Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m6A

Abstract

Background: Several epidemiological investigations demonstrated that maternal arsenic (As) exposure elevated risk of fetal growth restriction (FGR), but the mechanism remains unclear. Objectives: This study aimed to investigate the effects of gestational As exposure on placental and fetal development and its underlying mechanism. Methods: Dams were exposed to 0.15, 1.5, and 15 mg/L NaAsC>2 throughout pregnancy via drinking water. Sizes of fetuses and placentas, placental histopathology, and glycogen content were measured. Placental RNA sequencing was conducted. Human trophoblasts were exposed to NaAsC>2 (2 uM) to establish an in vitro model of As exposure. The mRNA stability and protein level of genes identified through RNA sequencing were measured. A^-Methyladenosine (m6A) modification was detected by methylated RNA immunoprecipitation-quantitative real-time polymerase chain reason (qPCR). The binding ability of insulin-like growth factor 2 binding protein 2 to the gene of interest was detected by RNA-binding protein immunoprecipitation-qPCR. Intracellular S-adenosylmethionine (SAM) and methyltransferase activity were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and colorimetry, respectively. In vitro As+3 methyltransferase (As3MT) knockdown or SAM supplementation and in vivo folic acid (FA) supplementation were used to evaluate the protective effect. A case-control study verified the findings. Results: Sizes of fetuses (exposed to 1.5 and 15 mg/L NaAs02) and placentas (exposed to 15 mg/L NaAsCy were lower in As-exposed mice. More glycogen+ trophoblasts accumulated and the expression of markers of interstitial invasion was lower in the 15 mg/L NaAs02-exposed mouse group in comparison with control. Placental RNA sequencing identified cysteine-rich angiogenic inducer 61 (Cyr61) as a candidate gene of interest. Mechanistically, mice and cells exposed to As had lower protein expression of CYR61, and this was attributed to a lower incidence of Cyr61 m6A. Furthermore, cells exposed to As had lower methyltransferase activity, suggesting that this could be the mechanism by which Cyr61 m6A was affected. Depletion of intracellular SAM, a cofactor for m6A methyltransferase catalytic domain, partially contributed to As-induced methyltransferase activity reduction. Either As3MT knockdown or SAM supplementation attenuated As-induced Cyr61 m6A down-regulation. In mice, FA supplementation rescued As-induced defective trophoblastic invasion and FGR. In humans, a negative correlation between maternal urinary As and plasma CYR61 was observed in infants who were small for gestational age. Discussion: Using in vitro and in vivo models, we found that intracellular SAM depletion-mediated Cyr61 m6A down-regulation partially contributed to As-induced defective trophoblastic invasion and FGR.

Headnote

Background: Several epidemiological investigations demonstrated that maternal arsenic (As) exposure elevated risk of fetal growth restriction (FGR), but the mechanism remains unclear.

Objectives: This study aimed to investigate the effects of gestational As exposure on placental and fetal development and its underlying mechanism.

Methods: Dams were exposed to 0.15, 1.5, and 15 mg/L NaAsC>2 throughout pregnancy via drinking water. Sizes of fetuses and placentas, placental histopathology, and glycogen content were measured. Placental RNA sequencing was conducted. Human trophoblasts were exposed to NaAsC>2 (2 uM) to establish an in vitro model of As exposure. The mRNA stability and protein level of genes identified through RNA sequencing were measured. A^-Methyladenosine (m6A) modification was detected by methylated RNA immunoprecipitation-quantitative real-time polymerase chain reason (qPCR). The binding ability of insulin-like growth factor 2 binding protein 2 to the gene of interest was detected by RNA-binding protein immunoprecipitation-qPCR. Intracellular S-adenosylmethionine (SAM) and methyltransferase activity were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and colorimetry, respectively. In vitro As+3 methyltransferase (As3MT) knockdown or SAM supplementation and in vivo folic acid (FA) supplementation were used to evaluate the protective effect. A case-control study verified the findings.

Results: Sizes of fetuses (exposed to 1.5 and 15 mg/L NaAs02) and placentas (exposed to 15 mg/L NaAsCy were lower in As-exposed mice. More glycogen+ trophoblasts accumulated and the expression of markers of interstitial invasion was lower in the 15 mg/L NaAs02-exposed mouse group in comparison with control. Placental RNA sequencing identified cysteine-rich angiogenic inducer 61 (Cyr61) as a candidate gene of interest. Mechanistically, mice and cells exposed to As had lower protein expression of CYR61, and this was attributed to a lower incidence of Cyr61 m6A. Furthermore, cells exposed to As had lower methyltransferase activity, suggesting that this could be the mechanism by which Cyr61 m6A was affected. Depletion of intracellular SAM, a cofactor for m6A methyltransferase catalytic domain, partially contributed to As-induced methyltransferase activity reduction. Either As3MT knockdown or SAM supplementation attenuated As-induced Cyr61 m6A down-regulation. In mice, FA supplementation rescued As-induced defective trophoblastic invasion and FGR. In humans, a negative correlation between maternal urinary As and plasma CYR61 was observed in infants who were small for gestational age.

Discussion: Using in vitro and in vivo models, we found that intracellular SAM depletion-mediated Cyr61 m6A down-regulation partially contributed to As-induced defective trophoblastic invasion and FGR. https://doi.org/10.1289/EHP12207

Introduction

Fetal growth restriction (FGR) is defined as fetal failure to reach its genetic growth potential, clinically manifested as small for gestational age (SGA).1'2 It has been widely accepted that FGR is a major cause of preterm birth, stillbirth, and neonatal death.3'4 Increasing evidence demonstrates that FGR may elevate the risk of certain diseases, including type 2 diabetes mellitus in mice,5 lower cognitive scores in a cohort of very preterm infants,6 and cardiovascular disease7 in adulthood. Maternal undernutrition during pregnancy, such as vitamin D8 and folate deficiency,9 was considered to be the major cause of FGR in some populations. Recently,numerous studies found that maternal exposure to environmental toxicants, including air pollutants,10'11 endocrine disrupters,12'13 and heavy metals14 were associated with an increased risk of FGR. Arsenic (As), a toxic metalloid, is widely present in the environment.15'16 The general population is usually exposed to As through drinking contaminated water or inhaling polluted air.15'16 Currently, it is estimated that 200 million people, including pregnant women, have been drinking water with As concentrations >10 ug/L, exceeding the World Health Organization (WHO) recommendation standard.1718 Several epidemiological reports indicated that maternal As exposure during pregnancy elevated the risk of SGA infants.19-21 Animal experiments found that gestational As exposure induced hepatic lipid accumulation in adult offspring.22 Nevertheless, the mechanism by which gestational As exposure impairs fetal development remains obscure.

The placentas play pivotal roles in maintaining fetal development. During the first trimester, human placental villous cytotropho-blast cells are differentiated into invasive extravillous trophoblasts (EVTs), accompanied by loss of epithelial features and gain of mesenchymal characteristics.23-25 Increasing evidence has demonstrated that insufficient invasion of EVTs during early pregnancy induces adverse pregnant outcomes, including preeclampsia,26 FGR,27 and spontaneous abortion.28 Several in vitro studies indicated that environmental xenobiotics, such as heavy metals29 and endocrine disrupters,30'31 disrupted EVT migration and invasion. An early in vitro experiment found that As suppressed human EVT migration and invasion.32 Epigenetic modifications play important roles in embryonic and placental development.33'34 A^-Methyladenosine (m6A) is one of abundant mRNA modifications in eukaryotes.35 In addition, m6A modification is dynamic and reversible, regulated by methyltransferases, demethylases, and m6A-bindingproteins.35'36 Methyltransferases contain the core components of methyltransferase-like (Mettl)3, Mettll4, and Wilms tumor 1-associated protein (WTAP), which are responsible for catalyzing m6A modification.35'36 Mettl3, a core enzyme, exerts methyl-transferase activity by combining with S-adenosylmethionine (SAM).37'38 Demethylases include a-ketoglutarate-dependent al-kylation repair homolog protein 5 (ALKBH5) and fat-mass and obesity-associated protein (FTO).35'36 The m6A-binding proteins specifically recognize target m6A-modified mRNA and subsequently regulate RNA fate, such as mRNA stability, translation, and splicing.35'36 The m6A-binding proteins include the insulinlike growth factor 2 mRNA-binding protein (IGF2BP) family.36 Several investigations show that maladjustment of m6A modification might contribute to placental trophoblast dysfunction.39'40 Here, we hypothesize that abnormality of placental m6A modification might be involved in As-induced FGR. To verify this hypothesis, an in vivo model of As-induced FGR was established. Subsequently, the key molecule of As-induced FGR was identified. Then, we established an in vitro model of arsenic exposure to explore the role of m6A modification of key molecules in As-induced FGR. Furthermore, we investigated the effect of folic acid (FA) supplementation on As-induced FGR. Finally, a case-control study was used to verify our findings.

