Identification of miR-210-5p in human placentae from pregnancies complicated by preeclampsia and intrauterine growth restriction, and its potential role in the pregnancy complications

Journal Pre-proofs Identification of microRNA 210-5p in Human Placentae from Pregnancies Complicated by Preeclampsia and Intrauterine Growth Restriction, and its Potential Role in the Pregnancy Complications Zain Awamleh, Victor K.M. Han PII: DOI: Reference:

S2210-7789(20)30003-9 https://doi.org/10.1016/j.preghy.2020.01.002 PREGHY 681

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Pregnancy Hypertension: An International Journal of Women's Cardiovascular Health

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26 August 2019 3 November 2019 12 January 2020

Please cite this article as: Awamleh, Z., Han, V.K.M., Identification of microRNA 210-5p in Human Placentae from Pregnancies Complicated by Preeclampsia and Intrauterine Growth Restriction, and its Potential Role in the Pregnancy Complications, Pregnancy Hypertension: An International Journal of Women's Cardiovascular Health (2020), doi: https://doi.org/10.1016/j.preghy.2020.01.002

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Title: Identification of microRNA 210-5p in Human Placentae from Pregnancies Complicated by Preeclampsia and Intrauterine Growth Restriction, and its Potential Role in the Pregnancy Complications

Running title: miR-210-5p in preeclampsia and IUGR Zain Awamleh1,2* and Victor K.M. Han1,2,3

1Children’s

Health Research Institute, London, ON N6C 2V5, Canada, 2Departments of

Biochemistry and 3Pediatrics, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON N6A 3K7, Canada

*To whom correspondence should be addressed at: Children’s Health Research Institute, 800 Commissioners Road East, London ON N6C 2V5, Canada tel: 5196858500 ext.55455, Email: [email protected]

(Grants sponsor: CIHR, grant numbers: 15579 and 15262)

Abstract Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy complications resulting from abnormal placental development. As epigenetic regulators, microRNAs can regulate placental development and contribute to the disease pathophysiology by influencing the expression of genes involved in placental development or disease. Our previous study revealed an increase in miR-210-5p expression in placentae from patients with early-onset pregnancy complications and identified candidate gene targets for miR-210-5p. The purpose of this study was to: (i) validate candidate gene targets predicted for miR-210-5p from microRNA-RNA expression data, and (ii) overexpress miR-210-5p in a trophoblast cell line (HTR-8/SVneo) to assess impact on trophoblast cell functions. Integration of the miRNA and RNA sequencing expression data revealed 8 candidate gene targets for miR-210-5p in patients with PE only or PE+IUGR. Luciferase reporter assays identified two gene targets for miR-210-5p, CSF1 and ITGAM. Realtime PCR confirmed the decreased expression of CSF1 and ITGAM in patients with PE+IUGR. Immunohistochemistry of placentae from late second trimester identified CSF1 and ITGAM in intermediate trophoblast cells in the decidua. Expression levels of CSF1 and ITGAM were reduced in HTR-8/SVneo cells following increased miR-210-5p expression. Concomitantly, HTR8/SVneo cells demonstrate an average 45% reduction in cell migration. These findings suggest that miR-210-5p may contribute to dysfunction of intermediate trophoblasts and potentially contribute to the disease process of these pregnancy complications.

Keywords: microRNA-210, placenta, trophoblast, preeclampsia, intrauterine growth restriction

1

Introduction

2

Micro(mi)RNAs are endogenous non-coding RNAs transcribed in the nucleus, exported to the

3

cytoplasm and processed into mature miRNAs of 20-22 nucleotides in length [1,2]. In the

4

cytoplasm, mature single-stranded miRNAs target messenger RNAs (mRNA) via perfect or

5

imperfect base pair complementarity to the 3´ untranslated region (3´UTR) of the mRNA and

6

decrease gene expression by enhanced mRNA degradation or impaired translation [1-3]. MiRNAs

7

are classified as epigenetic regulators, due to their ability to post-transcriptionally regulate gene

8

expression by sequence complementarity without alteration in genetic sequence [1,2]. Studies have

9

shown that miRNA expression can be tissue and developmental stage specific, implicating

10

miRNAs in important developmental processes [4,5]. MiRNAs participate in the regulation of a

11

wide spectrum of cell processes including proliferation, apoptosis, differentiation, and stress

12

response [2,3]. MicroRNAs are expressed highly in the human placenta, with two specific clusters

13

on chromosomes 14 and 19 and have been shown to differ in expression in the three trimesters of

14

pregnancy [4,5]. Placental miRNAs can enter the maternal circulation during pregnancy, as the

15

placenta sheds debris into the maternal circulation where they may be found as either cell free or

16

exosome-bound [6]. These findings have sparked interest in investigating the role of miRNAs in

17

placental development and disease, and their efficacy as biomarkers to predict pregnancy

18

complications potentially prior to the appearance of signs and symptoms [7,8]. Dysregulation of

19

miRNA expression has been shown in human placentae from pregnancy complications such as

20

preeclampsia (PE) and intrauterine growth restriction (IUGR) [16].

