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Elevated microRNA-520g in pre-eclampsia inhibits migration and invasion of trophoblasts
Placenta 51 (2017) 70e75
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Elevated microRNA-520g in pre-eclampsia inhibits migration and invasion of trophoblasts Liansheng Jiang a, Anxiong Long a, Longyi Tan a, Mao Hong a, Jingjing Wu b, Leiming Cai a, Qian Li a, * a b
Department of Clinical Laboratory, Shanghai First People's Hospital Baoshan Branch, Shanghai, PR China Department of Obstetrics and Gynecology, Shanghai First People's Hospital Baoshan Branch, Shanghai, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 December 2016 Received in revised form 30 January 2017 Accepted 1 February 2017
Introduction: Pre-eclampsia (PE) is a common and severe obstetric complication. MicroRNAs (miRs) have emerged as molecules that are associated with the disease. Methods: Quantitative reverse transcription PCR (RT-qPCR) was used for serum miR-520g characterization from 19 severe pre-eclamptic and 19 normal pregnancies. In situ hybridation was adopted to localize microRNA-520g (miR-520g). Migration and invasion of HTR-8/SVneo cells were evaluated after miR-520g mimic treatment with transwell system. MiR-520g target gene was verified in luciferase reporter system. Results: The expression of serum miR-520g displayed an upward trend as pregnancies progress. At firsttrimester, miR-520g in pre-eclampsia was significantly higher than that in the control, but no significant differences were found in the second and last trimesters. MiR-520g localized in cytoplasm of early trimester placental trophoblasts. The migration and invasion of HTR8/SVneo were inhibited by miR-520g mimic treatment. Matrix metalloproteinase 2 (MMP2) was verified as a direct target of miR-520g. Conclusions: Elevated maternal serum level of miR-520g level in first trimester was detected in patients with severe PE. By suppressing the migration and invasion of trophoblast via at least partial inhibition of MMP2 translation inhibition, miR-520g might play a role in the defective spiral artery remodeling, and thus contribute to pre-eclampsia pathophysiology. © 2017 Elsevier Ltd. All rights reserved.
Keywords: microRNA-520g Pre-eclampsia Trophoblast HTR8/SVneo Matrix metalloproteinase 2 Biomarker
1. Introduction Pre-eclampsia (PE) is a complex multi-system involved pregnancy syndrome that is characterized by hypertension and significant proteinuria after 20th week of pregnancy [1]. PE is one of the main diseases that threaten maternal and newborn health [1,2]. Delivery of the placenta instead of fetus is the sole effective treatment for PE. Thus the defective placenta formed before 20th gestation week due to impaired trophoblast invasion and aberrant spiral artery remodeling is regarded as the key event in the PE pathophysiology [3]. During the first 20 weeks of gestation, endothelial cells and beneath smooth muscle cells in uterine spiral arteries are lost and replaced by extravillous trophoblast cells. A number of mechanisms may account for undesirable spiral remodeling, including deficiency of extravillous trophoblast and
* Corresponding author. E-mail address: [email protected] (Q. Li). http://dx.doi.org/10.1016/j.placenta.2017.02.001 0143-4004/© 2017 Elsevier Ltd. All rights reserved.
decreased trophoblast invasion [4]. MicroRNAs (miRs) are endogenously expressed small (19e25 nucleotides) non-coding RNAs that regulate the expression of nearly 30% of all genes via posttranscriptional repression [5]. Accumulating evidence indicates that deregulated miRNAs is closely related to PE [6e12]. Our previous study has unveiled a panel of miRNAs that might serve as potential biomarker for predicting PE since the second trimester [13]. Further quantification of placenta-specific miR-520g in maternal serum was conducted. We found abnormal expression of miR-520g during first trimester but not in second or third trimester. The coincidence of the time between deregulated miR-520g and spiral arterial remodeling promoted our hypothesis that miR-520g might play an important role during placentation. In the current study, we quantified the expression of miR-520g in maternal serum from preeclamptic and normal pregnancies, and further examined the impact of miR-520g mimic on the human trophoblast cell line, HTR8/SVneo. We found that expression of
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miR-520g was upregulated during the first to early second trimesters in PE, and that miR-520g mimic inhibited HTR8/SVneo migration and invasion. Mechanistically, matrix metalloproteinase (MMP)-2 was identified as a direct target of miR-520g that may account for the observed phenotypes in HTR8/Svneo cells. 2. Materials and methods 2.1. Patient enrollment and sample processing This study was approved by the local ethical and internal review board (IRB) of Shanghai First People's Hospital Baoshan Branch. Peripheral blood samples were obtained from 1625 Chinese pregnancies who received routine obstetric examination in our hospital from Jan 2014 to Jun 2015. The first trimester (8e10 weeks) placenta tissues were obtained from selective legal abortion cases in our hospital. Informed consent was obtained from all the subjects. A total of 19 singleton pregnancies complicated with preeclampsia which had complete medical records from first trimester to delivery was found retrospectively, then 19 healthy pregnant women without complications in our serum bank were selected as control based on similar maternal age (±1 year) at delivery and the same gestational week of blood sampling. Severe PE were defined as systolic pressure >140 mmHg or diastolic blood pressure >90 mmHg on two different occasions with proteinuria (>0.3 g/ 24 h) after 20th gestation week. Sera were collected from different trimester pregnancies after at least 8 h fasting and kept in 80 C until used. Placental tissues were fixed in 4% paraformaldehyde overnight at 4 C for in situ hybridization. 