Materials and Methods

Reagents and Materials

Sodium arsenite (NaAs02; #SLBW7984) and FA (#F7876) were obtained from Sigma. SAM (#B9003S) was obtained from New England Biolabs. Actinomycin D (#GC16866) was obtained from GlpBio. Epigenase m6A methylase activity/inhibition assay kit (#P9019-96) and EpiQuik m6A methylation quantification kit (#P-9005-48) were purchased from Epigentek. NE-PER nuclear and cytoplasmic extraction reagents (#78835) were purchased from Thermo Scientific. TRI reagent (#TR-118) was purchased from Molecular Research Center, Inc. The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Study Design

The purpose of this study was to investigate the effects of gestational As exposure on placental and fetal development. To achieve this goal, we first established a mouse model of gestational exposure to As-induced FGR. Then, the mechanism was explored using mouse placentas and human trophoblasts. Finally, a case-control study was used to verify the association. For animal studies, pregnant mice were randomly assigned to experimental groups and exposed to different concentrations of NaAsC>2 by drinking water. FA, a precursor of intracellular SAM synthesis, was also used to prevent As-induced FGR. All animal experiments were performed in accordance with the guidelines set by the Association of Laboratory Animal Sciences and the Center for Laboratory Animal Sciences at Anhui Medical University (Ethical approval number: LLSC20190357). For in vitro studies, human trophoblasts (HT8/Svneo cell line) were exposed to NaAsC>2 after As3MT knockdown or exogenous SAM supplementation. For human studies, this case-control study was based on a cohort of pregnant women in the Wuxi Maternal and Child Health Hospital. This study was carried out with the understanding and written consent of each pregnant woman. This human study was approved by the Ethics Committee of Anhui Medical University (No. 20190295).

Animals and Treatments

Female and male CD-I mice (8 wk old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

Mice were maintained in a controlled environment (humidity: 50 ±5%; temperature: 20-25°C) on a 12-h light/dark cycle for a week. All mice had free access to food and water. For mating, females were mated overnight with males at the ratio of 2:1. All females were checked for vaginal plug, and the presence was defined as gestational day (GD) 0. In accordance with Charan et al.,41 the sample size calculation of our study takes into account the quantitative data at the end point, power of study at 80% and expecting 10% attrition. The sample size was range 7-11.

This study included two independent experiments. In experiment 1, we first observed whether gestational As exposure induced fetal FGR, so this experiment needed 11 mice or more in each group. Pregnant mice were administered different concentrations ofNaAsC>2 (0.15,1.5,or 15 mg/L) by drinking water from GD0 to GD18. Wang et al.42 used 15, 30, or 60 mg/L NaAs02 to investigate the impacts on d-serine metabolism in the hippocampus of offspring. Li et al.43 used 0.15, 1.5, and 15 mg/L As (III) to evaluate the effects on puberty in offspring. Hence, we chose 15 mg/L NaAsC>2 as the highest exposure concentrations because some naturally polluted areas have reached this level.44 In addition, we used three different concentrations (0.15, 1.5, 15 mg/L) of NaAsC>2 to observe whether concentration dependence existed. All pregnant mice were anesthetized using 2.5% avertin (#T48402, Sigma; total volume of the intraperitoneal injection was 1% of body weight) and then sacrificed on GD18 morning at 0900 hours (9:00 A.M.) The numbers of abortions (monitor vaginal bleeding and maternal weight, embryos loss), preterm delivery (dams delivered before GD18), resorption sites (mass without placenta and embryonic rudiments), dead fetuses (early dead fetus, fully formed placenta but without fully formed embryo; late-dead fetus, fully formed embryo but without signs of life), and live fetuses (fully formed with signs of life) were counted for each litter. In addition, for live fetuses, dry body weight, crown-rump length, placental dry weight, and placental diameter were measured. In accordance with Salavati et al.,45 in our mouse model, FGR was defined as a statistically significant decrease in fetal weight in comparison with the control group, accompanied by placental histopathological abnormalities. In addition, placentas were collected for hematoxylin and eosin (H&E) staining, periodic acid-Schiff (PAS) staining, realtime reverse transcription polymerase chain reaction (real-time RT-PCR), immunohistochemistry, and immunoblotting.

In experiment 2, to evaluate the effect of FA on As-induced FGR, 32 pregnant mice were divided into four groups: control, FA, As and As+FA groups. Li et al.46 reported that 120 ug/kg of FA (i.g.) inhibited amyloid (3-peptide accumulation by upregulating DNA methylation in mouse model. Health institutions around the world recommended47 that women who are able to conceive take FA supplements (400-1,000 ug/d). Hence, in the FA and As + FA groups, pregnant mice were administered with 150 Ug/kg FA by gavage daily from GD0 to GD17. The total volume of the gavage was 1% of body weight. In the As and As + FA groups, pregnant mice drank ultrapure water containing NaAsC>2 (15 mg/L). All pregnant mice were anesthetized in the morning on GDI8. Pregnancy outcomes were recorded as described in experiment 1. Then, placental H&E staining, immunoblotting, SAM content, and m6 A methylase activity were performed.

Placental Histopathology Examination

Mouse placentas were fixed in 4% paraformaldehyde for 24 h and then dehydrated in 75% ethanol for 24 h. Subsequently, placentas were embedded in paraffin. Paraffin-embedded placentas were cut 5 urn thick. The sections were stained with H&E. To analyze placental pathology, six sections of placentas were selected from six separate dams. We used the Image J Program from the National Institutes of Health (NIH) to analyze images.

Placental Glycogen Measurement

PAS staining was used to stain placental glycogen. Each group contained six placental sections from separate dams. PAS staining kit (#G1281; Solarbio) was used according to the manufacturer's protocol. In brief, deparaffinized sections were incubated for 10 min in Schiff's regent, and then stained with hematoxylin for 2 min. Subsequently, sections were differentiated in acidic differentiation solution for 20 s. Finally, sections were dehydrated through gradient ethanol (30%-100%) for 2 min each time. Using the Image J Program from the NIH to quantify placental glycogen cells, the number of glycogen-positive cells per field was calculated and expressed as a percentage of total cells in the junctional zone. Placental glycogen content was quantified by digestion and precipitation. Each group contained six placental tissues from separate dams. For digestion, placental tissues were boiled in 30% potassium hydroxide (KOH), 5% Na2SC>4 for 25 min to release the glycogen. After cooling on ice, glycogen was precipitated with 95% ethanol (1.1-1.2 volumes). Glycogen was dissolved in water and detected by a phenol sulfuric acid colorimetric method.48 In brief, glycogen solution was added with 5% phenol solution (CAS: 108-95-2; Sinopharm Chemical Reagent Co., Ltd.), and then 96%-98% H2S04 (CAS: 7664-93-9; Sinopharm Chemical Reagent Co., Ltd.) were added rapidly. We let the solution stand for 10 min, and then placed it in a water bath at 30°C for 20 min. Finally, absorbance was read at 490 nm by microplate reader (Synergy 4; BioTek). Glycogen content was normalized to placental weight.

Immunohistochemistry

The paraffin-embedded placentas of pregnant women and mice were cut 5 urn thick. Placental sections were deparaffinized by baking at 60°C for 30 min and soaking in xylene for 30 min. Subsequently, sections were rehydrated through gradient ethanol (100%-30%). Then, sections were placed in 0.01 M sodium citrate and boiled for 6 min and four times for antigen retrieval. Sections were incubated with primary antibodies against CYR61, vimentin, matrix metalloproteinases (MMP2), and MMP9 at 4°C overnight. Information of the primary antibodies is presented in Table SI. Finally, anti-rabbit immunohistochemistry kit (PV-6001; ZSGB-BIO) was used according to the manufacturer's protocol. Briefly, sections were added with anti-rabbit IgG polymer and incubated at 37°C for 45 min. Then, 3,3'-diaminobenzidine (DAB) reaction and counterstaining with hematoxylin were performed. Placental CYR61-, vimentin-, MMP2-, and MMP9-positive cells were counted in randomly selected fields from each slide. For mice, each group contained six placental sections from separate dams. For pregnant women, each group contained 18 placental sections from separate pregnant women.