21

Preeclampsia is a maternal hypertensive disorder of pregnancy, affecting 2-8% of pregnancies

22

worldwide [9,10]. IUGR is defined as poor fetal growth in utero, and patients present with an

23

expected fetal weight lower than the 10th centile for gestational age and gender combined with

24

abnormal Dopplers in uterine, fetal and/or umbilical vessels [11]. A subset of patients with

25

preeclampsia also develop IUGR and often present with symptoms prior to 34 weeks, classified as

26

early-onset (EO) PE and/or IUGR. In addition, placentae from PE and IUGR pregnancy

27

complications have similar histopathological features, such as villous infarctions, fibrin

28

deposition, and syncytial knotting, suggesting common pathophysiology [12,13]. While the

29

underlying pathophysiology of PE and IUGR is not fully understood, studies have shown that

30

placental maldevelopment in early gestation can contribute to the pathogenesis of the disease,

31

which usually presents after 20 weeks gestation [10,14]. More specifically, trophoblast invasion

32

of the spiral arteries for establishment of uteroplacental blood flow which occurs in mid-gestation

33

is thought to be impaired [10,14]. This can result in decreased uteroplacental blood perfusion and

34

subsequently a hypoxic intrauterine environment [10,14]. Recent evidence indicates that the

35

intrauterine environment and placenta in both PE and IUGR are hypoxic [15]. Expression levels

36

of the hypoxia-inducible miRNA miR-210 are consistently reported to be increased in placentae

37

and plasma samples from patients diagnosed with PE [16-18]. Upregulation of miR-210 has been

38

linked to impaired trophoblast cell functions such as proliferation and invasion [19].

39

In our previous study, we determined placental miRNA expression using miRNA sequencing of

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total RNA of placentae from early-onset pregnancy complications, and we identified an increase

41

in miR-210-3p and miR-210-5p in patients with PE (± IUGR) [20]. To identify candidate gene

42

targets, we utilized RNA sequencing to measure gene expression in the same placental samples

43

[20]. Integration of miRNA and gene expression results identified candidate gene targets for miR-

44

210-5p in EO-PE, EO-PE+IUGR group, or both. Candidate gene targets include Apelin (APLN),

45

Complement C3a Receptor 1 (C3AR1), Colony Stimulating Factor 1 (CSF1), Integrin Alpha M

46

(ITGAM), E-Selectin (SELE), Tyrosine Kinase 3 (TYRO3), Vav Guanine Nucleotide Exchange

47

Factor1 (VAV1), and Wnt Family Member 3 (WNT3) [20]. The purpose of this study was to

48

investigate the potential impact of increased miRNA 210-5p expression on the expression of

49

identified gene targets and on trophoblast cells in culture, to determine trophoblast cellular

50

functions that may have physiological or pathophysiological consequences. The objectives of this

51

study were three-fold (i) to examine whether miR-210-5p interacts with candidate gene targets

52

using luciferase reporter assays, (ii) to determine the cell types that express gene targets, and (iii)

53

to overexpress miR-210-5p in a cell line that expresses gene targets of interest to assess the impact

54

on target gene expression, migration, and proliferation of cells.

55

Materials and Methods

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Ethics statement. All women enrolled in this study gave written informed consent for the

57

collection of samples and information. This study was approved by the Office of Human Research

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Ethics at The University of Western Ontario in London, Ontario, Canada (reference # 102621,

59

approval date June 12, 2012).

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Sample collection. Preeclampsia was defined as hypertension (blood pressure > 140/90 mm Hg)

61

and proteinuria ( 300 mg in 24 hours) [9]. Severe PE is often associated with HELLP syndrome

62

characterized by the onset of edema, headache, elevated liver enzymes and low platelet count.

63

Patients diagnosed with PE and HELLP are indicated in Table 1. Intrauterine growth restriction

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was defined as estimated fetal weight by antenatal ultrasound below the 10th percentile for

65

gestational age and gender, associated with abnormal umbilical and uterine artery Dopplers [11]

66

and confirmed by the newborn birthweight. Patients with PE+IUGR presented with criteria

67

aforementioned for both diseases. Only patients diagnosed prior to 34 weeks (early-onset) were

68

included in this study. Patients with preterm labor and no other pregnancy complications before

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34 weeks of gestation were recruited as controls. Women with diabetes, gestational diabetes, pre-

70

existing hypertension, obvious chorioamnionitis (status confirmed after delivery by placental

71

pathology), alcohol/drug use, chromosomal or genetic abnormalities, congenital anomalies, or

72

infection were excluded.

73

Samples were collected from two central and two peripheral portions of the placenta within 30

74

minutes of delivery. Central samples were collected within a 5 cm radius from the umbilical cord

75

insertion site and the peripheral samples were collected 2-3 cm from the edge of the placenta. Full

76

depth 1 cm x 1 cm tissue samples were excised, and the maternal decidua was separated from the

77

chorionic villi using gross dissection. In this study, only the fetal components (chorionic villi) were

78

used for analysis. The tissue samples were flash frozen in liquid nitrogen and stored at -80C until

79

further analysis.

80

Reverse transcription and real-time PCR (placenta tissue). Total RNA was isolated from 80-

81

100 mg of tissue samples from each of the four regions of each placenta using the mirVana RNA

82

isolation kit (Invitrogen). Sample quantity and quality were checked using the Agilent Bioanalyzer

83

2100 (Agilent Technologies). Total RNA isolated from central and peripheral samples of each

84

placenta was pooled in equal quantities for one representative total RNA sample from each patient.

85

Total RNA was reverse transcribed using the High Capacity cDNA Synthesis Kit (Applied

86

Biosystems). Quantitative real-time PCR (qRT-PCR) of mRNAs was completed using TaqMan®

87

fast advanced PCR master mix (Applied Biosystems) in conjunction with TaqMan gene expression

88

assays. GAPDH was used as an endogenous control. Each sample was assayed in triplicate and

89

run on the ViiA7™ real-time machine. The 2-ΔΔCt method was used for fold change analysis. APLN

90

(Hs00175572_m1),

C3AR1

(Hs00269693_s1),

CSF1

(Hs00174164_m1),

ITGAM

91

(Hs00167304_m1),

SELE

(Hs00950409_g1),

TYRO3

(Hs03986773_m1),

VAV1

92

(Hs01041613_m1), WNT3 (Hs00902257_m1) (All assays from Applied Biosystems).

