Objective
The aim of this study was to characterize the molecular mechanism of preeclampsia (PE) development through miR-155.
Study Design
PE and normal placentas were used to measure miR-155 and cysteine-rich protein 61 (CYR61) expression. CYR61 3′ untranslated region was validated as the target of miR-155 using in vitro transfections. miR-155 and CYR61 expression levels were assessed by real-time reverse transcription polymerase chain reaction or Western blot.
Results
An inverse correlation was found between miR-155 and CYR61 expression levels, with miR-155 up-regulated and CYR61 down-regulated in PE tissues. Luciferase assays and CYR61 transfection assays experimentally validated that miR-155 efficiently targets the 3′ untranslated region of CYR61 .
Conclusion
This study reported for the first time that overexpression of miR-155 contributes to PE development by targeting and down-regulating angiogenic regulating factor CYR61, leading to pathological alterations. This finding not only characterizes a new mechanism for the disease but also provides a potential therapeutic target.
Preeclampsia (PE), a syndrome among pregnant women characterized by elevated maternal blood pressure and proteinuria >20 weeks of gestation, is one of the leading causes of pregnancy-related maternal and fetal morbidity and mortality. It has long been found that the placenta plays an important role in the development of PE, in which poor placentation, shallow invasion, and abnormal angiogenesis are the main pathological manifestations. In fact, more and more studies have identified these pathological processes to the results of local oxidative stress characteristic in PE placenta. Among these studies, soluble fms-like tyrosine kinase 1 and endoglin have been widely accepted as one classical mechanism through which placental ischemia and oxidative stress impair normal local angiogenesis process. Other mechanisms believed to contribute to PE are also being intensively investigated. Recently, another stress gene, cysteine-rich protein 61 ( CYR61 ), was reported to be significantly down-regulated in PE patients.
CYR61, also known as CCN1, a member of the CCN family, is a secreted matrix protein expressed by nearly all types of vascular cells and trophoblasts and implicated in diverse cellular processes such as proliferation, migration, differentiation, and adhesion. CYR61 has been demonstrated to be one of the important early angiogenic factors during pregnancy. Targeted knockout of CYR61 gene in mice results in embryonic death due to placental vascular insufficiency and compromised vessel integrity. It was also found that the expression level of CYR61 in human PE placenta was significantly lower than that of the normal control. Therefore, we hypothesized that the down-regulated expression of CYR61 might contribute to PE development. Since CYR61 can induce the expression of vascular endothelial growth factor (VEGF), it is likely that a decreased CYR61 level would cause insufficient expression of VEGF in PE placenta. The interesting issue is what is responsible for the decreased CYR61 expression in PE placenta.
MicroRNAs (miRNAs) are a class of highly conserved short noncoding RNA molecules (about 22 nucleotides) that are key players in the regulation of gene expression mainly at the posttranscriptional level. A recent miRNA expression profile in PE placenta showed 7 different miRNAs in higher expression status, indicating their potential roles in PE development. Among these miRNAs, miR-155 has been defined as an inflammation-related miRNA as it can be significantly up-regulated by exogenous, eg, tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS), and it can regulate various inflammation-related nuclear factors (eg, activator protein [AP]-1, nuclear factor [NF]-kB) as a feedback antiinflammation mechanism. Here, we show that the overexpressed miR-155 specifically down-regulates CYR61 in PE placenta, which may be an important pathway in the development of PE.
Materials and Methods
Patients and placental collection
Placental samples, delivered at 36-40 weeks, were collected from 20 severe PE women undergoing cesarean section in the Department of Obstetrics and Gynecology of Drum Tower Clinical Medical College, Nanjing Medical University, from March 2005 through April 2008. Placental tissues from 20 normotensive pregnancies with gestational age-matched groups also undergoing selective cesarean section were collected as controls. Severe PE was defined as having either severe hypertension (systolic blood pressure ≥160 mm Hg or diastolic blood pressure ≥110 mm Hg) or severe proteinuria (urinary protein excretion ≥2.5 g per 24 hours). All patients had normal platelet counts, normal functioning livers and kidneys, and normal fetal weights. The level of blood pressure of all patients returned to normal and symptoms of proteinuria disappeared postpartum 6 weeks. All placental samples were frozen within a half hour after delivery.
Patients with chronic hypertension, renal disease, collagen vascular disease, premature rupture of membrane, and other pregnancy complications such as fetal anomalies or chromosomal abnormalities were excluded from this study. Written consent was obtained from the patients before surgery. The Ethics Committee of Drum Tower Hospital approved the consent forms and procedures necessary to utilize the tissues. For RNA and protein isolation, only chorionic tissue from the central part of the placental maternal phase was collected. After cleansing in phosphate buffered saline, the tissues were frozen in liquid nitrogen and stored at –80°C until extraction of RNA and protein.
Cell culture and transfection
First trimester extravellous trophoblast cell line (HTR-8/SVneo) cells, human placental cell line derived from a choriocarcinoma (BeWo cells), and human embryonic kidney (HEK)-293T cells were grown in RPMI-1640, Ham’s F12, and Dulbecco’s modified Eagle media, respectively, all supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 100 U/mL penicillin, and 100 μg/mL (HyClone). All cells were maintained in standard culture conditions of 5% carbon dioxide at 37°C.