Immunoblotting

Immunoblotting was analyzed for placentas of mice and pregnant women and HTR8/SVneo cells. Total protein was extracted. In brief, placentas (50 mg) and trophoblast cells (2 X 106 per flask) were added with lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100,1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mmol/L phenylmethylsulfonyl fluoride, 1% protease inhibitors). Then, the homogenates were centrifuged at 12,000 X g for 15 min at 4°C to obtain supernatant. Supernatant (20^-0 ug protein) was electrophoretically separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were incubated at room temperature for 2 h with following antibodies: CYR61, E-cadherin, N-cadherin and vimentin, Occludin, MMP2, and MMP9. (3-Actin was used as a loading control. Information on the primary antibodies is presented in

Table SI. After washing in Dulbecco's phosphate-buffered saline (DPBS) containing 0.05% Tween-20 four times for 10 min each, the membranes were incubated with goat anti-rabbit IgG (#BL003A, 1:50,000 dilution; Biosharp) or goat antimouse antibody (#BL001A, 1:50,000 dilution; Biosharp) at room temperature for 2 h. The membranes were then washed for four times in DPBS containing 0.05% Tween-20 for 10 min each. Subsequently, the enhanced chemilumi-nescence (ECL) detection kit was used to develop signals on the automatic chemiluminescence image analysis system (Tanon Fine-do X6). For mice, each group contained three biologically independent repetitions. For pregnant women, each group contained 18 biologically independent replications. For HTR8/SVneo cells, each group contained three biologically independent replications. Relative quantification of protein was performed using densito-metric analysis of Image Pro Plus software and then standardized with the control protein.

RNA-Sequencing (RNA-seq) and Analysis

Total RNA from six sex-independent mouse placentas was extracted by Trizol regent (Thermo Fisher Scientific) and then treated with DNase I (#AM2222; Thermo Fisher Scientific) according to the manufacturer's instructions. Subsequently, RNA quality was evaluated by a 2100 Bioanalyzer (Agilent), which automatically measures the 28s: 18s rRNA ratio and computes an RNA integrity number (RIN). The 28s: 18s ratio of the sample should lie in 1.8 to 2.0, and the RIN number over 6.5 for sequencing. RNA-seq was done in Genesky using TruSeq Stranded mRNA Library Preparation (Illumina). In brief, mRNA was enriched with oligo-dT magnetic beads. Then, mRNA was sequentially treated as follows: fragmented mRNA, synthesized first strand cDNA, synthesized second strand cDNA, end repair, adenlylate 3' ends, ligate adapters, size selection, PCR amplification, and library quality check. The library was finally sequenced on the Illumina HiSeq 2000 in a 2 X 150 bp paired-end sequencing mode for obtain FastQ data. Data processing of raw reads was quality checked by using fastQC (version 0.11.8; http://www.bioinformatics.babraham.ac.uk/projects/ fastqc/) and trimmed for low-quality bases and adaptors by using TrimGalore (http://www.bioinformatics.babraham.ac.uk/projects/ trim_galore/). The amount of sequencing data of each sample was >8G, Q20 > 96%, and Q30 > 90%. After sequencing, the data were processed using STAR to generate reads alignments to the mouse reference genome (mmlO). Capture efficiency was computed by Picard. The command flagstat from SAMtools generates a quick summary of mapped, unmapped, discordantly mapped, and properly paired reads. In our study, the genome alignment rate of all sample data was >96%. Differential gene expression (DEG) analysis was performed with DEseq2 (version 1.10.1; https://bioconductor. org/packages/release/bioc/html/DESeq2.html). The resulting p-value was <0.05 by DESeq2 and with a fold change of >1.5 (|log2 fold change|>0.58) were considered to be differentially expressed. To find the potential roles of differentially expressed genes, GO and KEGG enrichment analyses were used with the clusterProfiler R language package [clusterProfiler (version 2.4.2; http://bioconductor. org/packages/release/bioc/html/clusterProfiler.html)]. We took the top 50 genes of p-values for clustering analysis.

Cell Culture and Treatment

The HTR-8/SVneo cell line is derived from human invasive extravillous trophoblasts and was purchased from ATCC (#CRL-3271). The cells were cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum and 1% 100 U/mL penicillin/streptomycin at 37°C in a 5% CO2 humidified atmosphere. To explore the effect of As on migration and invasion in HTR8/ SVneo cells, cells were incubated with NaAsC>2. We chose 2 uMas the final concentration of NaAsC>2, because no significant effect on cell viability was measured at this exposure using the CCK8 assay (Figure S2; Table S8). The dose of iAs in the cell line model was about 6 times higher than the upper limit of serum As content (Canada, 34.46 Ug/L; China, 43.52 ug/L) in the pregnant women,19'49 and approximately 12 times higher than the upper limit of human umbilical cord blood As content (Canada, 17.98 Ug/L; China, 23.6 Ug/L) in the Canada and China birth cohorts.49'50 As can accumulate in the placenta.51'52 Early data showed that the median concentration of placental As content was 34 ug/kg in the Argentina cohort in an area with high As concentration in drinking water (200 ug/L).52 To investigate whether SAM supplementation attenuates As-induced invasion inhibition, HTR8/SVneo cells were incubated with SAM (10 uM) during NaAs02 (2 liM) exposure. The final concentration of SAM was 10 uM according to CCK8 assay.

Cell Viability Assays

HTR8/SVneo cells (3.0 X 103 per well) were seeded in 96-well plates. For NaAsC>2 treatment, the cells were incubated with different concentrations of NaAs02 (0, 0.3125, 0.625, 1.25, 2.5, 5, and 10 liM). For SAM treatment, the cells were incubated with different concentrations of SAM (0, 5, 10, 20, 40, 80, 160, and 320 uM) during As exposure. Each concentration contained four biologically independent replications. After 24 h incubation, cell viability was determined by CCK-8 kit (BS350A; Biosharp) according to the manufacturer's protocol.

Wound-Healing Assay

Wound-healing assay was performed to evaluate cell migration ability. Each group contained three biologically independent replications. HTR-8/SVneo cells (2x 105 cells/well) were seeded in six-well plates and allowed them to adhere for 20 h. After adhesion, cells were cultured with 2 uM of NaAsC>2 for 24 h, and then the follow-up protocol was performed in accordance with previous studies.53

Transwell Migration and Invasion Assays

Migration and invasion capacity of HTR-8/SVneo cells were assessed. Each group contained three biologically independent replications. For migration assay, 2.5 X 104 cells in 200 uL complete medium were seeded in the upper chamber of 5 urn transwell filters. For invasion assay, 2.5 X 104 cells in 200 uL complete medium were seeded in the upper chamber of 5 urn transwell filters precoated with 50 uL Matrigel solution (100 ug/mL; #356234; BD Biosciences). For both assays, cells were allowed to adhere for 20 h at 37°C, 5% C02. After incubation for 20 h, 200 uL complete medium with 2 uM NaAsC>2 was added in upper chamber, and 600 uL complete medium with 2 uM NaAsC>2 was added in the lower compartment. Subsequently, plates were incubated for 24 h at 37°C, 5% CO2. We removed the remaining cells on the upper side of the chamber and the complete medium of the lower chamber. The membranes were fixed with methanol for 20 min and then stained with 0.1% crystal violet solution for 20 min. Because a large number of cells migrated to the bottom of the filter, we dissolved the cells that migrated to bottom of filter in decolorization solution of 30% acetic acid and measured the absorbance at 570 nm by microplate reader (Synergy 4; BioTek).

Immunofluorescence

HTR8/SVneo cells (4x 104 per well) were seeded into the slides (BS-14-RC; Biosharp) for 20 h, and we allowed them to adhere for 20 h. After adhesion, cells were cultured with 2 uM ofNaAsC>2 for 24 h. Each group contained three biologically independent replications. Before immunofluorescence staining, slides were washed twice with phosphate-buffered saline (PBS). HTR8/ SVneo cells were fixed with precold methanol for 5 min at room temperature. Subsequently, slides were incubated in blocking buffer [1% bis(trimethylsilyl)acetamide (BSA) in PBS] for 45 min and then with rabbit anti-CYR61 (ab228592, 1:600 dilution) overnight at 4°C. After being washed with PBS, the slides were incubated with Cy3-labeled goat anti-rabbit IgG (A0516; Beyotime; 1:400 dilution) for 60 min at room temperature. Slides were washed four times with PBS. The nucleus was counterstained with Hoechst 33342 (C1025; Beyotime; 1:400 dilution) for 5 min at room temperature. Slides were quickly washed three times in PBS and coverslipped with mounting medium. All slides were observed by fluorescence microscope (BX53F; Olympus).

Isolation of Total RNA and Real-Time RT-PCR

Total RNA was extracted from mouse placentas and trophoblasts (n = 6 biologically independent samples per group) using TRI reagent. Total RNA (1.0 ug) was treated with DNase without RNase and reverse-transcribed with Promega reverse transcription system (#A3500; Promega). Real-time RT-PCR was performed with a LightCycler 480 SYBR Green qPCR Master Mix (#04887352001; Roche Diagnostics GmbH). The PCR amplification reaction was the initial holding step (95°C for 5 min) and then 50 cycles of a three-step PCR (95°C for 15 s, 60°C for 15 s, 72°C for 30 s). The primers of all genes are listed in Table S2. All primers were synthesized from Invitrogen. Relative expression of target genes was calculated through 2 method and normalized against 18S mRNA value.