93

Target Prediction. A combination of target prediction tools was used to predict targets of miR-

94

210-5p. Software tools are: TargetScan Human (http://www.targetscan.org/vert_70/mirwalk2.0),

95

miRwalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/), miRDB (http://mirdb.org),

96

miRanda ((http://microrna.sanger.ac.uk/targets ). Gene ontology analysis was completed using

97

WebGestalt 2017.

98

Luciferase reporter assays. HTR-8/SVneo cells (generously provided by Dr. P. K. Lala, Western

99

University, London, ON) were cultured in RPMI-1640 media (Gibco) supplemented with 10%

100

fetal bovine serum at 37°C in 5% CO2. Cells were subcultured at a ratio of 1:3 when cells reached

101

80% confluency. Cells were seeded in 96-well plates 24-hours prior to co-transfection of vectors

102

and mimics at 37°C in 5% CO2. Vectors contain the firefly luciferase (Renilla) and the 3´UTRs of

103

candidate gene targets of interest pre-cloned under the control of a constitutive promoter. The

104

3´UTRs of candidate genes: C3AR1 (S803358), CSF1 (S807015), ITGAM (S808425), and

105

TYRO3 (S808004) were obtained from Active Motif (Carlsbad, CA, USA). DharmaFECT Duo

106

transfection reagent (GE Healthcare) was used to co-transfect the firefly vector (100 ng) with hsa-

107

miR-210-5p mimics or non-target control (NC) mimics, 100 nM each in serum-free media. Control

108

3´UTR reporter vectors were also used, empty 3´UTR (100 ng) (S890005) and 3´UTR of GAPDH

109

(100 ng) (S801378). After 24-hours incubation, luciferase activity was measured using the

110

LightSwitch™ Luciferase reporter assay reagent according to manufacturer’s instructions (Active

111

Motif).

112

Immunohistochemistry (IHC). Full thickness sections (0.5 cm x 0.5 cm) extending from the

113

basal plate decidua (BPD) to the chorionic plate (including maternal and fetal components) were

114

harvested at the same time the samples were collected for RNA analyses. Both central and

115

peripheral sites were collected. The specimens were immediately fixed in 10% formalin for a

116

minimum of 24 hours. Following fixation and washing, tissues were processed, and embedded in

117

paraffin. All tissues were then sectioned at 5 μm and mounted onto slides. Slides were then

118

deparaffinized, dehydrated, and processed for immunohistochemistry with antigen retrieval in

119

citrate buffer (pH 6.0). Slides were then blocked with a blocking agent, Background Sniper

120

(BS966, Biocare Medical). The primary antibody against CSF1 (1:75) or ITGAM (1:75)

121

(Supplementary Table 1) was applied and incubated overnight. The slides were then rinsed with

122

PBS and the secondary antibody, ImmPRESS Anti-Rabbit Peroxidase Polymer Detection Kit (MP-

123

7401, Vector Laboratories), was applied. The slides were rinsed again and labeled with a DAB

124

(3,3′–diaminobenzidine) stain (1718096001, Sigma Aldrich). Negative control slides underwent

125

the same procedures, without the primary antibody (Supplementary Figure 1). Finally, the sections

126

were counterstained with CAT Hematoxylin (CATHE, Biocare Medical). Imaging was performed

127

using a 200 x total magnification on a Zeiss AxioImager Z1 Microscope using Zen software and

128

an MRc5 camera (Zeiss Canada Ltd.).

129

Cell culture and treatment. HTR-8/SVneo cells were cultured in RPMI-1640 media (Gibco) with

130

10% fetal bovine serum in 24-well plates at 37°C in 5% CO2. Cells were subsequently transfected

131

using DharmaFECT 1 transfection reagent (GE Healthcare) in serum-free media. For miR-210-5p

132

experiments, cells were transfected with: 50 nM of miR-210-5p mimics (Invitrogen, MC27291),

133

100 nM of miR-210-5p inhibitors (Invitrogen, MH27291), or respective non-target control (NC)

134

mimics (50 nM, MIM9001), NC inhibitors (100 nM, INH9001) (Active Motif). After transfection,

135

cells were lysed for gene or protein expression analysis or used to measure cell functions.

136

Reverse transcription and real-time PCR (cells). HTR-8/SVneo cells were seeded and

137

transfected as described above (see ‘cell culture and treatment’ methods section). Total RNA was

138

isolated from HTR-8/SVneo cells using Qiagen’s RNeasy Mini kit (74104, Qiagen). Cells were

139

lysed using lysis buffer provided in the kit and further homogenized by passing lysate through a

140

20-gauge needle. Total RNA was then used for reverse transcription and real-time PCR as

141

described above.

142

Western blotting. HTR-8/SVneo cells were seeded and transfected as described above (see cell

143

culture and treatment methods section). Cells were then lysed using RIPA buffer containing

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protease and phosphatase inhibitors (Sigma-Aldrich). Bradford assay was used to assess protein

145

concentration. 30 ug of lysates were then resolved on 12.5% (CSF1) or 8% (ITGAM) SDS-PAGE

146

and transferred to a PDVF membrane using the Bio-Rad Trans-Blot Turbo transfer system (Bio-

147

Rad). Membranes were incubated with primary antibodies against CSF1 (1:500), ITGAM

148

(1:1000), or beta-actin (MS1295P, ThermoFisher) at 4°C overnight (Supplementary Table 1). The

149

membranes were then washed and incubated with horse radish peroxidase (HRP) conjugated

150

secondary antibody (170-6516, Bio-Rad). Resolved protein bands were detected using

151

chemiluminescence, and images were taken using the VersaDoc Imaging System (Bio-Rad).