2.2. In situ hybridization Formalin-fixed paraffin-embedded (FFPE) placenta tissue slices were used for miRNA in situ hybridization with miRCURY LNA MicroRNA ISH Optimization Kit (Exiqon) and double digoxigeninlabeled LNA probes for miR-520g (50 nM), scrambled probe (50 nM) and U6 snRNA probe (1 nM). Following the manufacturer's protocol, the hybridization was conducted at 55 C for 1 h. All the sections were counter stained with Nuclear Fast Red (Beijing Dingguo Changsheng biotechnology) for 1 min. 2.3. Cell culture and treatment The human HTR-8/SVneo trophoblast cell line was a generous gift from Dr Charles H. Granham. HTR-8/SVneo cells were maintained in RPMI-1640 (Gibco) medium supplemented with 10% fetal bovine serum on a 6-well plate. When the cells reached 60% confluence, double-stranded miR-520g mimic (Qiagen), or scramble control (Qiagen) at a final concentration of 20 nM were introduced into the cells using Lipofectamine RNAiMAX Reagent (Invitrogen). 6 h post-transfection, HTR-8/SVneo cells were re-fed with fresh RPMI-1640 with 10% FCS for subsequent experiments. 2.4. RNA extraction and real-time quantitative RT-PCR analysis Total RNAs were extracted from 200 ml serum using the miRNeasy Serum/Plasma Kit (Qiagen) as following the manufacturer's instructions. Synthetic spike-in control Cel_miR-39_1(Qiagen) is used to monitor miRNA purification and amplification. The RNAs were dissolved in 14 ml RNase-free water. Total RNAs were extracted from cells with TRIzol reagent (Ambion). MiR real-time quantitative RT-PCR was done as previously described. Briefly, total RNAs (4 ml) were reverse transcribed using the miScript ⅡRT Kit (Qiagen) in a 20 ml total volume reaction system containing miScript Primer Assay (Qiagen) on PCR
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system(Bioer). Then real-time PCR was performed on 7500 RealTime PCR System (Applied Biosystems) using miScript SYBR Green PCR kit (Qiagen) in a total reaction volume of 20 ml containing 2 ml cDNA(200 times diluted). The expression levels of miRs were determined utilizing the comparative CT method relative to cel_miR-39_1. For mRNA gene expression, cDNA was synthesized using PrimeScript RT reagent kit (Takara), and real time qPCR was performed with SYBR Fast qPCR Mix (Takara). Ribosomal protein L19 was amplified as an endogenous control [12]. All PCR reactions were run in triplicate. The expression levels of miRs and mRNAs were expressed as fold changes by the 2 -△△Ct method. PCR primers are listed in Table S1.
2.5. Transwell migration and matrigel invasion assay HTR-8/SVneo cells treated with miR-520g mimic or negative control were seeded into the upper chamber of a transwell (24-well plate, 8.0-mm pore size, Corning) coated with or without matrigel. Cells remained in the upper chamber were removed using a cotton swab, the membranes were inverted and stained with crystal violet and quantified.
2.6. Western blot analysis HTR-8/SVneo cells were lysed 24 h after miR-520g mimic or negative control transfection and quantified with a BCA kit (Thermo Fisher Scientific). 20 mg proteins were resolved by 8% SDSPAGE and transferred to a polyvinylidene fluoride membrane using standard methods. The membranes were incubated with the primary anti-MMP2 antibody (Abcam) overnight at 4 C, then incubated with the secondary horseradish peroxidase (HRP) conjugated antibodies and visualized using Pierce Western Blotting ECL substrate (Thermo Fisher Scientific).
2.7. Generation of luciferase reporter assay The 30 -UTR of the MMP2 (1003 bp) containing predicted binding sites of miR-520g (UGAAAC) were amplified by PCR from human genomic DNA and inserted into pmiR-RB-Report vector (Ribobio) downstream of the renilla luciferase. A further 2 sites mutant MMP2 30 -UTR (260ACTTTGA266 to 260TGAAACT266, 516CACTTTG522 to 516GTGAAAC522) vector was constructed and validated by sequencing. HEK 293 T cells were transfected with 250 ng/well pmiR-MMP2WT or pmiR-MMPMut with 50 nM miR-520g mimic or scramble control. Lipofectamine 2000 (Invitrogen) and OPTI-MEN were used for transfection. Luciferase activities were measured 48 h after transfection using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to renilla luciferase activity. MMP2WT and MMPMut PCR primers are listed in Table S2.
2.8. Statistical analysis Expression levels of serum miRs were compared using KruskalWallis H test. Because the Bonferroni correction was used to solve the problem of multiple comparison in serum miRs, the significance level was set at P < 0.01. For other comparison, a Student's ttest was used. A P value of less than 0.05 was considered statistically significant. All statistical analysis was performed with the SPSS 20.0 software.
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Table 1 Demographic features of the PE patients and healthy controls.
