Takuya Mishima

Human Villous Trophoblasts Express and Secrete Placenta-Specific MicroRNAs into Maternal Circulation via Exosomes

In this study, we performed small RNA library sequencing using human placental tissues to identify placenta-specific miRNAs. We also tested the hypothesis that human chorionic villi could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation. By small RNA library sequencing, most placenta-specific miRNAs (e.g., MIR517A) were linked to a miRNA cluster on chromosome 19. The miRNA cluster genes were differentially expressed in placental development. Subsequent validation by real-time PCR and in situ hybridization revealed that villous trophoblasts express placenta-specific miRNAs. The analysis of small RNA libraries from the blood plasma showed that the placenta-specific miRNAs are abundant in the plasma of pregnant women. By real-time PCR, we confirmed the rapid clearance of the placenta-specific miRNAs from the plasma after delivery, indicating that such miRNAs enter into maternal circulation. By using the trophoblast cell line BeWo in culture, we demonstrated that miRNAs are indeed extracellularly released via exosomes. Taken together, our findings suggest that miRNAs are exported from the human placental syncytiotrophoblast into maternal circulation, where they could target maternal tissues. Finally, to address the biological functions of placenta-specific miRNAs, we performed a proteome analysis of BeWo cells transfected with MIR517A. Bioinformatic analysis suggests that this miRNA is possibly involved in tumor necrosis factor-mediated signaling. Our data provide important insights into miRNA biology of the human placenta.

MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adult mouse testis and ovary

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that can regulate the expression of complementary mRNA targets. Identifying tissue-specific miRNAs is the first step toward understanding the biological functions of miRNAs, which include the regulation of tissue differentiation and the maintenance of tissue identity. In this study, we performed small RNA library sequencing in adult mouse testis and ovary to reveal their characteristic organ- and gender-specific profiles and to elucidate the characteristics of the miRNAs expressed in the reproductive system. We obtained 10,852 and 11 744 small RNA clones from mouse testis and ovary respectively (greater than 10,000 clones per organ), which included 6630 (159 genes) and 10,192 (154 genes) known miRNAs. A high level of efficiency of miRNA library sequencing was achieved: 61% (6630 miRNA clones/10,852 small RNA clones) and 87% (10,192/11,744) for adult mouse testis and ovary respectively. We obtained characteristic miRNA signatures in testis and ovary; 55 miRNAs were detected highly, exclusively, or predominantly in adult mouse testis and ovary, and discovered two novel miRNAs. Male-biased expression of miRNAs occurred on the X-chromosome. Our data provide important information on sex differences in miRNA expression that should facilitate studies of the reproductive organ-specific roles of miRNAs.

MiR-205 silences MED1 in hypoxic primary human trophoblasts

Acting through degradation of target mRNA or inhibition of translation, microRNAs (miRNAs) regulate development, differentiation, and cellular response to diverse cues. We analyzed changes in miRNA expression in human placental trophoblasts exposed to hypoxia, which may result from hypoperfusion and placental injury. Using an miRNA microarray screen, confirmed by Northern blot analysis, we defined a set of seven miRNAs (miR‐93, miR‐205, miR‐224, miR‐335, miR‐424, miR‐451, and miR‐491) that are differentially regulated in primary trophoblasts exposed to hypoxia. We combined in silico prediction of miRNA targets with gene expression profiling data to identify a series of potential targets for the miRNAs, which were further analyzed using luciferase reporter assays. Among experimentally confirmed targets, we found that the transcriptional coactivator MED1, which plays an important role in placental development, is a target for miR‐205. Using gain‐ and loss‐of‐function assays, we confirmed that miR‐205 interacts with a specific target in the 3′‐UTR sequence of MED1 and silences MED1 expression in human trophoblasts exposed to hypoxia, suggesting that miR‐205 plays a role in trophoblast injury.—Mouillet, J.‐F., Chu, T., Nelson, D. M., Mishima, T., Sadovsky, Y. MiR‐205 silences MED1 in hypoxic primary human trophoblasts. FASEB J. 24, 2030–2039 (2010). www.fasebj.org

RT-PCR-based analysis of microRNA (miR-1 and -124) expression in mouse CNS.