152

Cell viability assay. HTR-8/SVneo cells were seeded and transfected as described above (see cell

153

culture and treatment methods section). Cell proliferation was measured using cell proliferation

154

reagent WST-1 (Sigma-Aldrich) according manufacturer’s protocol. After 1-hour incubation with

155

the WST-1 reagent, absorbance was measured at 450 nm using Multiskan Ascent plate

156

reader (ThermoFisher). Reference wavelength of 650 nm was used, and culture medium was used

157

as a blank.

158

Wound healing (scratch) assay. An in vitro scratch assay was used as described previously [21].

159

After transfection (see cell culture and treatment methods section), HTR-8/SVneo cells were

160

grown to confluence, and scratches were made using a p200 pipette tip. The width of the scratch

161

was monitored by Leica DM IL microscope, images were captured along the scratch at 0 hours

162

and 24 hours using 40 x total magnification. Area of the scratch was then measured using Image J

163

Software, distance travelled is shown as migration level relative to control samples.

164

Transwell migration assay. Transwell compartments were prepared in a 24-well plate format,

165

with BD Falcon™ 8.0-µm pore Transwell cell culture inserts (353097; BD Biosciences). For the

166

lower compartment 0.8 mL of RPMI-1640 media with 10% FBS was added. For the upper

167

compartment, 1 x 105 cells transfected with miR-210-5p mimics, inhibitors, or respective non-

168

target control (see ‘cell culture and treatment’ methods section) in 0.2 mL serum-free RPMI-1640

169

media were gently added. After 24 hours incubation at 37°C and 5% CO2 non-migrated cells on

170

the top surface of the insert were carefully removed. Migrated cells on the bottom surface of the

171

insert were fixed with methanol and stained with 0.2% crystal violet. Cells on the bottom surface

172

of the inserts were imaged using Leica DM IL inverted microscope and 200 x total magnification.

173

Number of cells counted is shown as migration level relative to control samples.

174

Statistical Analysis. GraphPad Prism Software 6.0 was used to generate all graphs and analyses.

175

Statistical analysis was performed using the Mann-Whitney U-test or a two-tailed Student’s t-test,

176

a threshold of p-value < 0.05 was considered significant. Graphic representation values are

177

presented as mean ± SEM. For correlation analysis, Pearson correlation co-efficient was used for

178

graphical representation of correlation analysis between miRNA and gene expression values. Only

179

correlation with a r of  -0.5 and adjusted p-value  0.01 was considered significantly negatively

180

correlated. All experiments were repeated three times independently in triplicate at a minimum.

181

Results

182

Clinical Data. Clinical characteristics of the patient populations (EO-PE, EO-PE +IUGR, Control)

183

are shown in Table 1. These patient cohorts were the same cohorts used for the miRNA and RNA

184

sequencing study [20] with additional information. There were no differences in maternal age,

185

maternal BMI, or gestational age at delivery between patient groups. There were significant

186

differences in birth weights, placental weights, and blood pressure between patient groups with

187

complicated pregnancies and gestational age-matched controls. Birth weights and placental

188

weights were also significantly lower in the EO-PE+IUGR group compared to the EO-PE group.

189

These patients were selected using stringent inclusion and exclusion criteria to include patients

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with primarily placental factors underlying the diseases. Patients with known maternal and/or fetal

191

risk factors were not included (see Methods).

192

Candidate gene targets identified from sequencing study in placental samples from PE

193

pregnancies. In our previous study measuring miRNA expression using next generation

194

sequencing (NGS) in placentae from patients diagnosed with early-onset pregnancy complications,

195

we identified increased miR-210-5p expression in patient with EO-PE and EO-PE+IUGR

196

compared to gestational age matched controls. Integration of miRNA and gene expression data

197

identified a subset of predicted gene targets for miR-210-5p. Figure 1 A shows qRT-PCR results

198

for miR-210-5p predicted targets, to confirm gene expression results from prior NGS data [20].

199

The majority of candidate gene targets identified are in the PE + IUGR group (7/8), compared to

200

half in the EO-PE group (4/8) (Figure 1 A). All candidate gene targets were confirmed to be

201

decreased in their respective patient groups using qRT-PCR, with the exception of VAV1 and

202

WNT3 in the EO-PE group (Figure 1 A).

203

Validating miR-210-5p candidate targets using luciferase reporter assays. Based on qRT-PCR

204

results VAV1 and WNT3 were prioritized for validation using luciferase reporter assays. We

205

prioritized conducting luciferase reporter assays for C3AR1, CSF1, ITGAM and TYRO3 based on

206

enrichment of these 4 genes in the majority of the top 10 biological processes identified through

207

gene ontology (GO) analysis. Figure 1 B shows the top 10 biological processes miR-210-5p

208

candidate gene targets are enriched in, prevalent categories include the immune system and cell

209

migration/locomotion. Significant decrease in relative luciferase activity was observed in HTR-

210

8/SVneo cells containing 3´UTRs of either CSF1 or ITGAM (Figure 2 A). However, no changes

211

were observed in cells containing the 3´UTRs of C3AR1 or TYRO3 (data not shown). Both CSF1

212

and ITGAM were predicted targets by more than one software prediction tool at the same

213

nucleotide positions, including TargetScan and miRanda. miR-210-5p is predicted to target CSF1

214

at 2380-2387 nt region of the 3´UTR, and ITGAM at the 3887-3894 nt region of the 3´UTR (Figure

215

2 B). Inverse correlation analysis using sequencing data had previously shown significant negative

216

inverse correlation between the expression of miR-210-5p and CSF1 (r = - 0.81), and between

217

miR-210-5p and ITGAM (r = - 0.80) in the control and PE + IUGR groups (Figure 2 C, D).