Materal age (y) Gestational days at delivery (d) Neonate birthweight (g) Pregestation BMI (kg/m2) SBP (mm/Hg) DBP (mm/Hg) 24 h proteinuria (g)
PE (n ¼ 19)
CONTROL (n ¼ 19)
p value
31.3 ± 5.8 253.2 ± 7.2 2865.0 ± 730.9 24.9 ± 4.1 154.4 ± 11.6 103.6 ± 8.6 3.8 ± 1.2
30.9 ± 5.6 276.1 ± 4.8 3453.9 ± 311.2 20.3 ± 1.4 117.4 ± 8.8 73.2 ± 7.8 none
0.89 <0.05 <0.05 <0.05 <0.05 <0.05
3. Results 3.1. MiR-520g was aberrantly upregulated in the first trimester maternal serum in PE The demographic data of the case and control were shown in Table 1. Except for age, all the other factors, including the gestation days at delivery, neonate birthweight, pregestation BMI, 24-h proteinuria, systolic and diastolic blood pressure were significantly different between these two groups. With pregnancy progressing, the expression of serum miR-520g showed an increasing trend in both pre-eclamptic and health pregnancy, but the differences between second and third trimester in health pregnancy or between first and second in pre-eclamptic are not statistically significant (Fig. 1A). MiR-520g of first-trimester in pre-eclamptic group was
significantly higher than that in the control group (Fig. 1B), no statistically significant difference was found in the second and third-trimester between the two groups (Fig. 1C and D). 3.2. MiR-520g located in the cytoplasm of placental trophoblasts Human placenta is heterogeneous and consists of multiple cells. To clarify the source of miR-520g, we performed in situ hybridization using first trimester placenta tissues. MiR-520g was found in cytoplasm of trophoblasts from first trimester placenta (Fig. 2A and B). Both cytotrophoblasts (CTB) and syncytiotrophoblasts (STB) expressed miR-520g. No obvious difference in miR-520g expression was observed between CTB and STB. MiR-520g was not detected in either endothelial cells or mesenchymal cells. U6 and scramble miR were used as positive and negative control respectively (Fig. 2C and D). This result further verified miR-520g as a placenta-specific microRNA. 3.3. MiR-520g inhibited migration and invasion of HTR8/SVneo As HTR8/SVneo is a widely-used first trimester trophoblast cell line, we used it as a model to study the role of miR-520g in early pregnancy. Since HTR8/SVneo cells did not express high level of miR-520g, we transfected HTR8/Svneo cells with miR-520g mimic instead of using inhibitor. We found that both migration and invasion of HTR8/SVneo cells were inhibited following miR-520g mimic transfection (Fig. 3). MiR-520g mimic decreased the
Fig. 1. Relative expression of miR-520g in maternal serum from pre-eclamptic pregnancies and normal pregnancies. A. Box plot (from minimum to maximum) of relative level of serum miR-520g in three different gestation periods. B-D. Box plot of relative level of serum miR-520g in gestation week 10e14 (B) or gestation week 20e24 (C) or gestation week 30e34 (D) from pre-eclamptic pregnancies and normal pregnancies. PE means pre-eclampsia, CON means control, W means gestation week.
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Fig. 2. Location of miR-520g in first trimester placenta tissue. A-B. miR-520g staining in placenta tissue. C. U6 miR staining as a positive control in placenta tissue. D. Scramble control staining as a negative control in placenta tissue. fv, floating villi; st, syncytiotrophoblast; ct, cytotrophoblast. Scale bar, 50 mm. Blue color indicates the miRNA.
Fig. 3. The effect of miR-520g on HTR-8/Svneo migration (A) and invasion (B). ** means p < 0.01.
migration of HTR8/SVneo cells by 43% compared to the corresponding negative control (Fig. 3A). Meanwhile, miR-520g mimic suppressed the invasion of HTR8/SVneo cells by 45% compared to the corresponding negative control (Fig. 3B).
3.4. Validation of MMP2 as a direct target of miR-520g To find molecules responsible for miR-520g-related effects on cell migration and invasion, we used TargetScan (http://www. targetscan.org/vert_71/) to predict the potential targets of miR520g. MMP2, which is commonly involved in cell invasion, stood out as a promising candidate. We therefore focused on examining the relations between miR-520g and MMP2. We found that miR520g mimic transfection did not decrease MMP2 mRNA but
Fig. 4. Validation of MMP-2 as a direct target of miR-520g in luciferase assay. Vectors with wild type or mutated MMP2 30 -UTR were constructed and cotransfected with miR-520g mimic or scramble control. ** means p < 0.01.
downregulated MMP2 protein expression in HTR8/Svneo cells. To validate whether MMP2 was a direct target of miR-210, we constructed vectors which contained the wild type or mutated miR520g seeding site MMP2 30 -UTR region at the downstream of the reporter gene, renila luciferase. We then co-transfected HEK 293T cells with miR-520g mimic and vectors. MiR-520g mimic reduced the relative luciferase activity of the wide type MMP2 vector but not of the mutated MMP2 vector (Fig. 4). To examine the effect of miR-520g on HTR8/Svneo MMP-2 expression, we extracted RNA and protein from HTR8/Svneo after miR-520g mimic treatment. RTqPCR revealed a noticeable but not significant reduction of MMP2 mRNA (Fig. 5A), while western blotting showed lower pro-MMP2
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Fig. 5. The effect of miR-520g on MMP-2 expression. A. MMP-2 mRNA relative expression in HTR-8/Svneo after miR-520g mimic treatment as determined by RT-qPCR. B. Pro-MMP2 protein expression in HTR-8/Svneo after miR-520g mimic treatment as determined by western blot. C. Quantification analysis of MMP2 protein expression in HTR-8/Svneo after miR-520g mimic treatment.
protein expression after miR-520g mimic transfection (Fig. 5B and C).