Cyr61 mRNA Stability

To measure cellular Cyr61 mRNA stability, HTR8/SVneo cells (1.5X105 per well) were seeded in 6-well plates and allowed to adhere for 20 h, and then incubated with 2 uM NaAsC>2 for 24 h. Actinomycin D (addition l%o of medium volume, final concentration: 5 ug/mL) was added to HTR-8/SVneo cells. Cells were collected at different time points (0, 30, 60, 90 min). Then, cells were harvested, and RNA was extracted for real-time RT-PCR as described in section titled "Isolation of Total RNA and Real-Time RT-PCR." Each group contained three biologically independent replications. Cyr61 mRNA half-life (tl/2) was calculated using ln2/slope and normalized against 18S mRNA value.

m6A RNA Methylation Quantification

The m6A RNA methylation level of HTR8/SVneo cells were detected by EpiQuik m6 A methylation quantification kit. Total RNA was extracted using TRI reagent and purified by PolyATtract mRNA isolation systems (Z5310, Promega). Each group contained five biologically independent replications. The level of m6A in 100 ng mRNA was detected by EpiQuik m6A methylation quantification kit according to the manufacturer's instructions.

Methylated RNA Immunoprecipitation-Quantitative Real-Time Polymerase Chain Reaction (MeRIP-qPCR)

The m6A-modified Cyr61 level of HTR8/SVneo cells was measured by MeRIP-qPCR method. Each group contained three biologically independent replications. Total RNA was extracted using TRI reagent. Then, RNA was purified and fragmented. In brief, we added 100 uL RNA to the magnetic bead suspension (120 uL beads, 100 uL binding buffer). The suspension was placed in a 65 °C water bath for 5 min, on ice for 2 min and at room temperature for 10 min. We removed the supernatant andadded 40 uL water to wash the magnetic beads at 80°C for 3 min. The supernatant was removed to obtain mRNA. We next added 18 uL mRNA to 2 |±L fragmentation buffer (100 mM tris-HC1 PH 7.4, lOOmM MgCl2) and placed it on the PCR instrument at 95°C for 5 min. We added 0.5 uM EDTA to stop the reaction. Subsequently, NaAC, glycogen, and precooled ethanol were added and placed at -20°C for 30 min. The mixture was centrifuged at 15,000 Xg for 10 min. We removed the supernatant to obtain mRNA fragments. The fragments were incubated with anti-m6A antibody (202003; Affinity; 1:150 dilution) or anti-IgG (11203D; Invitrogen) in immunoprecipitation buffer (50 mM Tris PH7.4, 750 mM NaCl, 0.5% NP-40) at 4°C for 120 min. The m6A-bound RNA was precipitated by phenol-chloroform RNA extraction method. Subsequently, m6A-bound RNA was subjected to cDNA synthesis. The mixture was prepared (1 uL input RNA, 3 uL iP RNA, 0.8 uL oligo(dT)18, 0.5 uL random primer N9, 1.6 uL dNTPs mix, 6.6 uL H2O), and water bath at 65°C for 5 min. Then we added reaction solution (4 |±L 5 X first-strand buffer, 1 uL dithi-othreitol (DTT), 0.5 uL RNase Inhibitor, and 1 liL Superscript III RT) and placed the mixture in the PCR (Gene Amp PCR System 9700; Applied Biosystems) for 60 min at 50°C and 15 min at 70°C to obtain cDNA. The m6A-modified mRNA was detected by qRT-PCR method and normalized to the input. qRT-PCR was performed with a QuantStudio 5 Real-Time PCR System (Applied Biosystems). Next, 5 uL 2 X Master Mix (Arraystar Inc.), 0.5 uL 10 uM primers (Forward), 0.5 uL 10 uM primers (Reverse), 1 uL cDNA and 3 uL H20 were mixed for PCR reaction. The PCR amplification reaction was the initial holding step (95 °C for 10 min), followed by 40 cycles of PCR (95°C for 10 s, 60°C for 60 s). Cyr61 primers for MeRIP-qPCR were as follows: 1# F: 5' CAA GAA CGT CAT GAT GAT CCA 3', R: 5' CTT GTT TGT CTA GGT GTG CCC 3' (size: 154 bp); 2# F: 5' GAC TCA TTG TAG AAA GGA AGC C 3', R: 5' AAA GTA TTC TCC AAT CGTGGC3'(size:115bp).

RNA-Binding Protein Immunoprecipitation (RIP)-qPCR

The interaction between Cyr61 mRNA and IGF2BP1/2/3 was predicted by bioinformatics analysis website ENCORI (version 3.0; https://rnasysu.com/encori/index.php).54'55 RNA-binding protein (RBP)-mRNA of the RBP-target project was used to predict the interaction of IGF2BP1/2/3 targets with the binding sites of Cyr61 mRNA. Based onClipSiteNum from RBP-mRNA, we predicted whether there were potential binding sites between IGF2BP1/2/3 and Cyr61 mRNA. The binding ability of IGF2BP2 to Cyr61 mRNA was detected by RIP-qPCR assay. Each group contained three biologically independent replications. HTR8/SVneo cells (1 X 106/well) were seeded in 10 cm plates for 24 h. Then, the cells were incubated with 2 uM NaAsC>2 for 24 h. Cells were lysed in RIP lysis buffer at 4°C for 25 min. The mixture was centrifugated at 4°C for 20,000 X g for 10 min to obtain nuclear protein. We took 10 uL of nuclear protein as input, froze it at - 80° C, and finally extracted RNA. Then, nuclear protein (1 mg/mL) was incubated with anti-IGF2BP2 antibody (ab 128175; Abeam; 2 ug) or IgG-conjugated protein A/G magnetic beads overnight at 4°C. Then, the RNA-protein complexes were washed with 500 uL PBSN (PBS+1% NP-40) and 500 uL PBSE (PBS+2 mm EDTA) and then incubated with proteinase K digestion buffer to isolate the immunoprecipitated RNA. Finally, qRT-PCR was used to analyze the interaction between Cyr61 mRNA and IGF2BP2. qRT-PCR was performed with a ViiA 7 Real-time PCR System (Applied Biosystems). 5 liL 2 X Master Mix (Arraystar, Inc.), 0.5 liLIO uM primers (Forward), 0.5 uL 10 uM primers (Reverse), 2 uL cDNA, and 2 uL H2O were mixed for PCR reaction. The PCR amplification reaction was the initial holding step (95°C for 10 min), followed by 40 cycles of PCR (95 °C for 10 s, 60°C for 60 s). The primer was designedby Primer 5.0 software. Cyr61 primers for RIP-qPCR were as follows: F: 5' TGC TCA AAG ACC TGT GGA AC 3', R: 5' AAC ATC CAG CGT AAG TAA ACC T 3' (size: 196 bp). Calculate the % Input for each RIP fraction: %Input = 2(CtInPut-QRn>) x input dilution factor X 100%.

m6A Methylase Activity Assay

Epigenase m6A methylase activity/inhibition assay kit (colori-metric) was used to measure the m6A methylase activity. Nuclei were extracted from HTR-8/SVneo cells and mouse placentae using an NE-PER nuclear and cytoplasmic extraction reagents. Each group contained six biologically independent repetitions. The m6A methylase activity of nuclear extracts was analyzed with Epigenase m6A methylase activity/inhibition assay. Briefly, m6A methylase substrate was added into each sample well and incubated at 37°C for 90 min. A standard 50 uL of reaction mixture containing SAM, methylase buffer and nuclear extracts was incubated at 37°C for 90 min. The methylated m6A in the substrate was recognized with a high affinity m6A antibody and the immuno-signal was enhanced with enchanter solution. The signal was quantified through micro-plate reader at 450 nm with an optional reference wavelength of 655 nm. The methylated m6A signal intensity is proportional to m6A methylase activity.

Intracellular SAM Measurement

Intracellular SAM was detected by ExionLC liquid chromatography (SCIEX) coupled with a 3500 QTRAP tandem mass spectrometer (ABI). Each group contained six biologically independent replications. Intracellular SAM extraction referred to the previous study.56 In brief, the placenta (100 mg) was quickly frozen with liquid nitrogen and ground into fine powder. We added 600 uL 0.4 M perchloric acid. After centrifugation (4°C, 12,000 g, 20 min), the supernatant (30 uL) was taken, and 2.5 M K3PO4 was added to adjust the pH to 5-7. After standing at 4°C for 15 min, the supernatant was taken and injected into LC-MS/MS for analyzing SAM. Reverse-phase separation was performed using a 2x150 mm 4 urn Synergi Fusion-RP 80A column (Phenomenex), using a mobile phase consisting of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient program was as follows: 0-7 min, 25% B-99% B; 7.1-8 min, 75%-50% B; 8-10 min, 50%-25% B. Parameters: curtain gas, 10 psi; ionspray voltage, 5500V; ion source gas 1, 55 psi; ion source gas 2, 55 psi; temperature, 600°C; flow rate: 0.3 mL/min; sample volume: 5 uL; scanning mode: positive ion multireaction detection (MRM). Mother ion: 399.2 and daughter ion: 250.2; declustering potential, 68V; collision energy, 22V; Dwell time, 150 s.