218

Qualitative immunohistochemical (IHC) analysis of CSF1 and ITGAM in the human

219

placenta. Previous gene expression analysis in the placenta was conducted in homogenized

220

chorionic villi containing various cell types. Therefore, to identify which cell types in the placenta

221

that most prominently express CSF1 and ITGAM, IHC analysis was conducted for cellular

222

localization purposes. Staining was completed in PE + IUGR samples and gestational-age matched

223

preterm control samples for localization of each gene target in both patient groups. For each sample

224

both chorionic villus (CV) and basal plate decidua (BPD) were stained from whole sections

225

obtained from central and peripheral regions of the placenta. CSF1 strongly localized to Hofbauer

226

cells in tertiary chorionic villi, meanwhile lighter staining was observed in the cytotrophoblast

227

(CT) and syncytiotrophoblast (SCT) cells (Figure 3). ITGAM localized to SCT cells in tertiary

228

villi, and both ITGAM and CSF1 were expressed in intermediate CT cells within the basal plate

229

decidua (Figure 3). To verify the identity of intermediate trophoblast cells, staining for pan

230

cytokeratin and IGFBP1, positive markers for trophoblast and decidual cells of the placenta

231

respectively was also performed (Supplementary Figure 2).

232

Expression of CSF1 and ITGAM in HTR-8/SVneo cells. HTR-8/SVneo cells were transfected

233

with either miR-210-5p mimics or inhibitors. Following treatment, mRNA and protein expression

234

levels of CSF1 and ITGAM were measured in these cells and compared to cells transfected with

235

NC mimics or NC inhibitors. Transfection of miR-210-5p mimics into HTR-8/SVneo cells resulted

236

in a decrease in CSF1 and ITGAM mRNA expression (Figure 4 A, B). Conversely, transfection of

237

miR-210-5p inhibitors into HTR-8/SVneo cells increased CSF1 and ITGAM mRNA expression

238

(Figure 4 A, B). Similar trends in expression were observed for CSF1 protein after transfection

239

(Figure 4 C, D). ITGAM protein in HTR-8/SVneo cells was undetectable using two different

240

commercially available antibodies, although detectable in placental tissues using the same

241

antibodies (data not shown). ITGAM protein was also undetectable in BeWo cells, another

242

trophoblast cell line (data not shown).

243

Impact of miR-210-5p on cell functions. To investigate the impact of miR-210-5p on cell

244

functions, HTR-8/SVneo cells were transfected with miR-210-5p mimics, inhibitors, or

245

corresponding NC and cell proliferation and migration were assessed. Cell proliferation was

246

measured using spectrophotometric quantification, after addition of WST-1 directly to cell culture.

247

WST-1 (Sigma-Aldrich, St. Louis, MO, USA), is a tetrazolium salt added to culture is cleaved by

248

mitochondrial dehydrogenase into a colored dye, absorbance measured is directly proportional to

249

net metabolic activity of cells. Cell migration was measured using a wound healing assay and a

250

transwell assay. Transfection of HTR-8/SVneo cells with miR-210-5p mimics decreased

251

proliferation and migration of cells (Figure 5). Transfection of HTR-8/SVneo with miR-210-5p

252

mimics decreased proliferation and migration of cells. The fraction of viable cells was 20% less

253

in cells treated with miR-210-5p mimics compared to cells treated with NC mimics. Meanwhile

254

the wound healing assay showed a 30% decrease in relative migration levels, and the transwell

255

assay showed a 60% reduction. Transfection of HTR-8/SVneo with miR-210-5p inhibitors had no

256

impact on proliferation but promoted migration of cells (Figure 5). After treatment with miR-210-

257

5p inhibitors the wound healing assay showed a 35% increase in relative migration levels,

258

meanwhile the transwell assay showed a 65% increase.

259

Discussion

260

In our previous study investigating miRNA expression in placentae from patients diagnosed with

261

early-onset pregnancy complications, we identified increased expression of miR-210-3p in 3

262

patient groups (EO-PE, EO-IUGR, and EO-PE+IUGR), and miR-210-5p in patients with EO-PE

263

and EO-PE+IUGR [20]. MicroRNA-210 is one of the most widely identified miRNAs in placentae

264

from complicated pregnancies, and it is identified to be upregulated in placenta from patients with

265

PE only and in patients with PE and small-for-gestational age babies [16, 22, 23]. In the human

266

placenta, using in situ hybridization, miR-210 expression has been localized to the villous

267

trophoblast and the extravillous interstitial trophoblast [22]. In addition, miR-210 has been widely

268

investigated for its potential use as a diagnostic biomarker, both miR-210-3p and miR-210-5p

269

expression levels were found to be significantly higher in maternal plasma from PE patients [17,

270

18]. As previously described, the intrauterine environment in pregnancies complicated by PE

271

and/or IUGR can be hypoxic due to decreased perfusion of maternal blood into the uteroplacental

272

unit [15]. It is now known that under hypoxic conditions, miR-210 is upregulated in expression,

273

and the upregulation is mediated by the transcription factors HIF-1 or NF-κB [19,24].

274

To assess the impact of miR-210 upregulation in PE placenta, previous studies have identified

275

gene targets that are downregulated and are implicated in processes important for placental

276

development and/or function [19,22]. Gene targets validated using cell culture methods include:

277

EFNA3, HOXA1, ISCU, KCMF1, and THSD7A [19, 22, 25, 26]. In our previous study, gene

278

expression data from the same placental samples allowed us to identify candidate gene targets. We

279

identified 8 candidate targets for miR-210-5p across the two patient groups (EO-PE and EO-PE +

280

IUGR), and in this study, we confirmed the expression of these genes using qRT-PCR in the

281

respective patient groups (Figure 1A). Luciferase reporter assays identified CSF1 and ITGAM as

282

gene targets for miR-210-5p (Figure 2 A). In this study, CSF1 and ITGAM are decreased in patients

283

with early-onset PE + IUGR compared to gestational age-matched controls (Figure 1A).