More than 700 microRNAs (miRNAs) have been cloned, and the functions of these molecules in developmental timing, cell proliferation, and cancer have been investigated widely. MiRNAs are analyzed with Northern blot and sequential colony evaluation; however, reverse transcription-polymerase chain reaction (RT-PCR)-based miRNA assay remains to be developed. In this report, we describe improved real-time RT-PCR methods using specific or non-specific RT primer for the semi-quantitative analysis of miRNA expression. The use of the new methods in a model study revealed differential expression of miRNA-1 (miR-1) and miR-124 in mouse organs. Specifically, our methods revealed that miR-124 concentrations in the mouse central nervous system (CNS; cerebral cortex, cerebellum, and spinal cord) were more than 100 times those in other organs. By contrast, miR-1 expression in the CNS was 100-1000 times lower than that in skeletal muscle and heart. Furthermore, we revealed anatomically regional differences in miR-124 expression within the CNS: expression ratios versus the cerebral cortex were 60.7% for the cerebellum and 35.4% for the spinal cord. These results suggest that our RT-PCR-based methods would be a powerful tool for studies of miRNA expression that is associated with various neural events.

Sequencing and bioinformatics-based analyses of the microRNA transcriptome in hepatitis B-related hepatocellular carcinoma.

MicroRNAs (miRNAs) participate in crucial biological processes, and it is now evident that miRNA alterations are involved in the progression of human cancers. Recent studies on miRNA profiling performed with cloning suggest that sequencing is useful for the detection of novel miRNAs, modifications, and precise compositions and that miRNA expression levels calculated by clone count are reproducible. Here we focus on sequencing of miRNA to obtain a comprehensive profile and characterization of these transcriptomes as they relate to human liver. Sequencing using 454 sequencing and conventional cloning from 22 pair of HCC and adjacent normal liver (ANL) and 3 HCC cell lines identified reliable reads of more than 314000 miRNAs from HCC and more than 268000 from ANL for registered human miRNAs. Computational bioinformatics identified 7 novel miRNAs with high conservation, 15 novel opposite miRNAs, and 3 novel antisense miRNAs. Moreover sequencing can detect miRNA modifications including adenosine-to-inosine editing in miR-376 families. Expression profiling using clone count analysis was used to identify miRNAs that are expressed aberrantly in liver cancer including miR-122, miR-21, and miR-34a. Furthermore, sequencing-based miRNA clustering, but not individual miRNA, detects high risk patients who have high potentials for early tumor recurrence after liver surgery (P = 0.006), and which is the only significant variable among pathological and clinical and variables (P = 0,022). We believe that the combination of sequencing and bioinformatics will accelerate the discovery of novel miRNAs and biomarkers involved in human liver cancer.

Relationship between altered expression levels of MIR21, MIR143, MIR145, and MIR205 and clinicopathologic features of esophageal squamous cell carcinoma

In spite of the undisputed importance of altered expression patterns of microRNAs (miRNAs) in various cancers, there is little information on the clinicopathologic significance of cancer-related miRNAs (MIR21, MIR143, MIR144, MIR145, and MIR205) in esophageal squamous cell carcinoma (ESCC). We examined the expression levels of the precursor and mature miRNA genes in ESCC using real-time polymerase chain reaction (PCR). We also investigated the mRNA expression levels of processing elements (RNASEN, DGCR8, and DICER1) that participate in miRNA-biogenesis pathway. Furthermore, we analyzed the relationships between the expression levels of these five miRNAs and the clinicopathologic parameters of ESCC patients. The expression levels of mature MIR21 and mature MIR145 were higher in ESCC than those in normal epithelium (P < 0.05). The mature/pre ratio of MIR21 in ESCC was higher than that in normal epithelium (P < 0.05). With regard to miRNA-processing elements, the expression level of RNASEN was higher in ESCC than in normal epithelium (P < 0.05). Furthermore, altered expression of these miRNAs was related to the clinicopathologic features of ESCC patients. The high expression of mature MIR21 and mature MIR205 was associated with lymph node positivity in ESCC patients (P < 0.05). The high levels of expression of mature MIR143 and mature MIR145 were associated with recurrence of metastasis in ESCC patients (P < 0.05). The findings may imply that miRNA biogenesis is aberrantly accelerated in ESCC. Analysis of the expression levels of miRNAs should provide useful information for evaluation of the staging, prognosis, and treatment of ESCC patients.