4. Discussion MiR-520g belongs to the chromosome 19 miRNA cluster (C19MC) that is barely detectable in tissues other than placenta [14]. C19MC expression including miR-520g is upregulated significantly from first to third trimester trophoblast [15,16]. MiRNAs originated from placenta might be released into the maternal circulation in the form of exosomes [17]. Excessive apoptosis of trophoblasts related to pregnancy complications such as preeclampsia might induce a rise of miRNAs in the circulation [18]. Our miR-520g in situ hybridization showed cytoplasmic localization in trophoblast, which is consistent with previous results. The result that miR-520g expression in maternal circulation increased with gestation progression is reasonable, considering the expanding trophoblasts number, and is consistent with an earlier report [15]. Intriguingly, we found elevated expression of miR-520g in first trimester of severe pre-eclamptic pregnancy. This discovery, to our knowledge, represents the first report of upregulation of miR-520g expression in PE first trimester. This pilot study The timing of the aberrant rise of miR-520g in first trimester coincides with the time window of spiral artery remodeling. We speculate that the elevation of miR-520g in first trimester in PE might due to aberrant excessive trophoblast apoptosis and might play a role in trophoblast invasion or migration. Spiral artery remodeling starts from 8th to 10th gestation week in human pregnancy, whose hallmark is the invasion of the uterine spiral arteries by the extravillous trophoblast [3,4,19]. Inadequate spiral artery remodeling in the first trimester is widely considered as the first major step to drive PE. Spiral artery remodeling is a large network that is tightly and delicately regulated by a number of factors, including trophoblasts and endothelial cells. Given the crucial role of trophoblast in pre-eclampsia and its first trimester extravillous trophoblast origin [20], we chose to use HTR-8/SVneo cells, an immortalized cell line that mimics trophoblast cells as a
model cellular system in our study. We found that HTR-8/SVneo cells expressed very low amount of miR-520g and that miR-520g mimic treatment inhibited the migration and invasion of HTR-8/ SVneo cells. In addition to its placental origin, miR-520g has also been reported to be an oncogenic miRNA [21,22] and be upregulated in hepatocellular carcinoma (HCC) tissues and highly invasive HCC cell lines [23,24]. Kan H et al. showed that miR-520g facilitated HCC cell migration and invasion by inducing epithelialmesenchymal transition (EMT) via SMAD7 targeting, as reflected in the increase of E-cadherin and reduction of vimentin) [24]. Although MMPs have been repeatedly associated with tumor metastasis, the consensus view is, in general, that MMPs are not produced by epithelial cells but by the surrounding stromal cells [25]. Hence, the mechanisms involved in migration/invasion of trophoblasts and HCC cells might be different despite of some similarities between trophoblasts and tumor cells. The difference of the role of miR-520g in different cells indicates that cell type dependent mechanisms might be involved in regulating different pathways under varying disease contexts. Through target prediction analysis, we focused on MMP2 due to its 30 UTR seed binding sites with miR-520g and its well-known role in trophoblast cell invasion [26]. MMP2 is a zinc metalloendopeptidase secreted from cells as a zymogen (pro-MMP2). Our luciferase report assays clearly identified MMP2 as a direct target of miR-520g. PCR and western blot results showed that miR520g mimic transfection did not significantly reduce MMP2 mRNA level but downregulated pro-MMP2 protein expression in HTR8/ Svneo, which indicated miR-520g's posttranscriptional regulation on MMP2 might via translation inhibition rather than through mRNA degradation. A decrease in the MMP2 might reduce the invasiveness of trophoblast, which contributes to the pathogenesis of PE [27]. Tsai et al. reported miR-520g repressed MMP2 mRNA and propeptide expression in a dose dependent manner in primary endometrial stromal cells [28]. The inconsistency between the two studies is probably due to the use of different cell lines and different miR-520g mimic concentrations. Considering that the inhibition MMP2 by miR-520g may be reflected in circulation, we detected
L. Jiang et al. / Placenta 51 (2017) 70e75
plasma MMP-2 from first trimester pregnancy using ELISA. Surprisingly we found a relatively higher MMP2 in pre-eclamptic group when compared with healthy group (225.0 ± 34.2 versus 203.5 ± 24.5 ng/ml, n ¼ 10, P ¼ 0.04). This result was consistent with Myers et al.’, in which they reported elevation of MMP2 in the plasma of pre-eclamptic women as early as 22 gestation week and before delivery [29]. However, a reduction of MMP2 in preeclamptic placenta was also reported in two other studies [27,30]. This discrepancy might arise from the varying degrees of endothelial activation in PE, which is expected to cause the elevation of MMP2 in circulation [31]. To conclude, we reported here that elevated maternal serum miR-520g level in first trimester was detected in patients with PE. We further provided evidence for the role of miR-520g in PE development by targeting MMP2. Given the limited sample size of the current study, we are full aware of the necessity to include more patients in future studies to draw a definite conclusion. Further investigations on the mechanisms involved in miR-520g upregulation in the first trimester of PE are required for understanding the pathophysiology of PE.
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
Conflict of interest [18]
The authors declare that they have no conflict of interests in this article content. Liansheng Jiang, Anxiong Long, Longyi Tan, Mao Hong, Jingjing Wu, Leiming Cai, Qian Li. Acknowledgements This study program was supported by a grant (14411973600) from the Shanghai Municipal Science and Technology Commission in China. We thank Xiuqin Xiao for serum sample collection and storage.