284

Colony-stimulating factor-1 (CSF1) is a growth factor that is known to regulate proliferation,

285

migration and differentiation of mononuclear phagocytes, through a transmembrane tyrosine

286

kinase receptor, CSF-1R [27]. In our search for targets, we noted that CSF1 receptor (CSF1R) was

287

also a predicted target of miR-210-5p. CSF1 and its receptor CSF-1R have been shown to be

288

expressed in the placenta [28-30]. CSF-1R immunoreactivity (IR) is detected in placental

289

trophoblast in the first trimester, whereas CSF1 IR is detected in the cytotrophoblasts lining the

290

villous core [30,31]. In early third trimester placental samples from our patients, CSF1 IR was

291

localized to SCT and CT layers but was the strongest in the Hofbauer cells of the CV, and in the

292

intermediate CT cells of the basal plate decidua (BPD) (Figure 3 A-D).

293

Previous studies have shown that extravillous trophoblast (EVT) cells propagated in cell culture

294

continue to express CSF1 and CSF1R mRNA and protein, and the addition of exogenous CSF1 to

295

EVT cell cultures significantly stimulated proliferation but had no impact on the invasiveness of

296

cells [32]. Another study suggests a role for CSF1 in trophoblast cell proliferation and showed

297

CSF1 could be acting in part through HLX1 to regulate cell proliferation [33]. In addition,

298

treatment of term placental CTs with exogenous CSF1 in culture, increases the number and size

299

of multinucleated structures forming extended stretches of syncytium, thereby implicating CSF1

300

in syncytialization of trophoblast cells [34,35]. There is previous evidence that CSF1 can be

301

regulated by miRNAs in ovarian cancer cells, where CSF1 is a target of miR-128 and miR-152,

302

and the overexpression of miRNAs correlates with a decrease in CSF1 expression and impacts cell

303

migration and adhesion [36]. Reported expression of macrophage-CSF (M-CSF) and granulocyte-

304

macrophage-CSF (GM-CSF) in blood and placenta from PE pregnancies is conflicting. While

305

some studies report an increase in M-CSF levels in the maternal sera and an increase in GM/M-

306

CSF in the placenta, others report no change [31, 37-39]. On the other hand, a study in patients

307

diagnosed with IUGR, found M-CSF levels to be significantly lower in amniotic fluid samples

308

[40]. Conflicting reports can be attributed to lack of standardization of the patient selection

309

process, for example grouping early- and late-onset PE patients together or grouping patients with

310

PE ± IUGR together. In our study placental CSF1 mRNA expression is decreased in patients with

311

early-onset preeclampsia and intrauterine growth restriction, but not in patients with EO-PE or

312

EO-IUGR.

313

Integrin subunit alpha M, also known as ITGAM or CD11b, binds noncovalently to a β2 subunit

314

(CD18) to form integrin ⍺Mβ2, that is expressed in monocytes, granulocytes, and macrophages

315

[41,42]. CD11b/CD18 have the capacity to recognize a number of ligands, such as fibrinogen,

316

complement fragment iC3b and ICAM-1 to mediate leukocyte adhesion and migration, and are

317

therefore implicated in inflammation [41]. Studies have shown the independent role of CD11b and

318

CD18. Cells expressing only the ⍺M subunit (ITGAM) can recognize ligands, normally recognized

319

by the integrin ⍺Mβ2, independently of the β2 subunit, and subsequently mediate firm cell adhesion

320

and spreading in response to these ligands [41]. Previous reports of CD11b expression in maternal

321

sera or macrophages of the placenta have been conflicting [43-46]. In a trophoblast cell culture,

322

ITGAM is increased two-fold upon treatment with chemokines [47]. In a more recent study

323

utilizing a microarray approach for the transcriptional profiling of placentae from women with

324

severe PE, RNA profiles show increased expression of ITGAM in the endovascular

325

cytotrophoblast compared to the syncytiotrophoblast and invasive cytotrophoblast samples from

326

both PE and preterm placentae [48]. However, there were no differences in ITGAM expression

327

between PE and preterm placenta [48]. In pregnant mice, ITGAM expression is localized to the

328

spongiotrophoblast layer and was shown to increase with gestation [49]. In the current study,

329

ITGAM immunoreactivity was localized to the intermediate CT cells in the BPD (Figure 3 E, F).

330

We therefore chose an intermediate trophoblast cell line, HTR-8/SVneo cells to determine the

331

functional role of miR-210-5p.

332 333

Transfection of HTR-8/SVneo cells with miR-210-5p mimics and inhibitors, impacted CSF1 and

334

ITGAM mRNA expression (Figure 4 A, B). Only changes in CSF1 protein levels corresponded to

335

changes observed in mRNA levels (Figure 4 C). ITGAM protein was not detectable in HTR-

336

8/SVneo cells, although it was strongly expressed in the chorionic villi homogenates. It is possible

337

that HTR-8/SVneo cells may have lost the capacity to translate ITGAM mRNA into a full

338

functional protein during the transformation from a primary cell to a cell line, or that the translation

339

may be dependent on the environment. Culturing trophoblast cells in the presence of other

340

placental cell types such as endothelial or Hofbauer cells may trigger mRNA translation into

341

protein. Previous reports have shown that miR-210 impacts gene targets that are important in cell

342

functions such as migration, invasion, growth/proliferation, and mitochondrial metabolism

343

[19,25,26,50,51]. Based on evidence implicating miR-210-3p in important trophoblast cell

344

functions [25,26,50,51], we sought to assess the impact of miR-210-5p on HTR-8/SVneo cell

345

proliferation and migration. Transfection of cells with miR-210-5p mimics reduced proliferation

346

and migration of cells, while inhibition of miR-210-5p only had an effect on cell migration (Figure

347

5).