The Expression and Function of Fatty Acid Transport Protein-2 and -4 in the Murine Placenta

Background The uptake and trans-placental trafficking of fatty acids from the maternal blood into the fetal circulation are essential for embryonic development, and involve several families of proteins. Fatty acid transport proteins (FATPs) uniquely transport fatty acids into cells. We surmised that placental FATPs are germane for fetal growth, and are regulated during hypoxic stress, which is associated with reduced fat supply to the fetus. Methodology/Principal Findings Using cultured primary term human trophoblasts we found that FATP2, FATP4 and FATP6 were highly expressed in trophoblasts. Hypoxia enhanced the expression of trophoblastic FATP2 and reduced the expression of FATP4, with no change in FATP6. We also found that Fatp2 and Fatp4 are expressed in the mouse amnion and placenta, respectively. Mice deficient in Fatp2 or Fatp4 did not deviate from normal Mendelian distribution, with both embryos and placentas exhibiting normal weight and morphology, triglyceride content, and expression of genes related to fatty acid mobilization. Conclusions/Significance We conclude that even though hypoxia regulates the expression of FATP2 and FATP4 in human trophoblasts, mouse Fatp2 and Fatp4 are not essential for intrauterine fetal growth.

The Cytotrophoblast Layer of Human Chorionic Villi Becomes Thinner but Maintains Its Structural Integrity During Gestation

Abstract Chorionic villi in the human placenta serve as essential structures in fetomaternal exchanges. According to the embryology and placentology literature, during the first trimester, the cytotrophoblast (CTB) layer that is subjacent to the syncytiotrophoblast (STB) and supported by a basal lamina is nearly complete, but later, it becomes discontinuous. In the present study, we investigated the structural integrity of the CTB layer in the normal villous tree by advanced microscopy techniques using an antibody to hepatocyte growth factor (HGF) activator inhibitor type 1 (SPINT1), a potent inhibitor of HGF activators expressed exclusively on villous CTB. In full-term placenta, the cell surface of the CTB layer was spread over the basal lamina but was not interrupted. Morphometric analysis showed that throughout the villous tree, 80% of the continuity of the CTB layer of full-term placenta and 90% of that of first-trimester placenta were preserved. Gestation was accompanied by unique structural change in the basal domain of the trophoblast layer. The initially cuboidal-shaped CTB cells were transformed to flat cells with many cellular processes that, together with those of the adjacent STB, eventually covered the trophoblast basal lamina in a complex network of interdigitations. In addition, the expression levels of SPINT1, ST14, HGF, and MET mRNAs in the villous tree increased over the course of gestation. These results suggest that the structural integrity of the SPINT1-positive CTB layer may play an important role in villous differentiation and in maintenance of the villous tree via the HGF signaling system during gestation.

The Unique Expression and Function of miR-424 in Human Placental Trophoblasts

ABSTRACT Placental hypoperfusion causes cellular hypoxia and is associated with fetal growth restriction and preeclampsia. In response to hypoxia, the repertoire of genes expressed in placental trophoblasts changes, which influences key cellular processes such as differentiation and fusion. Diverse miRNAs were recently found to modulate the cellular response to hypoxia. Here we show that miR-424, which was previously shown to be upregulated by hypoxia in nontrophoblastic cell types, is uniquely downregulated in primary human trophoblasts by hypoxia or chemicals known to hinder cell differentiation. We also identify FGFR1 as a direct target of miR-424 in human trophoblasts. This effect is unique to miR-424 and is not seen with other members of this miRNA family that are expressed in trophoblasts, such as miR-15 and miR-16. Our findings establish a unique role for miR-424 during differentiation of human trophoblasts.

Sexually Dimorphic Expression of the Novel Germ Cell Antigen TEX101 During Mouse Gonad Development

Abstract Prospermatogonia, or gonocytes, are the cells that differentiate from primordial germ cells to the first mature type of spermatogonia in the developing testis. Although prospermatogonia play a central role in this stage (i.e., prespermatogenesis), the details regarding their characterization have not been fully elucidated. Recently, we identified a novel mouse testicular germ cell-specific antigen, TES101 reactive protein (TES101RP), in the adult mouse testis. The protein TES101RP is also designated as protein TEX101. In the present study, we investigated the expression of TEX101 on germ cells in developing mouse gonads using histochemical techniques (i.e., immunohistochemistry, BrdU labeling, and TUNEL staining) and reverse transcription-polymerase chain reaction. TEX101 appeared on germ cells in both male and female gonads after the pregonadal period. In the testis, TEX101 was expressed constitutively on surviving prospermatogonia during prespermatogenesis. After the initiation of spermatogenesis, the prospermatogonia differentiated into spermatogonia. TEX101 expression disappeared from the spermatogonia, but reappeared on spermatocytes and spermatids. In the ovary, TEX101 was expressed on germ cells until the start of folliculogenesis; TEX101 was not detected on oocytes that were surrounded by follicular cells. These findings indicate that TEX101 is a specific marker for both male and female germ cells during gonadal development. Because the on and off switching of TEX101 expression in germ cells almost parallels the kinetics of gametogenesis, TEX101 may play an important physiological role in germ cell development.