[19]
[20]
[21]
[22]
Appendix A. Supplementary data
[23]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.placenta.2017.02.001.
[24]
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Placenta journal homepage: www.elsevier.com/locate/placenta
Elevated microRNA-520g in pre-eclampsia inhibits migration and invasion of trophoblasts Liansheng Jiang a, Anxiong Long a, Longyi Tan a, Mao Hong a, Jingjing Wu b, Leiming Cai a, Qian Li a, * a b
Department of Clinical Laboratory, Shanghai First People's Hospital Baoshan Branch, Shanghai, PR China Department of Obstetrics and Gynecology, Shanghai First People's Hospital Baoshan Branch, Shanghai, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 December 2016 Received in revised form 30 January 2017 Accepted 1 February 2017
Introduction: Pre-eclampsia (PE) is a common and severe obstetric complication. MicroRNAs (miRs) have emerged as molecules that are associated with the disease. Methods: Quantitative reverse transcription PCR (RT-qPCR) was used for serum miR-520g characterization from 19 severe pre-eclamptic and 19 normal pregnancies. In situ hybridation was adopted to localize microRNA-520g (miR-520g). Migration and invasion of HTR-8/SVneo cells were evaluated after miR-520g mimic treatment with transwell system. MiR-520g target gene was verified in luciferase reporter system. Results: The expression of serum miR-520g displayed an upward trend as pregnancies progress. At firsttrimester, miR-520g in pre-eclampsia was significantly higher than that in the control, but no significant differences were found in the second and last trimesters. MiR-520g localized in cytoplasm of early trimester placental trophoblasts. The migration and invasion of HTR8/SVneo were inhibited by miR-520g mimic treatment. Matrix metalloproteinase 2 (MMP2) was verified as a direct target of miR-520g. Conclusions: Elevated maternal serum level of miR-520g level in first trimester was detected in patients with severe PE. By suppressing the migration and invasion of trophoblast via at least partial inhibition of MMP2 translation inhibition, miR-520g might play a role in the defective spiral artery remodeling, and thus contribute to pre-eclampsia pathophysiology. © 2017 Elsevier Ltd. All rights reserved.
Keywords: microRNA-520g Pre-eclampsia Trophoblast HTR8/SVneo Matrix metalloproteinase 2 Biomarker
1. Introduction Pre-eclampsia (PE) is a complex multi-system involved pregnancy syndrome that is characterized by hypertension and significant proteinuria after 20th week of pregnancy [1]. PE is one of the main diseases that threaten maternal and newborn health [1,2]. Delivery of the placenta instead of fetus is the sole effective treatment for PE. Thus the defective placenta formed before 20th gestation week due to impaired trophoblast invasion and aberrant spiral artery remodeling is regarded as the key event in the PE pathophysiology [3]. During the first 20 weeks of gestation, endothelial cells and beneath smooth muscle cells in uterine spiral arteries are lost and replaced by extravillous trophoblast cells. A number of mechanisms may account for undesirable spiral remodeling, including deficiency of extravillous trophoblast and
* Corresponding author. E-mail address: [email protected] (Q. Li). http://dx.doi.org/10.1016/j.placenta.2017.02.001 0143-4004/© 2017 Elsevier Ltd. All rights reserved.
decreased trophoblast invasion [4]. MicroRNAs (miRs) are endogenously expressed small (19e25 nucleotides) non-coding RNAs that regulate the expression of nearly 30% of all genes via posttranscriptional repression [5]. Accumulating evidence indicates that deregulated miRNAs is closely related to PE [6e12]. Our previous study has unveiled a panel of miRNAs that might serve as potential biomarker for predicting PE since the second trimester [13]. Further quantification of placenta-specific miR-520g in maternal serum was conducted. We found abnormal expression of miR-520g during first trimester but not in second or third trimester. The coincidence of the time between deregulated miR-520g and spiral arterial remodeling promoted our hypothesis that miR-520g might play an important role during placentation. In the current study, we quantified the expression of miR-520g in maternal serum from preeclamptic and normal pregnancies, and further examined the impact of miR-520g mimic on the human trophoblast cell line, HTR8/SVneo. We found that expression of
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miR-520g was upregulated during the first to early second trimesters in PE, and that miR-520g mimic inhibited HTR8/SVneo migration and invasion. Mechanistically, matrix metalloproteinase (MMP)-2 was identified as a direct target of miR-520g that may account for the observed phenotypes in HTR8/Svneo cells. 2. Materials and methods 2.1. Patient enrollment and sample processing This study was approved by the local ethical and internal review board (IRB) of Shanghai First People's Hospital Baoshan Branch. Peripheral blood samples were obtained from 1625 Chinese pregnancies who received routine obstetric examination in our hospital from Jan 2014 to Jun 2015. The first trimester (8e10 weeks) placenta tissues were obtained from selective legal abortion cases in our hospital. Informed consent was obtained from all the subjects. A total of 19 singleton pregnancies complicated with preeclampsia which had complete medical records from first trimester to delivery was found retrospectively, then 19 healthy pregnant women without complications in our serum bank were selected as control based on similar maternal age (±1 year) at delivery and the same gestational week of blood sampling. Severe PE were defined as systolic pressure >140 mmHg or diastolic blood pressure >90 mmHg on two different occasions with proteinuria (>0.3 g/ 24 h) after 20th gestation week. Sera were collected from different trimester pregnancies after at least 8 h fasting and kept in 80 C until used. Placental tissues were fixed in 4% paraformaldehyde overnight at 4 C for in situ hybridization. 