Real-time RT-polymerase chain reaction
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. A total of 1 μg of total RNA from placental samples was used for reverse transcription. To detect miR-155 expression, complementary DNA was synthesized using a miR-155-specific stem-loop primer: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCCCTA-3′. Quantitative polymerase chain reaction (PCR) analysis used the following primers: forward, 5′-CTGTTAATGCTAATCGTGATAG-3′; reverse, 5′-GCAGGGTCCGAGGT-3′. Small unclear RNA U6 (U6) small RNA was used as internal control with the following primers: forward, 5′-CTCGCTTCGGCAGCACA-3′; reverse, 5′-AACGCTTCACGAATTTGCG T-3′. The reverse primer of U6 was used for reverse transcription. CYR61 was analyzed using random primer in reverse transcription, and PCR primers as follows: forward, 5′-ACTTCATGGTCCCAGTGCTC-3′; reverse, 5′-AAATCCGGGTTTCTTT CACA-3′. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was coamplified as internal control in each experiment with the following primers: forward, 5′-AACGGATTTGGTCGTATTG-3′; reverse, 5′-GGAAGAT GGTGATGGGATT-3′. The expression of miR-155 and CYR61 , relative to the control, was detected using an SYBR green-based real-time quantitative PCR assay (Toyobo Co., LDT, Osaka, Japan) as previously described.
Western blot analysis
Protein extracts were prepared from placental tissues and cells using lysis buffer supplemented with EDTA-free complete protease inhibitors (Roche, Penzberg, Germany). Tissue or cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The primary antibodies used were goat antihuman polyclonal CYR61 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit antihuman tubulin antibody (Chemicon, Hampshire, UK). Protein bands were visualized via enhanced chemiluminescence detection reagents (Amersham Biosciences, Buckinghamshire, UK).
Plasmids and constructs
miRNA expression constructs
A 409–base pair DNA fragment encompassing the has-mir-155 gene was PCR amplified from genomic DNA using primers: 5′-CCTTTCAGATTTACTA TATGCTG-3′ and 5′-CCTTACTTTCAAAACTTAGTTTA-3′. The PCR product was cloned into pEGFP-C1 (Clontech) by Bgl II and Hind III (Promega, Shanghai, China) digestion.
Luciferase reporter vector
Sequences of the complete 3′ untranslated region (UTR) of human CYR61 (nt 2634-3298, accession no. NM_001554) were amplified using PCR and then introduced downstream of the Luciferase reporter gene in the Xba I (a DNA restriction endonuclease) cloning sites of the pGL3 promoter vector (Promega).
CYR61 expression vector
Full-length CYR61 complementary DNAs, which encompassed the entire open reading frame with or without the 3’UTR, were amplified using PCR and the products were inserted into the eukaryotic expression pCS2 vector (Clontech) with 6Myc (a protein coding by Myc gene) tags. All of the constructs were sequenced to ensure authenticity.
Luciferase assays
Luciferase (an oxidative enzyme used for reporter) assays were carried out with the Dual-Luciferase Reporter Assay System (Promega). To check for transfection efficiency, pRL-SV40 (Promega), a control plasmid, was used. Transfection was performed using Lipofectamine 2000 (Invitrogen). HEK-293T cells were cotransfected with Luciferase reporter plasmids (40 ng) and miR-155 expression vector (1-2 μg). Luciferase activities were measured via Luminometer (Promega) 36 hours after transfection.
Cell migration assay
Wound-healing experiments were performed as described previously. HTR-8/SVneo cells were transfected with 2.5 μg of miR-155 plasmid. When the cells grew up to 90% confluence, a single wound was made in the center of the cellular monolayer and cell debris was removed by washing and a reticule mark was made on the bottom of every dish. After 9, 24, and 32 hours of incubation, the wound closure areas were visualized under an inverted microscope. The widths of the wounds were measured in 5 different locations averaged over 3 fields of view per well from a 3-4 replicate set of samples.
Measurement of free VEGF
Conditioned media were collected and centrifuged to remove cellular debris. VEGF165 secretion was detected by a human VEGF colorimetric enzyme-linked immunosorbent assay kit (Shenggong Biotech Co., Ltd, Shanghai, China) according to the manufacturer’s instructions. HTR-8/SVneo cells were plated in 24-well plates (5×10 5 cells/well) in culture medium with 1% FBS containing various concentrations of CYR61 (20-80 ng/mL) and incubated for 24 hours at 37°C with 5% carbon dioxide atmosphere. Sample or standard VEGF165 was added to each well, previously coated with human monoclonal anti-VEGF antibody. After 1.5 hours of incubation, wells were washed and incubated with an enzyme-linked polyclonal anti-VEGF antibody. Tetramethylbenzidine substrate solution was added to each well and the color developed in proportion to the amount of VEGF bound in the initial step. The plate was scanned at a wavelength of 450 nm.
Statistical analysis
All experiments were repeated at least 3 times. Results were expressed as mean ± SEM or mean ± SD. For nonparametric independent 2-group comparisons, the data were analyzed for statistical significance by 2-tailed Student t test or the Mann-Whitney test when data did not follow the Gaussian distribution with the program SPSS 11 (SPSS, Inc, Chicago, IL). Analysis of variance was used for comparing the data from 3 groups. Differences with a P value < .05 were considered statistically significant.
Results
miR-155 was up-regulated whereas CYR61 was down-regulated in severe PE placenta
We examined the differences in miR-155 and CYR61 expression levels between the control and PE placentas by quantitative real time reverse transcriptase-polymerase chain reaction (qRT-PCR) ( Figure 1 , A). The expression of miR-155 in severe PE placenta is 2.58-fold higher in comparison to the control placentas ( P < .001). The transcript level of CYR61 was significantly lower in severe PE placenta compared to the control group ( Figure 1 , A) ( P < .01). Expression of miR-155 and CYR61 was negatively correlated (r = –0.66; P < .01). Western blots were also used to investigate the expression of CYR61 protein in severe PE and normal placentas. Protein level of CYR61 decreased in PE placenta, similar to the results obtained from quantitative RT-PCR ( Figure 1 , B). These findings suggest an inverse relationship between miR-155 and CYR61 expression, and an increased miR-155 expression associated with the down-regulation of CYR61 in PE placenta.