Plasmid Transfection

Plasmid transfection was used in HTR8/SVneo cells. Two arsenic (+3) methyltransferase (As3MT) shRNA plasmids (shAs3MT 1# and shAs3MT 2#) were obtained from GenePharma. CYR61 shRNA plasmid (shCYR61) and CYR61 overexpression plasmid (CYR61 OE) were obtained from HanBio. Plasmids were transfected in HTR8/SVneo cells using GP-Transfect-Mate kit (GenePharma) following the manufacturer's instructions. In short, cells were grown to 60%-80% confluence and began to transfect. GP-Transfect-Mate reagent was added to serum-free medium. Plasmids were added to serum-free medium. Then, the mixture of GP-Transfect-Mate reagent was added to plasmids mixture and placed for 20 min. Cells were transfected for 6 h and further replaced with fresh culture medium. After transfection, As3MT knockdown in cells were selected by G418 at 400 ug/mL. Cyr61 knockdown or overexpression in cells was selected by puromycin at 3 ug/mL. Theknockdown or overexpression efficiency was determined by real-time RT-PCR. The primers of Cyr61 and As3MT were presented in Table S2. The primers were synthesized from Invitrogen. The target sequences of As3MT-, CYR61-shRNA, and CYR61-overexpression plasmid are shown in Table S3 and Table S4, respectively.

Case-Control Study

This case-control study was based on a cohort of pregnant women in the Wuxi Maternal and Child Health Hospital. According to research objective, the exclusion criteria were as follow: unavailable informed consent, cigarette smoking during pregnancy, alcohol drinking during pregnancy, hypertension syndrome, preeclampsia, gestational diabetes, and recurrent miscarriages. In this study, SGA infant was defined as newborn weight below 10th percentile for the same gender and gestational age. Appropriate for gestational age (AGA) subjects were matched to SGA subjects regarding maternal age, prepregnancy BMI, monthly income, level of education, delivery mode, infant sex, FA supplement, birth weight, birth length, and head circumference. Finally, 45 mother-child pairs per study arm were matched. The demographic characteristics are presented in Table S5. The first-morning urine and blood of pregnant women in the first trimester were collected. Blood was centrifuged at 3,500 g for 15 min at 4°C to obtain plasma. Urine was mixed and packed. Urine and plasma were stored at -80°C for measurement of As and CYR61 level, respectively. Placental tissues were immediately collected after delivery. Placental tissue of 2 X 2 cm2 were collected from the fetal side of the longest axis of the placenta. The placental membrane was trimmed and then washed by saline. Partial placental tissue was fixed in 4% paraformaldehyde for immunohistochemistry. The remaining placental tissue was frozen in liquid nitrogen and then stored in a - 80° C refrigerator for immunoblotting.

As Measurement

Hydride generation-atomic fluorescence spectrometry (PF-7) was used to determine As in the urine of pregnant women (n = 45/group), serum, and placenta of dams (ra = 8/group). In short, urine (100 |±L) of pregnant women, mouse serum (100 uL), and placenta (100 mg) were added into the mixed solution of HNO3: H2O2 (V: V = 3:l) overnight. The mixture was dried at 200°C to 200 uL volume, and then we added 1 mL 1% HNO3 for resuspension. Suspension was diluted using the mixture of 5% ascorbic acid, 5% thiourea and 3% hydrochloricacid for detection. All glass products were soaked in 10% HNO3 for at least 24 h. All reagents were guaranteed reagent grade. The As standard element solution was from the Chinese Academy of Metrological Sciences (GBW080117). The detection limit of As was 0.02 ug/dL.

Enzyme-Linked Immunosorbent Assay (ELISA)

A commercial ELISA kit was used to detect plasma CYR61 content in the pregnant women according to the manufacturer's protocol. Each group contained 45 biologically independent replications.

Measurement of Maternal Plasma Folate

The blood of pregnant women in the first trimester were collected. Blood was centrifuged at 3,500 Xg for 15 min at 4°C to obtain plasma and stored at -80°C. Maternal plasma folate content was measured using folate detection kit (chemiluminescence immunoassay) (Lot no. 2021040100; Mindray) by the Mindray CL-6000i system. Each group contained 45 biologically independent replications.

Statistical Analysis

The data from animal and cell experiments were expressed as means ± standard error of the mean (SEM) with dots indicating individual values. Population data were expressed as mean± standard deviation (SD) with dots indicating individual values. According to a previous study,57 confounding factors that might affect the association among maternal urinary As concentration and SGA infants were selected as follows: maternal age (<24y, 25-29 y, and >30 y), prepregnancy body mass index (BMI) (<18.5, 18.5-24.9, and >25.0kg/m2), monthly income (<3,000, 3,000-5,999 and >6,000 CNY), level of education (<9, 9-15, and >15 y), delivery mode (natural labor and cesarean section), and infant sex (male and female). The sample size was shown in each method and figure legend. Normally distributed data were analyzed by Student's f-test for the differences between two groups. Analysis of variance (ANOVA) was used for analyzing the differences among four groups. When ANOVA showed p<0.05, the Student-Neuman-Keuls (SNK) multiple comparisons tests were used. The Spearman correlation was used for analyzing the correlation between two groups; p < 0.05 was considered statistically significant.

Results

Serum As Content in Pregnant Mice

Pregnant mice were exposed to different concentrations of NaAsC>2 [0 (control), 0.15, 1.5, or 15 mg/L] by drinking water from GD0 to GD18. Serum As content of all dams was measured. As shown in Table 1, mean As content in maternal serum was 7.45 ± 0.57 ug/L in control dams. Mean As content in maternal serum was higher by some 5-folds (36.08 ±4.31 vs. 7.45 ± 0.57 ug/L) in As-L group. Moreover, mean As content in maternal serum was higher by some 7-folds (55.47 ±4.26 vs. 7.45 ± 0.57 ug/L) in As-M group. Finally, mean As content in maternal serum was higher by some 20-folds (152.94 ±21.79 vs. 7.45 ± 0.57 ug/L) in As-H group.

Effects of Gestational As Exposure on Fetal and Placental Development in Mice

The effects of gestational As exposure on the pregnant outcomes were investigated. As shown in Table 2, neither dam death nor preterm delivery was observed throughout pregnancy. No statistically significant difference in the number of dead fetuses per litter, the number of live fetuses per litter, and sex ratio was observed among different groups. As shown in Table 2, the number of resorptions per litter was higher in As-exposed mice. The effects of gestational As exposure on fetal weight and crown-rump length were furtheranalyzed. Fetal weight and crown-rump length were lower in the As-M and As-H groups than the control group (Table 2). Placental As contents were then analyzed. Results found that placental As contents were higher by 4.3-folds (53.08 ±8.80 vs. 12.32 ± 5.87 ng/g), 15-folds (183.38 ± 17.84 vs. 12.32 ± 5.87 ng/g), and 57-folds (703.81 ± 34.29 vs. 12.32 ±5.87 ng/g) in the As-L, As-M, and As-H groups, respectively (Table 1), suggesting that As could accumulate in the placenta. The effects of gestational As exposure on placenta development were evaluated. As expected, placental weight and diameter were markedly lower in the As-H group (Table 2). H&E staining revealed an obviously thicker junctional zone and thinner labyrinth zone in As-exposed mouse placentas (Figure SI A). As shown in Table 2, the percentage of labyrinth zone area was lower in the As-H group. Moreover, the ratio of cross-sectional thickness of labyrinth zone to junctional zone was lower in the As-M and As-H groups than in that of control group (Table 2). The expansion of junctional zone seems to be due primarily to the increased numbers of vacuolated glycogen trophoblast cells. Placental glycogen accumulation was confirmed with PAS staining (Figure SIB) and quantification of tissue glycogen content. As shown in Table 2, glycogen-positive trophoblast cells in junctional zone were obviously higher in the As-M and As-H groups than in that of control group. In addition, placental glycogen content was accordingly higher in the As-M and As-H groups (Table 2).

