MicroRNA expression profiles of trophoblastic cells
Introduction
Since the discovery of the first microRNA lin-4 in 1993 [1], the study of microRNAs (miRNAs) has generated great interest due to their vast potential in the regulation of protein-coding genes. MiRNAs are highly conserved sequences of single-stranded RNA (∼19–22 nt) which repress gene expression by a mechanism involving the RNA interference pathway [2]. Depending on the complementary grade between the miRNA and its mRNA target, the pathway results in inhibition of translation, or cleavage of the target mRNA, when partially or fully complimentary, respectively [3]. This characteristic allows targeting of several genes simultaneously and therefore, it is expected that 30% of the human genome may be regulated by miRNAs [4].
Remarkably, miRNA genes are frequently located at fragile sites and cancer-related genomic regions [5], and tend to be organized into clusters suggesting that miRNAs belonging to a same cluster are likely to have similar functions and be regulated by the same promoter and in the same transcriptional orientation [6], [7]. The analysis of the miRNA signature (miRNome) in normal human tissues revealed some universally expressed miRNAs but also several groups of miRNAs exclusively or preferentially expressed in a tissue-specific manner [8]. Likewise, the miRNA expression signature is frequently altered in cancer [9], [10], and can be used to distinguish between cancer and normal tissues [11], [12] or even to identify poorly differentiated tumors [13].
Recent reports have described two large miRNA clusters expressed in placenta: The chromosome 19 miRNA cluster (C19MC), which maps to chromosome 19q13.41 and comprises 54 predicted miRNAs, 43 of which have been already cloned and sequenced (reviewed in Ref. [3]); and the C14MC located in the 14q32 domain and which contains 34 miRNAs [14]. C19MC is exclusively found in primates while C14MC appears to be conserved among all eutherian species [15]. Both are encoded within imprinted domains: C19MC is expressed from the paternally inherited chromosome whilst C14MC is expressed from the maternally inherited chromosome [14], [15]. Imprinted genes are known to be involved in human embryonic development and to play important roles in the regulation of cellular differentiation and fate [16].
The signature of miRNAs in trophoblast cells is largely unknown. Initial investigations identified some miRNAs specifically expressed in placenta tissue and released into maternal plasma [17]. Further analyses of the expression of small RNAs in placenta by small RNA library sequencing confirmed that most placenta-specific miRNAs were linked to the C19MC cluster and demonstrated that villous trophoblast cells express such miRNAs [18]. Recent reports have quantitatively analyzed the expression of up to 820 miRNAs in placenta tissue samples collected in the first or third trimester [19], [20]. Interestingly, the concentration of pregnancy-associated miRNAs increased throughout pregnancy [19] and was altered in placentas from pregnancies with preeclampsia or preterm labor [20]. These results suggest miRNAs as potential serum markers for the normal function of the placenta. However, little is known about the cellular origin of these placental miRNAs.
The study of the miRNome of isolated trophoblast cells is restricted by the limitations associated with primary cells such as relatively low number of isolated cells, short lifespan or lack of proliferation in vitro [21]. Therefore, during the last three decades, several trophoblastic cell lines have been established attempting to resemble primary trophoblasts and avoiding the limitations of isolation. Two main methodologies have been used: Introduction of the gene encoding simian virus 40 large T (sv40T) antigen [22] or establishment of human choriocarcinoma cell lines [23]. Therefore, the different genetic background and the methods used for immortalization should be taken into consideration for interpretation and discussion of results obtained from the respective cell line.
To our knowledge, there is a very limited number of publications on miRNA expression profiles in trophoblastic cell lines, or their comparison with primary isolated trophoblast cells [24]. To overcome this lack of knowledge, we assessed the miRNA expression patterns of four cell lines and isolated trophoblast cells from first and third trimester placentae. We included the immortalized human first trimester trophoblast cell line HTR-8/SVneo [22], the choriocarcinoma cell line JEG-3 and the two hybrids cell lines, ACH-3P and AC1-M59, which resulted of fusion of the AC-1 choriocarcinoma cell line with first and third trimester isolated trophoblast cells, respectively [23], [25].
For confirmation of array results, expression of 9 individual miRNAs was assessed by qPCR. Instead of random choice, we selected five miRNAs representing the C19MC (miR-518a-5p and miR-519e), the C14MC (miR-411 and miR-539) and the miR-371-3 cluster (miR-373); as well as four miRNAs that have been previously described to be expressed in placenta or tumors: miR-9, miR-21, miR-141, and let-7g [3], [14], [17], [18], [26].
By fingerprinting miRNA gene expression we aimed to contribute to better understanding of differences and resemblances of these frequently used cell lines and primary trophoblast cells. Concluding from our observations, the above mentioned cluster C14MC and C19MC may play key roles in regulating their phenotypical and functional diversity.
Section snippets
Cell lines
Four cell lines were investigated in this study: the immortalized extravillous first trimester trophoblast cell line HTR-8/SVneo (kind gift from CH Graham, Kingston Canada) [22], the choriocarcinoma cell line JEG-3 (DSMZ, Braunschweig, Germany), and two hybrids of AC-1 choriocarcinoma cells (derived from JEG-3 by mutagenesis) with human first and third trimester trophoblast cells, ACH-3P and AC1-M59 cells, respectively (kind gift from G Desoye, Graz, Austria) [23], [25], [27].
Cell culture
Cell cultures were
Expression profiles of miRNAs in isolated trophoblast cells and cell lines
We assessed the complete (miRBase v13.0) microRNA expression profile of the four trophoblastic cell lines HTR-8/SVneo, JEG-3, AC1-M59 and ACH-3P as well as that of trophoblast cells isolated from first and third trimester placentae (complete array data are accessible trough GEO Series accession number GSE32346; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32346).
The detection of the total of 762 miRNAs was performed on two different array cards A and B containing 381 miRNAs each. Around
Expression of selected miRNAs confirmed by qPCR
For confirmation of array results, we analyzed by qPCR individually the expression of 2 miRNAs representing C14MC (miR-411 and miR-539), 2 miRNAs representing C19MC (miR-519e and miR-518a-5p) and miR-373, a member of the miR-371-3 cluster. In agreement with the arrays, the expression of the miRNAs belonging to C19MC increases from first to third trimester, while the expression of members of C14MC decreases.
As observed in the arrays, HTR-8/SVneo cells differ significantly in the expression of
Discussion
Recent studies indicate that miRNA expression signatures may be useful for the characterization and prediction of cancer [13], but the investigation of their physiological role in pregnancy and their possible involvement in pregnancy disorders are still incipient. The expression of individual miRNAs as well as miRNA clusters is regulated in a tissue-specific manner. Within its complex organ specific miRNA signature, the placenta expresses the two microRNA clusters C19MC and C14MC. Both are
Conclusion
Our study provides a comprehensive encyclopedia of the microRNA expression profile of four cell lines widely used as models of trophoblast cells, and their comparison with primary isolated trophoblast cells from first and third trimester. In regard to the current international discussion about the nature of HTR-8/SVneo cells, this study confirms their close relationship with primary trophoblast cells, but it also exhibits large inequalities. Simultaneously, it demonstrates that also
Acknowledgment
The project has been supported by the German Research Foundation (DFG, project Ma1550/7-1). DMMP had a Ph.D. grant from the regional graduate academy of the Friedrich-Schiller-University Jena, Germany. The Boehringer Ingelheim Fonds provided her grants for starting in Jena and learning methods at the “Istituto Clinico Humanitas”, Milan, Italy. WC receives a Ph.D. grant from the German Academic Exchange Service (DAAD).
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