2.2. In situ hybridization Formalin-fixed paraffin-embedded (FFPE) placenta tissue slices were used for miRNA in situ hybridization with miRCURY LNA MicroRNA ISH Optimization Kit (Exiqon) and double digoxigeninlabeled LNA probes for miR-520g (50 nM), scrambled probe (50 nM) and U6 snRNA probe (1 nM). Following the manufacturer's protocol, the hybridization was conducted at 55 C for 1 h. All the sections were counter stained with Nuclear Fast Red (Beijing Dingguo Changsheng biotechnology) for 1 min. 2.3. Cell culture and treatment The human HTR-8/SVneo trophoblast cell line was a generous gift from Dr Charles H. Granham. HTR-8/SVneo cells were maintained in RPMI-1640 (Gibco) medium supplemented with 10% fetal bovine serum on a 6-well plate. When the cells reached 60% confluence, double-stranded miR-520g mimic (Qiagen), or scramble control (Qiagen) at a final concentration of 20 nM were introduced into the cells using Lipofectamine RNAiMAX Reagent (Invitrogen). 6 h post-transfection, HTR-8/SVneo cells were re-fed with fresh RPMI-1640 with 10% FCS for subsequent experiments. 2.4. RNA extraction and real-time quantitative RT-PCR analysis Total RNAs were extracted from 200 ml serum using the miRNeasy Serum/Plasma Kit (Qiagen) as following the manufacturer's instructions. Synthetic spike-in control Cel_miR-39_1(Qiagen) is used to monitor miRNA purification and amplification. The RNAs were dissolved in 14 ml RNase-free water. Total RNAs were extracted from cells with TRIzol reagent (Ambion). MiR real-time quantitative RT-PCR was done as previously described. Briefly, total RNAs (4 ml) were reverse transcribed using the miScript ⅡRT Kit (Qiagen) in a 20 ml total volume reaction system containing miScript Primer Assay (Qiagen) on PCR
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system(Bioer). Then real-time PCR was performed on 7500 RealTime PCR System (Applied Biosystems) using miScript SYBR Green PCR kit (Qiagen) in a total reaction volume of 20 ml containing 2 ml cDNA(200 times diluted). The expression levels of miRs were determined utilizing the comparative CT method relative to cel_miR-39_1. For mRNA gene expression, cDNA was synthesized using PrimeScript RT reagent kit (Takara), and real time qPCR was performed with SYBR Fast qPCR Mix (Takara). Ribosomal protein L19 was amplified as an endogenous control [12]. All PCR reactions were run in triplicate. The expression levels of miRs and mRNAs were expressed as fold changes by the 2 -△△Ct method. PCR primers are listed in Table S1.
2.5. Transwell migration and matrigel invasion assay HTR-8/SVneo cells treated with miR-520g mimic or negative control were seeded into the upper chamber of a transwell (24-well plate, 8.0-mm pore size, Corning) coated with or without matrigel. Cells remained in the upper chamber were removed using a cotton swab, the membranes were inverted and stained with crystal violet and quantified.
2.6. Western blot analysis HTR-8/SVneo cells were lysed 24 h after miR-520g mimic or negative control transfection and quantified with a BCA kit (Thermo Fisher Scientific). 20 mg proteins were resolved by 8% SDSPAGE and transferred to a polyvinylidene fluoride membrane using standard methods. The membranes were incubated with the primary anti-MMP2 antibody (Abcam) overnight at 4 C, then incubated with the secondary horseradish peroxidase (HRP) conjugated antibodies and visualized using Pierce Western Blotting ECL substrate (Thermo Fisher Scientific).
2.7. Generation of luciferase reporter assay The 30 -UTR of the MMP2 (1003 bp) containing predicted binding sites of miR-520g (UGAAAC) were amplified by PCR from human genomic DNA and inserted into pmiR-RB-Report vector (Ribobio) downstream of the renilla luciferase. A further 2 sites mutant MMP2 30 -UTR (260ACTTTGA266 to 260TGAAACT266, 516CACTTTG522 to 516GTGAAAC522) vector was constructed and validated by sequencing. HEK 293 T cells were transfected with 250 ng/well pmiR-MMP2WT or pmiR-MMPMut with 50 nM miR-520g mimic or scramble control. Lipofectamine 2000 (Invitrogen) and OPTI-MEN were used for transfection. Luciferase activities were measured 48 h after transfection using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to renilla luciferase activity. MMP2WT and MMPMut PCR primers are listed in Table S2.
2.8. Statistical analysis Expression levels of serum miRs were compared using KruskalWallis H test. Because the Bonferroni correction was used to solve the problem of multiple comparison in serum miRs, the significance level was set at P < 0.01. For other comparison, a Student's ttest was used. A P value of less than 0.05 was considered statistically significant. All statistical analysis was performed with the SPSS 20.0 software.
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Table 1 Demographic features of the PE patients and healthy controls.