Effects of As Exposure on Interstitial Migration and Invasion in Mouse Placentae and Human Placental Trophoblasts

It was widely accepted that murine glycogen trophoblast cells were similar to human extravillous cytotrophoblast cells.58 Mouse glycogen trophoblast cells break through junctional zone and invade interstitially into maternal decidua starting at GDI2 and continuing until GD18.59'60 All pregnant mice were exposed to the control or NaAsC>2 by drinking water from GDO to GDI8. To enable analysis of the effects of gestational As exposure on interstitial invasion of placental trophoblasts, all pregnant mice were sacrificed on GD18 to obtain the placenta. Based on the histological evaluation of the placenta after gestational exposure to NaAsC>2, specifically the observation that mice developmentally exposed to 15 mg/L NaAsC>2 had a smaller labyrinth zone relative to whole placenta and abnormal accumulation of glycogen-positive cells in junctional zone (Figure S1; Table 2), the following experiments were conducted onthe As-H group. As shown in Figure 1A, protein expression of E-cadherin, an epithelial marker, was higher in the As-H group than in the control group. By contrast, protein expression of vimentin and N-cadherin, two mesenchymal markers, were lower in the As-H group. Protein expression of MMP2 and MMP9 were lower in the As-H group (Figure IB). To further explore the mechanism, we established an in vitro model of arsenic exposure. As shown in Figure S2, HTR8/S Vneo cells were treated with different concentrations of NaAs02 (0, 0.3125, 0.625, 1.25, 2.5, 5, and 10 uM) for 24 h. Cell viability was lower in a concentration-dependent manner (0.3125 uM, 95.51 ±0.64% of control; 0.625 uM, 93.82 ± 1.36% of control; 1.25 uM, 86.86 ±2.95% of control; 2.5 liM, 85.23 ±0.88% of control; 5 uM, 62.33 ±2.05% of control; and 10 uM, 49.06 ±0.46% of control). To avoid excessive cell death, we selected exposure dose as 2 uM for subsequent experiments. Cell migration ability was determined by wound-healing assay and transwell migration assay. Wound-healing assay showed that the percentage of wound closing was lower in As-exposed HTR8/S Vneo cells (Figure 1C). Transwell migration assay showed that migration ability was lower in As-exposed HTR8/SVneo cells (Figure ID). Cell invasion was further analyzed using transwell invasion assay. As expected, invasion ability was lower in As-exposed HTR8/SVneo cells (Figure IE). Finally, the protein expression of occludin, a marker of tight junction, was higher in As-exposed HTR8/SVneo cells. Protein expression of N-cadherin and vimentin, two mesenchymal markers, was lower in As-exposed HTR8/SVneo cells (Figure IF), whereas MMP2 and MMP9 were lower expressed in As-exposed HTR8/SVneo cells (Figure 1G).

Identification of a Candidate Gene to Study Further

We performed transcriptome RNA sequencing to screen key genes which were involved in As-evoked inhibition of migration and invasion in mouse placental trophoblasts. As shown in Figure 2A, a total of 2,632 genes were found to be differentially expressed in As-exposed placentas, of which 743 were up-regulated and 1,889 were down-regulated by at least 1.5-fold. KEGG analysis revealed the enriched biological pathways related to cell invasion, including cytokine-cytokine receptor interaction pathway, cell adhesion molecules pathway, and ECM-receptor interaction pathway (Figure 2B). To further gain the key genes involved in the regulation of cell invasion, we screened the top 100 genes based on the p-value ranking of all differential genes and then screened 40 candidate genes among them, with the average fragments per kilobase per million (FPKM) >10 of allsamples as the filter condition. As shown in Figure 2C, 25 genes were up-regulated, and 15 genes were down-regulated in As-exposed placentas. Five genes are involved in the regulation of trophoblast function, including SPARC-like protein 1 (Sparcll),61 carcinoembryonic antigen-related cell adhesion molecule (Ceacam) 13,62 Ceacam3,62 transforming growth factor beta 2 (TGF-(32),63 and cysteine-rich angiogenic inducer 61 (Cyr61).M To avoid the false positive results, p-value was subject to multiple comparisons. The adjusted p-values of five candidate genes are shown in Table S6. Furthermore, five candidate genes were verified using real-time RT-PCR. In comparison with the control group, Sparcll, Ceacaml3, and Ceacam3 mRNAs were up-regulated in the As-H group. In contrast, Cyr61 mRNA was down-regulated in the As-H group (Figure 2D). There was no difference in placental Tgffil mRNA between the As-H group and control group (Figure 2D). Immunoblotting showed no difference on TGF-(32 protein between the As-H group and the control group (Figure S3 A). The mRNA expression of TGF-(3 superfamily members, such as Tgffil, Tgffi2, Tgffirl, Bmp2, Bmp6, Nodal, Acvr2a, and Acvrl, were analyzed by RNA-seq. Unexpectedly, above eight genes were not altered in As-exposed mouse placentas (Figure S3B), suggesting that TGF(3 was not involved in As-induced suppression of placental trophoblast migration and invasion. To screen the fold change of candidate genes exceeding 1.5, we compared the fold change of five candidategenes between RNA-seq and RT-PCR. As shown in Figure 2E, only Sparcll and Cyr61 had > 1.5-fold changes in both the RNA-seq and RT-PCR. Indeed, both Sparcll61 and Cyr6164 are associated with trophoblast migration. In comparison with Sparcll gene, the fold change of Cyr61 was more obvious (Figure 2E). Next, CYR61 in mouse placentas was further detected. Accordingly, placental CYR61 protein expression was lower in the As-H group (Figure 2F). Immunohistochemistry showed that CYR61-positive cells were located in placental labyrinth zone and junctional zone of both control and As-H mice (Figure 2G). The percentage of CYR61-positive cells in both labyrinth zone and junctional zone was lower in the As-H group than those control group (Figure S4A,B). Furthermore, CYR61 expression was also analyzed in As-exposed HTR8/SVneo cells. As shown in Figure 2H,I, Cyr61 mRNA and protein levels were lower in As-exposed HTR8/SVneo cells. Immunofluorescence showed that CYR61-positive cells were lower in As-exposed HTR8/SVneo cells than in that of control group (Figure 2J).

Role ofCYR61 in Migration and Invasion of As-Exposed Human Placental Trophoblasts

To explore the role of CYR61, we established HTR8/SVneo cells with CYR61 overexpression and knockdown, respectively. Efficiency of Cyr61 overexpression was verified by real-timeRT-

PCR (Figure 3A). As expected, CYR61 overexpression attenuated As-induced down-regulation of vimentin, N-cadherin, and MMP2 (Figure 3B). Moreover, CYR61 overexpression partially restored As-evoked down-regulation of migration and invasion abilities, determined by transwell migration and invasion assays, respectively (Figure 3C,D). Oppositely, Cyr61 knockdown was performed, as verified by real-time RT-PCR (Figure 3E). In comparison with the control group, cells with knockdown of CYR61 had lower protein expression of vimentin, N-cadherin, and MMP2 (Figure 3F). Hence, CYR61 knockdown aggravated As-induced downregulation of vimentin, N-cadherin, and MMP2 (Figure 3F). Correspondingly, transwell migration and invasion assays showed that CYR61 knockdown inhibited migration and invasion abilities in HTR8/SVneo cells (Figure 3G,H). CYR61 knockdown exacerbated As-induced inhibition of migration and invasion (Figure 3G,H).

Role ofm6A Modification on Cyr61 mRNA Stability and CYR61 Protein in As-Exposed Human Placental Trophoblasts

The mRNA stability affects mRNA levels which in turn, impact protein production.65-67 Actinomycin D chase experiment was used to evaluate Cyr61 mRNA stability. As shown in Figure 4A, Cyr61 mRNA stability was lower following As exposure. Them6A modification influences mRNA stability.68 The m6A modification was analyzed in As-exposed HTR8/SVneo cells. Unexpectedly, a higher m6 A modification was shown in total RNAs of As-exposed HTR8/SVneo cells (Figure 4B). Using the SRAMP tool, a prediction tool of mammalian m6A sites,69 we analyzed m6 A modification sites of Cyr61 mRNA and found five RRACH sequences sites (Figure 4C). The m6A modification of Cyr61 mRNA was further determined by MeRIP-qPCR. As shown in Figure 4D, Cyr61mRNA was enriched in the anti-m6 A group in comparison with the IgG group. Of interest, enrichment of Cyr61 mRNA m6A-specific antibodies was obviously down-regulated in As-exposed HTR8/ SVneo cells (Figure 4D). IGF2BP family members (IGF2BP1/2/ 3) recognize m6A modification sites to enhance mRNA stability.68 Hence, the interaction between Cyr61 mRNA and IGF2BP1/2/3 was then predicted by the bioinformatics analysis website ENCORI (https://rnasysu.com/encori/index.php).54'55 As shownin Figure 4E, there were 1, 21, and 2 potential binding sites between Cyr61 mRNA and IGF2BP1, IGF2BP2 and IGF2BP3, respectively. Hence, we speculate that IGF2BP2 is most likely to bind to Cyr61 mRNA in comparison with IGF2BP1 and IGF2BP3. RIP-qPCR assay revealed that IGF2BP2 bound to Cyr61 mRNA. Of interest, binding ability between Cyr61 mRNA and IGF2BP2 was markedly lower in As-exposed HTR8/SVneo cells (Figure 4F).