348

This study contributes to accumulating evidence supporting the role of miRNAs in important

349

cellular functions in the placenta such as cell migration, invasion, and proliferation. However, it is

350

important to note that the increased expression of miR-210-5p demonstrated in this study is in the

351

chorionic villi of placentae from PE and IUGR at the time of birth when the disorders have already

352

manifested clinically. During the early second trimester, cytotrophoblast cells proliferate and

353

differentiate into extravillous trophoblast (EVT) cells that migrate and invade into the maternal

354

decidua through anchoring chorionic villi to remodel the maternal uterine spiral arteries [52].

355

Poorly remodelled uterine arteries result in poor perfusion of the placenta and leads to hypoxic and

356

oxidative stress, which is hypothesized to be the underlying pathophysiologic process in PE and

357

IUGR [15]. It is possible that the same miRNAs, such as miR-210, that are identified at the end of

358

pregnancy are increased in the developing placenta in the early second trimester and influence

359

trophoblast proliferation, migration, and invasion. These miRNAs can be detected and quantified

360

in the maternal circulation at this stage of pregnancy and potentially serve as biomarkers of PE

361

and/or IUGR prior to the manifestations of the diseases. Recent reports show increased exosome-

362

mediated transfer of miR-210 from hypoxic tumor cells to nearby tumor cells and to sera of patients

363

with clear-cell renal cell carcinoma (ccRcc) [53,54]. The latter and our study suggest that the

364

determination of exosome-bound and cell-free miR-210 levels during the early 2nd trimester prior

365

to clinical presentation of PE or IUGR is an important future study.

366

Gene ontology analysis also revealed enrichment of predicted gene targets in immune system

367

processes (Figure 1 B). Gene expression studies in preeclamptic placenta often identify pathways

368

and processes linked to immune and inflammatory responses [55,56]. Recent reports by Leavey et

369

al., (2015; 2016) identified a subclass of PE that is severe and can co-occur with IUGR but is likely

370

due to poor maternal-fetal compatibility (“immunologic PE”) [57,58]. Future studies are required

371

to elucidate the role of miR-210 and other miRNAs in regulating immune responses at the

372

maternal-fetal interface.

373

In summary, in this study, we confirmed increased miR-210-5p expression in placentae from

374

patients with severe early-onset PE ( IUGR); predicted and validated CSF1 and ITGAM, as gene

375

targets; and demonstrated the impact of miR-210-5p on trophoblast migration and proliferation in

376

vitro, which are potential pathophysiological processes in PE and/or IUGR. The next step is to

377

demonstrate the increase of miR-210-5p in the circulation of early second trimester patients as a

378

predictive biomarker for PE and/or IUGR, which may lead to potential interventions to reduce the

379

severity of these pregnancy complications.

380

Abbreviations

381

CSF1: Colony stimulating factor- 1; CT: Cytotrophoblast; EO: Early-onset; EVT: Extravillous

382

Trophoblast; GO: Gene Ontology; IHC: Immunohistochemistry; IR: Immunoreactivity; ITGAM:

383

Integrin subunit alphaM; IUGR: Intrauterine Growth Restriction; miRNA: microRNA; NGS: Next

384

Generation Sequencing; PE: Preeclampsia; qRT-PCR: Quantitative Real time PCR; SCT:

385

Syncytiotrophoblast

386

Acknowledgements

387

We would like to thank all the donors and the Research Centre for Women’s and Infants Health

388

(RCWIH) BioBank for placental samples used in this project. We would also like to acknowledge

389

Karen Nygard (Biotron Facility, Western University) for assistance with immunohistochemical

390

staining of placental tissues.

391

Contributions of authorship

392

ZA made substantial contributions to design, acquisition of data, analysis and interpretation of

393

data, and in writing and revising the article. VKMH made substantial contributions to design,

394

interpretation of data and revising the article. All authors approved final version of the article.

395

Funding

396

This study was funded by grants from the Canadian Institutes of Health Research (15579 and

397

15262 to VKMH) and The Douglas and Vivian Bocking Chair in Fetal and Newborn Growth (to

398

VKMH). ZA is supported through Western University’s Graduate Research Scholarship and the

399

Graduate Student Grant from Western University’s Department of Paediatrics.

400

Competing Interests

401

Authors have no competing interests to declare. References

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Figure Legends Figure 1. mRNA expression levels of candidate gene targets for miR-210-5p. To find relative mRNA expression of candidate gene targets in placental samples the 2-CT method was used and values were normalized to GAPDH expression. (A) Candidate targets identified in patients with EO-PE [N=20] or EO-PE+IUGR [N=20] compared to controls [N=21]. Each sample was assayed three times. Data is shown as the mean  SEM, ** indicates p-value <0.01 by Mann-Whitney U test, ns – no significant difference between PE group and gestational age matched controls. (B) Gene ontology analysis identified a substantial list of biological processes that miR-210-5p candidate gene targets are significantly (Adj. p-value < 0.01) implicated in, the top 10 are shown here, analysis was conducted using WebGestalt 2017.

