassay CVs for hsamiR-520b, -miR-520f, and -520c3p) in the absolute RT-qPCR assays were 8.1%, 6.4%, and 6.8%, respectively. Absolute RT-qPCR results for villous tissues and plasma samples are shown in Fig. 1. The concentrations of candidate CHM pregnancy-associated miRNAs were significantly higher in the CHM group than in the control group, for both villous tissues and plasma samples. These CHM-associated miRNAs were identified in both CHM tissues and plasma samples. Placenta-specific C19MC is imprinted in the placenta, with expression from the paternally inherited chromosome (5). Therefore, the increased expression of genes encoding CHM pregnancy-associated miRNAs in CHM tissues and plasma samples may reflect the overexpression of the paternally expressed imprinted miRNA-encoding genes at C19MC in androgenic CHM pregnancies.
Finally, to identify plasma CHM pregnancy-associated miRNAs, we measured circulating concentrations of plasma CHM-associated miRNAs from 14 CHM pregnant women before and after evacuation. The median (range) plasma concentrations (in copies per milliliter) for the 3 CHM-associated miRNAs before and after evacuation, respectively, were as follows: 16 102.2 (8212.961264.0) and 2117.65 (593.567321.2) for hsa-miR-520b; 27 237.99 (11 298.2-73 268.12) and 3623.0 (718.1-7237.2) for -520f; and 24473.11 (12 726.1274 321.94) and 2157.65 (923.24729.8) for -520c-3p. Circulating plasma concentrations of all CHM-associated miRNAs were significantly decreased after evacuation (Wilcoxon signed rank tests, P = 0.001).
Although our data are still preliminary because of the small sample sizes, measurement of hsa-miR520b, -520f, and -520c-3p could prove useful as diagnostic, prognostic, or follow-up molecular markers in the clinical management of CHM, in addition to the current biochemical marker, human chorionic gonadotropin. Future studies of the biological pathway of these 3 plasma CHM pregnancy-associated miRNAs may clarify the molecular pathogenesis of CHMs and may lead to the discovery of novel therapeutic targets.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared. Honoraria: None declared.
Research Funding: K. Miura, Japan Society for the Promotion of Science (KAKENHI grant nos. 23592406 and 24791712); H. Masuzaki, Japan Society for the Promotion of Science (KAKENHI grant no. 25462563). Expert Testimony: None declared.
Patents: None declared.
Acknowledgments: We thank N. Sugimoto (Hokkaido System Science, Hokkaido, Japan), and Drs. A. Higashijima, S. Miura, S. Abe, O. Jo, A. Yoshida, H. Mishima, and A. Kinoshita for their technical assistance.
(1.) Miura K, Miura S, Yamasaki K, Higashijima A, Kinoshita A, Yoshiura K, et al. Identification of pregnancy-associated microRNAs in maternal plasma. Clin Chem 2010;56:1767-71.
(2.) Higashijima A, Miura K, Mishima H, Kinoshita A, Jo O, Abe S, et al. Characterization of placenta-specific microRNAs in fetal growth restriction pregnancy. Prenatal Diagn 2013;33:214-22.
(3.) Hansen KD, Irizarry RA, Wu Z. Removing technical variability in RNA-Seq data using conditional quantile normalization. Biostatistics 2012; 13:204-16.
(4.) Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008; 5:621-8.
(5.) Noguer-Dance M, Abu-Amero S, Al-Khtib M, Lefevre A, Coullin P, Moore GE, et al. The primate-specific microRNA gene cluster (C19MC)
is imprinted in the placenta. Hum Mol Genet 2010;19:3566-82.
Yuri Hasegawa 
Kiyonori Miura  *
Kenichi Furuya 
Koh-ichiro Yoshiura 
Hideaki Masuzaki 
 Departments of Obstetrics and Gynecology and
 Human Genetics Nagasaki University Graduate School of Biomedical Sciences Nagasaki, Japan  Department of Obstetrics and Gynecology National Defense Medical College Saitama, Japan
* Address correspondence to this author at: Department of Obstetrics and Gynecology Nagasaki University Graduate School of Biomedical Sciences 1-7-1 Sakamoto Nagasaki 852-8501, Japan Fax 95-819-7365 E-mail email@example.com
 Nonstandard abbreviations: miRNA, microRNA; CHM, complete hydatidiform mole; NGS, nextgeneration sequencing; DDBJ, DNA Data Bank of Japan; RPKM, reads per kilobase of exon model per million mapped reads; hsa-miR, Homo sapiens miRNA; C19MC, chromosome 19 miRNA cluster; RT-qPCR, reverse-transcription quantitative realtime PCR.
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|Title Annotation:||Letters to the Editor|
|Author:||Hasegawa, Yuri; Miura, Kiyonori; Furuya, Kenichi; Yoshiura, Koh-ichiro; Masuzaki, Hideaki|
|Article Type:||Letter to the editor|
|Date:||Sep 1, 2013|
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