Role ofmA Methylase on Cyr61 m A Modification in As-Exposed Human Placental Trophoblasts

The expression of m6A methyltransferases and demethylase was first detected using real-time RT-PCR. No difference in mRNAs of m6A methyltransferases, including Mettl3, MettU4, Wtap, Rbml5, and Rbml5b, was observed between As-exposed HTR8/ SVneo cells and controls (Figure 4G). The mRNA expression of Alkbh5, an m6A demethylase gene, was lower in As-exposed HTR8/SVneo cells, whereas of Fto, another m6A demethylase gene was not significantly different between As-exposed and vehicle-exposed cells (Figure 4G). Of interest, m6A methyltrans-ferase activity was lower in As-exposed HTR8/SVneo cells (Figure 4H). SAM is not only a methyl donor for methylation reactions but also a cofactor for the m6A methyltransferase catalytic domain,37'38 as visualized in Figure 41. Intracellular SAM, determined by LC-MS/MS, was obviously lower in As-exposed HTR8/SVneo cells than in that of control group (Figure 4J).

Effect of As3MT Knockdown on Cyr61 m A Modification in As-Exposed Human Placental Trophoblasts

As3MT catalyzes methylation of iAs3+ using SAM as a substrate.70 To test the influence of SAM depletion on As-induced inhibition of cell migration of invasion, two shRNA constructs were used to selectively knockdown As3MT (Figure 5A, shAs3MT 1# and shAs3MT 2#). As expected, cells with As3MT knockdown exhibited levels of SAM that were similar to control cells, even after exposure to As (Figure 5B). Correspondingly, cells with As3MT knockdown had similar levels of m6 A methylase activity as control after exposure to As (Figure 5C). Moreover, cells with As3MT knockdown had levels of Cyr61 mRNA stability (Figure 5D,E) and CYR61 protein (Figure 5F) similar to that of the control, even after exposure to As. In addition, cells with As3MT knockdown exposed to As had similar protein levels of vimentin, N-cadherin, and MMP2 as control (Figure 5F), suggesting that knockdown reversed As-induced down-regulation of these proteins. Transwell migration and invasion assays showed that As-exposed HTR8/SVneo cellswith As3MT knockdown were similar in migration and invasion capabilities to the control, suggesting that knockdown attenuated the As-induced inhibition of these processes (Figure 5G,H).

Effect of SAM Supplementation on Cyr61 m6A Modification in As-Exposed Human Placental Trophoblasts

To further observe the role of SAM depletion on As-induced inhibition of Cyr61 mRNA m6A modification, exogenous SAM was supplemented in an in vitro model of arsenic exposure. As shown in Figure S5, HTR8/S Vneo cells were treated with different concentrations of SAM (0,5,10,20,40,80,160, and 320 uM) for 24 h. Cell viability was reduced in a concentration-dependent manner (5 uM, 96.52 ±3.16% of control; 10 uM, 93.70 ±3.74% of control; 20 uM, 84.77 ± 6.11% of control; 40 uM, 81.85 ± 2.87% of control; 80 uM, 78.93 ± 2.90% of control; 160 uM, 74.43 ± 8.71% of control; and 320 uM, 79.62 ± 4.58% of control). To avoid excessive cell death, we selected exposure dose as 10 uM for subsequentexperiments. As expected, supplementation with SAM reversed As-induced reduction of intracellular SAM content (Figure 6A). Accordingly, supplementation with SAM reversed m6A methyl-ase activity in As-exposed HTR8/SVneo cells (Figure 6B). Moreover, SAM supplementation attenuated As-induced downregu-lation of Cyr61 mRNA stability and CYR61 protein (Figure 6C,D). In addition, SAM supplementation attenuated As-induced down-regulation of vimentin, N-cadherin, and MMP2 in HTR8/S Vneo cells (Figure 6E). Finally, SAM supplementation reversed As-induced inhibition of cell migration and invasion in HTR8/ S Vneo cells (Figure 6F,G).

Effect of FA Supplementation on Gestational Placental Development and Fetal Growth Restriction in As-Exposed Mice

To enable observation of the effect of supplementation with FA on As-induced placental development impairment and fetalgrowth restriction, pregnant mice in the FA and As+FA groups were administered 150 ug/kg FA by gavage from GDO to GD17. As shown in Table S7, neither dam death nor preterm delivery was observed throughout pregnancy. No significant difference in the number of dead fetuses per litter, the number of live fetuses per litter, and sex ratio was found among different groups. As shown in Table S7, although the number of resorptions per litter was higher in As-exposed mice than in that of the control group, the number of resorptions was not significantly different between As and As+FA-exposed mice. The effects of FA supplementation on As-induced fetal growth restriction were further analyzed. As we expected, there were differences in fetal weight and crown-rump length between As-exposed and As+FA-exposed mice, with mice exposed to As+FA having greater weight and longer crown-rump length, more similar to control (Figure 7A,B). Placental weight and diameter were then detected. Accordingly, placental weight and diameter was higher in the As+FA-exposed group in comparison with the As-exposed group, similar to control (Figure 7C,D). The pathological morphology of the placenta is shown in Figure 7E. As expected, the percentage of labyrinth zone area in the entire placenta area was lower in As-exposed mice (Figure 7F). Moreover, the ratio of cross-sectional thicknessof labyrinth zone to junctional zone was lower in As-exposed mice (Figure 7G). Of interest, FA supplementation reversed As-evoked pathological damage in mouse placenta (Figure 7F,G). Next, the effects of FA supplementation on placental SAM content and m6A methylase activity were analyzed in As-exposed mice. As expected, placental SAM content and m6A methylase activity were lower in As-exposed mice (Figure 7H,I). Of interest, As-induced reduction of placental SAM content and m6A methylase activity was reversed in mice pretreated with FA (Figure 7H,I). Finally, placental CYR61 and MMP2 protein expression was higher in the As+FA-exposed mice in comparison with the As-exposed mice, suggesting that FA attenuated As-associated differences in these proteins (Figure 7J).

Based on a Case-Control Study: Association among Maternal Urinary As Concentration, Plasma Folate Content, Plasma CYR61 Content, and FGR

Maternal urinary As concentration and plasma CYR61 content in AGA and SGA infants were compared. As shown in Figure 8A, maternal urinary As concentration was higher in SGA infants than in matched AGA infants. By contrast, maternal plasma

CYR61 content in the first trimester was lower in SGA infants than in AG A infants (Figure 8B). The correlation between maternal urinary As concentration and plasma CYR61 content was then analyzed in AGA infants and SGA infants, respectively. In AGA infants, there was a weak negative correlation between maternal urinary As concentration and plasma CYR61 content (Figure 8C; r = - 0.0760, p = 0.0636). In SGA infants, there was a significant negative correlation between maternal urinary As concentration and plasma CYR61 content (Figure 8D; r= -0.2551, p = 0.0004).

Immunohistochemistry showed that the percentage of placental CYR61-positive cells was lower in SGA infants than in AGA infants (Figure 8E). Next, several invasion-related proteins in AGA and SGA placentas were compared. As shown in Figure 8F, the levels of vimentin, MMP2, and MMP9 were lower in SGA placentas than in AGA placentas. In addition, immunohistochemistry showed that vimentin-, MMP2-, and MMP9- positive cells were lower in SGA placentas than in AGA placentas (Figure S6A-S6C). The effect of maternal plasma folate between maternal urinary Asconcentration and plasma CYR61 content was further analyzed. Maternal plasma folate content in AGA and SGA infants was compared. As shown in Figure S7A, no significant difference in plasma folate content was observed between AGA and SGA infants. In AGA infants, there was no correlation between maternal urinary As concentration and plasma folate content (Figure S7B; r = 0.0034, p = 0.7026). In SGA infants, there was a significant negative correlation between maternal urinary As concentration and plasma folate content (Figure S7C; r= -0.0914, p = 0.0435). Moreover, the correlation between maternal plasma folate and CYR61 contents was evaluated. However, there were no correlation between maternal plasma folate and CYR61 contents in AGA (Figure S7D; r = 0.0637, p = 0.0945) and SGA (Figure S7E; r = 0.0768, p = 0.0652) infants, respectively.