Materal age (y) Gestational days at delivery (d) Neonate birthweight (g) Pregestation BMI (kg/m2) SBP (mm/Hg) DBP (mm/Hg) 24 h proteinuria (g)
PE (n ¼ 19)
CONTROL (n ¼ 19)
p value
31.3 ± 5.8 253.2 ± 7.2 2865.0 ± 730.9 24.9 ± 4.1 154.4 ± 11.6 103.6 ± 8.6 3.8 ± 1.2
30.9 ± 5.6 276.1 ± 4.8 3453.9 ± 311.2 20.3 ± 1.4 117.4 ± 8.8 73.2 ± 7.8 none
0.89 <0.05 <0.05 <0.05 <0.05 <0.05
3. Results 3.1. MiR-520g was aberrantly upregulated in the first trimester maternal serum in PE The demographic data of the case and control were shown in Table 1. Except for age, all the other factors, including the gestation days at delivery, neonate birthweight, pregestation BMI, 24-h proteinuria, systolic and diastolic blood pressure were significantly different between these two groups. With pregnancy progressing, the expression of serum miR-520g showed an increasing trend in both pre-eclamptic and health pregnancy, but the differences between second and third trimester in health pregnancy or between first and second in pre-eclamptic are not statistically significant (Fig. 1A). MiR-520g of first-trimester in pre-eclamptic group was
significantly higher than that in the control group (Fig. 1B), no statistically significant difference was found in the second and third-trimester between the two groups (Fig. 1C and D). 3.2. MiR-520g located in the cytoplasm of placental trophoblasts Human placenta is heterogeneous and consists of multiple cells. To clarify the source of miR-520g, we performed in situ hybridization using first trimester placenta tissues. MiR-520g was found in cytoplasm of trophoblasts from first trimester placenta (Fig. 2A and B). Both cytotrophoblasts (CTB) and syncytiotrophoblasts (STB) expressed miR-520g. No obvious difference in miR-520g expression was observed between CTB and STB. MiR-520g was not detected in either endothelial cells or mesenchymal cells. U6 and scramble miR were used as positive and negative control respectively (Fig. 2C and D). This result further verified miR-520g as a placenta-specific microRNA. 3.3. MiR-520g inhibited migration and invasion of HTR8/SVneo As HTR8/SVneo is a widely-used first trimester trophoblast cell line, we used it as a model to study the role of miR-520g in early pregnancy. Since HTR8/SVneo cells did not express high level of miR-520g, we transfected HTR8/Svneo cells with miR-520g mimic instead of using inhibitor. We found that both migration and invasion of HTR8/SVneo cells were inhibited following miR-520g mimic transfection (Fig. 3). MiR-520g mimic decreased the
Fig. 1. Relative expression of miR-520g in maternal serum from pre-eclamptic pregnancies and normal pregnancies. A. Box plot (from minimum to maximum) of relative level of serum miR-520g in three different gestation periods. B-D. Box plot of relative level of serum miR-520g in gestation week 10e14 (B) or gestation week 20e24 (C) or gestation week 30e34 (D) from pre-eclamptic pregnancies and normal pregnancies. PE means pre-eclampsia, CON means control, W means gestation week.
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Fig. 2. Location of miR-520g in first trimester placenta tissue. A-B. miR-520g staining in placenta tissue. C. U6 miR staining as a positive control in placenta tissue. D. Scramble control staining as a negative control in placenta tissue. fv, floating villi; st, syncytiotrophoblast; ct, cytotrophoblast. Scale bar, 50 mm. Blue color indicates the miRNA.
Fig. 3. The effect of miR-520g on HTR-8/Svneo migration (A) and invasion (B). ** means p < 0.01.
migration of HTR8/SVneo cells by 43% compared to the corresponding negative control (Fig. 3A). Meanwhile, miR-520g mimic suppressed the invasion of HTR8/SVneo cells by 45% compared to the corresponding negative control (Fig. 3B).
3.4. Validation of MMP2 as a direct target of miR-520g To find molecules responsible for miR-520g-related effects on cell migration and invasion, we used TargetScan (http://www. targetscan.org/vert_71/) to predict the potential targets of miR520g. MMP2, which is commonly involved in cell invasion, stood out as a promising candidate. We therefore focused on examining the relations between miR-520g and MMP2. We found that miR520g mimic transfection did not decrease MMP2 mRNA but
Fig. 4. Validation of MMP-2 as a direct target of miR-520g in luciferase assay. Vectors with wild type or mutated MMP2 30 -UTR were constructed and cotransfected with miR-520g mimic or scramble control. ** means p < 0.01.
downregulated MMP2 protein expression in HTR8/Svneo cells. To validate whether MMP2 was a direct target of miR-210, we constructed vectors which contained the wild type or mutated miR520g seeding site MMP2 30 -UTR region at the downstream of the reporter gene, renila luciferase. We then co-transfected HEK 293T cells with miR-520g mimic and vectors. MiR-520g mimic reduced the relative luciferase activity of the wide type MMP2 vector but not of the mutated MMP2 vector (Fig. 4). To examine the effect of miR-520g on HTR8/Svneo MMP-2 expression, we extracted RNA and protein from HTR8/Svneo after miR-520g mimic treatment. RTqPCR revealed a noticeable but not significant reduction of MMP2 mRNA (Fig. 5A), while western blotting showed lower pro-MMP2
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Fig. 5. The effect of miR-520g on MMP-2 expression. A. MMP-2 mRNA relative expression in HTR-8/Svneo after miR-520g mimic treatment as determined by RT-qPCR. B. Pro-MMP2 protein expression in HTR-8/Svneo after miR-520g mimic treatment as determined by western blot. C. Quantification analysis of MMP2 protein expression in HTR-8/Svneo after miR-520g mimic treatment.
protein expression after miR-520g mimic transfection (Fig. 5B and C).