Figure 2. Validation of miR-210-5p candidate gene targets. (A) Relative luciferase activity measured 24 hours after co-transfection of HTR-8/SVneo cells with luciferase constructs containing 3´UTR of CSF1, or ITGAM, or control constructs, along with miR-210-5p mimic or non-target control (NC) mimic. Data is shown as mean ± SEM; *** indicates p-value < 0.001 by a two-tailed Student’s t-test, n=3 performed in triplicate (B) Schematic of the luciferase construct and sequence alignment between miR-210-5p and gene targets. Significant negative correlation between the expression values of (C) miR-210-5p and CSF1 in the PE + IUGR group, and (D) miR-210-5p and ITGAM in the PE + IUGR group obtained from miRNA and RNA-seq expression datasets. Figure 3. Immunohistochemical staining for gene targets CSF1 and ITGAM. Staining for CSF1 in preterm control placenta (A,B) and in early-onset PE + IUGR (C,D), gestational age 29 weeks + 4 days and 29 weeks + 6 days respectively. Staining for ITGAM in preterm control placenta (E,F) and in early-onset PE + IUGR (G,H), gestational age 31 weeks + 5 days and 32 weeks + 1 day respectively. Black arrows show positivity in SCT cells, white arrows show positivity in CT cells, red arrows show positivity in Hofbauer cells, green arrows show positivity in intermediate CT cells. All images were captured at 200 x total magnification. Figure 4. Impact of miR-210-5p on gene expression in human trophoblast cells. (A) CSF1 and (B) ITGAM mRNA expression in HTR-8/SVneo cells transfected with miR-210-5p mimics or inhibitors and compared to the corresponding control (NC mimic or inhibitor) as detected by qRTPCR and normalized to GAPDH expression using the 2-CT method. (C) Western blot analysis showed CSF1 protein levels in HTR-8/SVneo cells following transfection with miR-210-5p

mimics (top panel) or inhibitors (lower panel) and compared to the corresponding control (NC mimic or inhibitor) (D) Summary graph from three independent experiments, CSF1 density was normalized to -actin in the same blot. Values represent mean ± SEM; ** indicates p-value < 0.01 by a two-tailed Student’s t-test, n=3 performed in triplicate. Figure 5. miR-210-5p impact on cell functions in human trophoblast cells. (A) Effect of miR210-5p on cell proliferation was investigated using WST-1 reagent. Cells were incubated with WST-1 reagent following transfection with miR-210-5p mimic or inhibitor and compared to the corresponding control (NC mimic or inhibitor). (B) To investigate the effect of miR-210-5p overexpression and inhibition on cell migration, HTR-8/SVneo cells were transfected with miR210-5p mimics, inhibitor, or the corresponding control, scratches were then created and the width of the scratch in each experimental group was measured at time 0 and at 24 hours using 40 x total magnification. Migration level is the distance traveled in 24 hours relative to the control group. bar= 100 m. (C) Transfected HTR-8/SVneo cells were transferred into a transwell chamber to assess impact on migration; images were taken 24 hours after seeding using 200 x total magnification. Migration level is the number of cells migrated through the membrane relative to the control group. bar= 25 m. All experiments were repeated three times independently, data is shown as mean ± SEM ** indicates P < 0.01 by a two-tailed Student’s t-test, n=3 performed in triplicate.

Supplementary Figure 1. Negative control images for immunohistochemical analysis. Slides designated negative controls underwent the same procedures, with the exception of the application of the primary antibody. (A) Stem villus, (B) Chorionic villus section, and (C) Basal plate decidua

section from preterm control, gestational age 29+4. Blue staining is CAT Hematoxylin counterstain. All images were captured at 20 x, bar = 50 m. Supplementary Figure 2. Pan cytokeratin and IGFBP1 immunohistochemical analysis. Slides designated negative controls underwent the same procedures, with the exception of the application of the primary antibody. Pan cytokeratin in (A) Preterm control placenta and (B) earlyonset PE+IUGR placenta. IGFBP1 in (C) Preterm control placenta and (D) early-onset PE+IUGR placenta. Green arrows show positivity in trophoblast cells, and black arrow show positivity in decidual cells. Blue staining is CAT Hematoxylin counterstain. All images were captured at 20 x, bar = 50 m.

402

Table 1. Clinical characteristics of the patient groups with complicated pregnancies and

403

gestational age matched controls. Characteristic (Mean ± SD)

PE N=20

PE + IUGR N=20

Control N=21

Maternal Age (years)

28.6 ± 7.0

32.6 ± 5.7

28.2 ± 5.0

BMI (kg/m )

28.9 ± 7.4

28.7 ± 5.3

28.6 ± 7.5

GA at Delivery (weeks)

29.6 ± 3.1

29.4 ± 2.5

30.6 ± 2.6

Sex (Females)

10 (50%)

10 (50%)

11 (52%)

Mode of Delivery: C-Section (%)

15 (75%)

19 (95%)

4 (19%)

C-Section with Labor (%)

6 (40%)

5 (26%)

4 (100%)

2

Birth Weight (grams)

1300 ± 499.7

Placental Weight (grams)

342.9 ± 135.4

Birth Weight Percentile

30.4 ± 19.2

Systolic BP (mm Hg)

173.5 ± 19.1

Diastolic BP (mm Hg)

1

933.9 ± 342.2

2,4

3

244.7 ± 75.2

2,4

4.8 ± 2.0

1803 ± 623.5 462.7 ± 136.3 63.4 ± 26.5

2

116.5 ± 15.8

104.3 ± 8.6

2

69.24 ± 13.1

6 (30%)

8 (40%)

NA

Ethnicity: Caucasian

17

15

18

Indigenous/ Native

1

1

1

African

1

1

-

Asian

1

3

1

Hispanic

-

-

1

HELLP Syndrome

2

170 ± 14.6

108.0 ± 10.3

2

1) p-value < 0.05 vs. control 2) p-value < 0.001 vs. control 3) p-value <0.0001 vs. control 4) p-value <0.01 vs. PE only

404

405 406 407 408 409 410 411 412 413

Highlights: 

Placental expression of miR-210-5p is increased in patients with PE and IUGR



miR-210-5p targets CSF1 and ITGAM that may play a role in placental development



miR-210-5p impacts cell proliferation and migration in trophoblast cells in vitro