In this study, we observed the differentially miRNAs expression profiles in villus and decidua of RM patients compared to that of normal pregnant women by using deep sequencing analysis. A total of 32 miRNAs were screened out to be significantly up-regualted in decidua of RM patients, while nine miRNAs were identified to be differentially expressed in placental villi of RM patients, including four up-regulated (hsa-miR-191-5p, -24-3p, -100-5p and -146a-5p) and five down-regulated (hsa-miR-1, -372, -371a-5p, -376c-3p and -486-5p) miRNAs, compared to that of normal pregnant women. We further confirmed by qRT-PCR the up-regulation of hsa-miR-516-5p, -517a-3p, -519a-3p and -519d in decidua of RM patients, and hsa-miR-100-5p and -146a-5p in villus of RM patients; as well as the down-regulation of hsa-miR-1 and -372 in villus of RM patients. Furthermore, the increased expressions of Bcl-2, a predicted target of miR-1, and Pten, a predicted target of miR-372 were observed in villus of RM patients.
Recurrent miscarriage is presently difficult to be prevented and treated for the lack of knowledge on molecular mechanisms of this disease. Thus, in order to identify novel potential targets for the clinical diagnosis or treatment of RM, we tried to establish the miRNAs expression profiles in the decidua and villus from RM patients in this study. Although we have successfully recruited 18 RM patients and 15 normal pregnant women, and all of the 33 total RNA samples from decidual tissues were qualified for deep-sequencing analysis; however, unfortunately, only three samples of villus total RNA in RM group and four samples of villus total RNA in normal pregnancy group met the requests on RNA integrity for construction of cDNA libraries. We thought that this might be resulted from a relatively long storage time of villous tissue samples (more than 9 months), and it seems that, under the same storage conditions, human decidua tissue samples should be much more stable than villus tissue samples. The size of villus samples was so small that we wonder whether or not the results of the deep-sequencing analysis were reliable. Six pairs of decidua and villus from RM patients and normal pregnancy controls were used for validation of eight selected miRNAs by qRT-PCR, and the results of qPCR were extremely consistent with deep sequencing data, indicating that the deep sequencing data were reliable.
Since each miRNA has been predicted to have a broad range of target mRNAs based on the degree of sequence homology, these 32 miRNAs in decidua and 9 miRNAs in villus could undoubtedly be involved in different cellular functions, and we wonder whether or not these cellular functions would be important for establishment and maintenance of pregnancy. The GO ananlysis provides a comprehensive source for functional genomics, and is an effective bioinformatics research tool to unify the representation of gene and gene product, and creates evidence-supported annotations to describe the biological roles of individual genomic products (e.g. genes, proteins, ncRNAs, complexes) . Thus, we carried out the GO analysis for the 1533 predicted target genes (252 in decidua and 1281 in villus) of these differentially expressed miRNAs. It was revealed that, in decidua, these predicted target genes are mainly participated in cell death, apoptosis, cell proliferation and hormone stimulus, and the major KEGG pathways were cancer, ErbB signaling, focal adhesion, p53-signaling and apoptosis. Meanwhile, in villus, these target genes are mostly involved in the regulation of cell proliferation, apoptosis, blood vessel development and angiogenesis; and the major KEGG pathways analysis were apoptosis, p53-signaling and cell cycle. The pathologies that lead to RM must ultimately, either directly or indirectly, affect the interaction between the maternal endometrial (decidual) and trophoblastic tissue . According to the GO analysis results, the aberrant expression of these target genes in villus might affect trophoblast invasion and placentation, while aberrant expression of these target genes in decidua could adversely impact trophoblast invasion . More interestingly, given the p53-signaling pathway plays critical roles in apoptosis, and an advisable apoptosis of decidual cells, trophoblast cells and decidual immune cells are thought to be essential for the establishment and maintenance of pregnancy [21, 22], we speculated that the differentially expressed miRNAs (such as hsa-miR-519a, hsa-miR-517a, hsa-miR-205, hsa-miR-1, hsa-miR-372 and has-miR-486) which target apoptosis related gene, might involved in the regulation of p53-signaling and apoptosis might participate in the pathogenesis of RM.