4. Discussion MiR-520g belongs to the chromosome 19 miRNA cluster (C19MC) that is barely detectable in tissues other than placenta [14]. C19MC expression including miR-520g is upregulated significantly from first to third trimester trophoblast [15,16]. MiRNAs originated from placenta might be released into the maternal circulation in the form of exosomes [17]. Excessive apoptosis of trophoblasts related to pregnancy complications such as preeclampsia might induce a rise of miRNAs in the circulation [18]. Our miR-520g in situ hybridization showed cytoplasmic localization in trophoblast, which is consistent with previous results. The result that miR-520g expression in maternal circulation increased with gestation progression is reasonable, considering the expanding trophoblasts number, and is consistent with an earlier report [15]. Intriguingly, we found elevated expression of miR-520g in first trimester of severe pre-eclamptic pregnancy. This discovery, to our knowledge, represents the first report of upregulation of miR-520g expression in PE first trimester. This pilot study The timing of the aberrant rise of miR-520g in first trimester coincides with the time window of spiral artery remodeling. We speculate that the elevation of miR-520g in first trimester in PE might due to aberrant excessive trophoblast apoptosis and might play a role in trophoblast invasion or migration. Spiral artery remodeling starts from 8th to 10th gestation week in human pregnancy, whose hallmark is the invasion of the uterine spiral arteries by the extravillous trophoblast [3,4,19]. Inadequate spiral artery remodeling in the first trimester is widely considered as the first major step to drive PE. Spiral artery remodeling is a large network that is tightly and delicately regulated by a number of factors, including trophoblasts and endothelial cells. Given the crucial role of trophoblast in pre-eclampsia and its first trimester extravillous trophoblast origin [20], we chose to use HTR-8/SVneo cells, an immortalized cell line that mimics trophoblast cells as a
model cellular system in our study. We found that HTR-8/SVneo cells expressed very low amount of miR-520g and that miR-520g mimic treatment inhibited the migration and invasion of HTR-8/ SVneo cells. In addition to its placental origin, miR-520g has also been reported to be an oncogenic miRNA [21,22] and be upregulated in hepatocellular carcinoma (HCC) tissues and highly invasive HCC cell lines [23,24]. Kan H et al. showed that miR-520g facilitated HCC cell migration and invasion by inducing epithelialmesenchymal transition (EMT) via SMAD7 targeting, as reflected in the increase of E-cadherin and reduction of vimentin) [24]. Although MMPs have been repeatedly associated with tumor metastasis, the consensus view is, in general, that MMPs are not produced by epithelial cells but by the surrounding stromal cells [25]. Hence, the mechanisms involved in migration/invasion of trophoblasts and HCC cells might be different despite of some similarities between trophoblasts and tumor cells. The difference of the role of miR-520g in different cells indicates that cell type dependent mechanisms might be involved in regulating different pathways under varying disease contexts. Through target prediction analysis, we focused on MMP2 due to its 30 UTR seed binding sites with miR-520g and its well-known role in trophoblast cell invasion [26]. MMP2 is a zinc metalloendopeptidase secreted from cells as a zymogen (pro-MMP2). Our luciferase report assays clearly identified MMP2 as a direct target of miR-520g. PCR and western blot results showed that miR520g mimic transfection did not significantly reduce MMP2 mRNA level but downregulated pro-MMP2 protein expression in HTR8/ Svneo, which indicated miR-520g's posttranscriptional regulation on MMP2 might via translation inhibition rather than through mRNA degradation. A decrease in the MMP2 might reduce the invasiveness of trophoblast, which contributes to the pathogenesis of PE [27]. Tsai et al. reported miR-520g repressed MMP2 mRNA and propeptide expression in a dose dependent manner in primary endometrial stromal cells [28]. The inconsistency between the two studies is probably due to the use of different cell lines and different miR-520g mimic concentrations. Considering that the inhibition MMP2 by miR-520g may be reflected in circulation, we detected
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plasma MMP-2 from first trimester pregnancy using ELISA. Surprisingly we found a relatively higher MMP2 in pre-eclamptic group when compared with healthy group (225.0 ± 34.2 versus 203.5 ± 24.5 ng/ml, n ¼ 10, P ¼ 0.04). This result was consistent with Myers et al.’, in which they reported elevation of MMP2 in the plasma of pre-eclamptic women as early as 22 gestation week and before delivery [29]. However, a reduction of MMP2 in preeclamptic placenta was also reported in two other studies [27,30]. This discrepancy might arise from the varying degrees of endothelial activation in PE, which is expected to cause the elevation of MMP2 in circulation [31]. To conclude, we reported here that elevated maternal serum miR-520g level in first trimester was detected in patients with PE. We further provided evidence for the role of miR-520g in PE development by targeting MMP2. Given the limited sample size of the current study, we are full aware of the necessity to include more patients in future studies to draw a definite conclusion. Further investigations on the mechanisms involved in miR-520g upregulation in the first trimester of PE are required for understanding the pathophysiology of PE.
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
Conflict of interest [18]
The authors declare that they have no conflict of interests in this article content. Liansheng Jiang, Anxiong Long, Longyi Tan, Mao Hong, Jingjing Wu, Leiming Cai, Qian Li. Acknowledgements This study program was supported by a grant (14411973600) from the Shanghai Municipal Science and Technology Commission in China. We thank Xiuqin Xiao for serum sample collection and storage.
[19]
[20]
[21]
[22]
Appendix A. Supplementary data
[23]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.placenta.2017.02.001.
[24]
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