Bcl-2, a key regulator of cell apoptosis, was identified as a predicted target of miR-1, a down-regulated miRNA in villus of RM patients. MiR-1 is a member of the muscle-specific miR-1 family (myomiRs) that currently consists of six members . It has previously been reported that, miR-1 inhibits cell proliferation but promotes cell differentiation, and is involved in tumorigenesis as a tumor suppressor. Its expression is abnormally down-regulated in several types of cancers, including lung, prostate, colorectal cancers and rhabdomyosarcoma . The abnormalities of Bcl-2 function have been implicated in many diseases including cancer, neurodegenerative disorders and autoimmune diseases . It was observed in this study that, the expression level of Bcl-2 protein was significantly increased in RM villus, suggesting an inhibitory effect of miR-1 on Bcl-2, and the miR-1/Bcl-2 signaling might be involved in the progression of RM. We also noticed that, two out of six bands of Bcl-2 were higher expressed in normal pregnancy than in RM, this might be resulted from the individual differences or/and the gestational week variation. As the cell proliferation and death of trophoblast are active during the early placental development , the heterogeneity of Bcl-2 expression might be observed in different human villus tissues, calling for the validation by a larger sample size. Meanwhile, it is confusing that, the expression level of Bcl-2 in trophoblast cells increased during placenta development , and a reduced expression of Bcl-2 was in association with pregnancy loss . This may be due to the multiple and complex effects of differentially expressed miRNAs on the microenvironment of decidua and villus of RM patients. In order to improve the clinical management of RM patients, great efforts have been made to search for biomarkers potentially could be used to predict adverse outcome of pregnancy at the early stage, and miRNAs present promising biomarker candidates of RM because the serum concentration of miRNA could be measured in maternal peripheral blood samples [1, 29]. MiR-1 has been identified as potential diagnostic biomarker for colorectal cancer . Thus, the serum concentrations of miR-1 in RM patients should be measured and compared with normal pregnant women to explore the possibility of their acting as biomarker candidate for RM.
Pten, another important factor involved in p53-signaling pathway and apoptosis, was predicted as a target gene of miR-372, which was down-regulated in villus of RM patients. MiR-372 belongs to the mir-371-372 gene cluster, which is located on chromosome 19q13.42 . Although the role of miR-372 itself in reproductive regulation has not been clear, it has been reported that, the expression level of miRNA-371 was increased from first trimester trophoblast cells to term trophoblast cells . Further evidence reinforced that miR-371 cluster was up-regulated in the first trimester placentas compared to the third trimester placentas, indicating that miR-371 might play critical roles in placental development . Meanwhile, it has been demonstrated that miR-372 regulated the cell cycle, apoptosis, invasion and proliferation in several types of human cancers , thus, it would be reasonable for us to speculate that miR-372 might also be involved in placental development. To study the correlation of miR-372 and Pten, we detected the expression of Pten in villus of RM patients, and found that the expression level of Pten was increased in RM villus, suggesting an inhibitory effect of miR-372 on Pten, and the miR-372/Pten signaling might be involved in the progression of RM. Consistently, it has been reported that, the villous expression of Pten was decreased as the pregnancy advanced, and an up-regulated expression of Pten was observed in early pregnancy loss [35, 36]. It has been shown that circulating serum mir-372 could serve as testicular germ cell cancer biomarker . So, miR-372 presents the another biomarker candidate for RM that is needed to be validated in larger size studies in the future.
In this study, we have screened out a number of intriguing miRNAs expression differences between RM and normal pregnancy. To further authenticate the association between these differentially expressed miRNAs and the pathologic process of RM, a case–control cohort of RM with a considerably large size (at least 100 pairs) should be established to collect the tissue and peripheral blood samples. The aberrant expressions of miRNAs might be linked to the abnormal cellular processes in RM patients, but the hypotheses about the roles of each specific miRNA in the progression of RM are needed to be further investigated. Also, pregnancy complications are notoriously hard to study in the laboratory because of the absence of appropriate models of human pregnancy. Furthermore, we found many changes in miRNAs expression potentially affecting many different processes, and it should to be note that, as the miscarried embryos were dead, necrosis or inflammation might have occurred in the dead embryonic tissues, whereas the embryonic tissue of induced abortion was fresh, therefore the differentially expressed miRNAs identified here might be the result of miscarriage but not the causes.