Discussion

The current study aimed to investigate the effects of gestational As exposure on placental and fetal development and its underlying mechanism. The major findings were as follows: First, mice exposed to As during gestation were smaller (lower fetal weightand shorter crown-rump length) than those from control dams, suggesting FGR; second, placental trophoblasts from humans and mice demonstrated a lower ability to migrate and invade when exposed to As; third, trophoblast cells exposed to As had lower CYR61 expression and results suggested that CYR61 was involved in As-evoked inhibition of interstitial migration and invasion in trophoblast cells; fourth, CYR61 down-regulation was partially attributed to As-evoked reduction of Cyr61 m6A modification and Cyr61 mRNA stability; last, we concluded that SAM depletion-mediated suppression of m6A methyltransferase activity contributed to As-induced reduction of Cyr61 m6A modification. These results suggest that SAM depletion-mediated down-regulation of Cyr61 m6A modification was partially involved in As-induced defective trophoblastic invasion and FGR.

Numerous epidemiological investigations have suggested an association between gestational As exposure and FGR.19-21 In our study, an animal model of gestational As exposure was established, during which maternal serum As contents in control dams were similar with mean serum As content in Chinese pregnant women (5.10 ng/L).19 Maternal serum As contents in As-L group were close to the upper limit of serum As content in the pregnant women (Canada, 34.46 ug/L; China, 43.52 ug/L).19'49 Moreover, maternal serum As contents in As-M group were slightly higher than the upper limit of serum As content in the pregnant women (Canada, 34.46 ug/L; China, 43.52 ug/L).19'49 Finally, maternal serum As contents in As-H group was four times higher than the upper limit of serum As content in the pregnant women (Canada, 34.46 ug/L; China, 43.52 ug/L).19'49 Francisca et al.44 reported that the arsenic concentrations in groundwater is up to 1.8 mg/L in Cordoba Province, Argentina. It is incredible that the highest As concentration in La Franica town was 12 mg/L. In present study, animal experiments found that fetal weight and crown-rump length were lower in As-exposed mice. These results provide novel evidence supporting the hypothesis that gestational As exposure can induce FGR. Accumulating evidence has demonstrated that placental dysplasia, caused by insufficient invasion of EVTs, is a frequent etiology of FGR.71'72 Several studies in mice showed that gestational exposure to environmental toxicants led to FGR by impairing placental development.73'74 In this study, we found that placental sizes were lower and the thickness of placental labyrinth zone was lower in As-exposed mice. Glycogen-positive cells, a marker of insufficient trophoblast invasion, were higher in the junctional zone of As-exposed mice. A series of in vitro tests demonstrated that E-cadherin, an epithelial marker, was up-regulated in As-exposed trophoblast cells. In contrast, vimentin and N-cadherin, two mesenchymal markers, were down-regulated in As-exposed trophoblast cells. Moreover, migration and invasion were suppressed in As-exposed trophoblast cells. These results suggest that gestational As exposure impairs placental and fetal development through inhibiting trophoblast migration and invasion.

It is widely accepted that TGF- (3 superfamily play pivotal roles in the modulation of EVT invasion during human placentation.25 In the current study, transcriptome RNA-seq was used to screen key genes involved in As-induced inhibition of trophoblast migration and invasion. Unexpectedly, mRNA expression of TGF-(3 superfamily members, such as Tgffil, Tgffi2, Tgffirl, Bmp2, Bmp6, Nodal, Acvr2a, and Acvrl, was not altered in As-exposed mouse placentas. Of interest, placental Cyr61 mRNA was down-regulated in As-exposed mouse. In the placenta, Cyr61 is expressed in trophoblastic giant cells, ectoplacental cone, and endothelial cells.75 Kipkeew et al.76 found that CYR61 might promote the migration ability of SGHPL-5 trophoblasts by the focal adhesion kinase (FAK) signaling pathway. Moreover, CYR61 is involved in modulation of EVT invasion in the process of human placentation.77'78 In the present study, we showed that CYR61 overexpression partially restored As-induced down-regulation of migration and invasion abilities. Oppositely, CYR61 knockdown exacerbated As-evoked inhibition of migration and invasion in HTR8/SVneo cells. Several studies demonstrated that placental CYR61 down-regulation was associated with preeclampsia.79'80 In this study, we measured CYR61 protein in human samples, mouse placentas, and trophoblast cells. In human samples, plasma CYR61 content was lower in SGA mothers than that in the AGA mothers. There were fewer CYR61-positive cells in SGA placentas. Moreover, there was a negative association between plasma CYR61 and urinary As in SGA subjects. The in vivo and in vitro experiments found that CYR61 protein was down-regulated in As-exposed mouse placentas and human trophoblast cells. Therefore, it is reasonable to assume that down-regulation of CYR61 might contribute, at least partially, to As-evoked inhibition of trophoblast migration and invasion.

The mRNA stability affects mRNA level, which in turn impacts protein production.65-67 The current results showed that Cyr61 mRNA stability was lower in As-exposed trophoblast cells. Recently, it is accepted that m6A modification influences mRNA stability.68 A recent report indicated that placental m6A down-regulation reduced Cyr61 mRNA stability, thereby inhibiting trophoblast invasion in patients with recurrent miscarriages.81 Next, we analyzed the influence of As on Cyr61 m6A modification. Of interest, Cyr61 m6A modification was down-regulated in As-exposed trophoblast cells. Recently, IGF2BPs, a family of m6A readers, recognized m6A modification sites to enhance mRNA stability.68 In this study, the interaction between Cyr61 mRNA and IGF2BP2 was determined using RIP-qPCR. As expected, a strong binding ability was observed between Cyr61 mRNA and IGF2BP2. Of interest, the binding ability of Cyr61 mRNA with IGF2BP2 was suppressed in As-exposed trophoblast cells. These results indicate that reduction of Cyr61 mRNA stability and CYR61 protein may be attributed to As-induced down-regulation of Cyr61 m6 A in trophoblast cells.

Accumulating data have demonstrated that methyltransfer-ases, such as METTL3 and METTL14, and demethylases, mainly FTO and ALKBH5, dynamically regulate RNA m6A modification.35'36 Several studies showed that environmental toxicants altered RNA m6A modification by regulating the expression of methyltransferases.82'83 Unexpectedly, our results showed that mRNA expression of RNA methyltransferases, including MettB, Mettlli, Wtap, RbmlS, and RbmlSb, was not altered in As-exposed trophoblast cells. The mRNA level of Alkbh5, an RNA demethylase gene, was down-regulated in As-exposed trophoblast cells. These results cannot explain As-induced down-regulation of Cyr61 m6A modification. Several studies have indicated that MettB, the catalytic subunit of RNA methyltransferases, contains a catalytic domain with a cavity that can accommodate SAM.37'38 Indeed, SAM provides a methyl donor for RNA m6A modification.84 In this study, SAM content and m6A methyltransferase activity were measured. As expected, SAM content was lower and m6A methyltransferase activity was inhibited in As-exposed trophoblast cells. It is well known that As3MT catalyzes iAs3+ methylation using SAM as a substrate.70 To determine the role of As-evoked SAM depletion on RNA m6A modification, the As3MT gene was knocked down in trophoblast cells. In addition, exogenous SAM was supplemented in As-exposed trophoblast cells. Of interest, either As3MT knockdown or SAM supplementation reversed As-evoked reduction of SAM content and m6A methyltransferase activity in trophoblast cells. Moreover, either As3MT knockdown or SAM supplementation restored Cyr61 mRNA stability in As-exposed trophoblast cells. Finally, either As3MT knockdown or SAM supplementation attenuated inhibition of migration and invasion in As-exposed trophoblast cells. These results provide evidence that As-evoked CYR61 down-regulation may be attributed to reduction of Cyr61 m6A modification and mRNA stability. Intracellular SAM depletion-mediated suppression of m6A methyltransferase activity may be involved in As-induced reduction of Cyr61 m6A modification.

It is well known that folate is a precursor of intracellular SAM synthesis. SAM is the universal methyl donor for methylation reactions, including histone,85 DNA,86 and RNA87 methylation. However, inorganic As is methylated by As3MT, also using SAM as a methyl group to form monomethylarsonic acid and dimethy-larsonic acid, promoting the excretion of As.88 Two randomized double-blind controlled trials demonstrated that FA supplementation promoted As methylation metabolism and thereby reduced blood As concentration in As-exposed adults.89'90 An animal experiment showed that gestational FA supplementation improved As-evoked hyperglycemia and insulin resistance in adult male offspring.91 Moreover, FA supplementation prevented As-evoked abnormal cardiac development in zebrafish embryos.92 We investigated the effects of supplementation with FA on placental and fetal development in As-exposed mice. As expected, As-induced reduction of placental SAM content, and m6A methyltransferase activity was reversed by FA supplementation. Moreover, As-induced down-regulation of placental CYR61 was attenuated in mice pretreated with FA. In addition, As-induced inhibition of trophoblast migration and invasion was alleviated in FA-pretreated mice. Finally, As-induced defective trophoblastic invasion and FGR were prevented by FA supplementation. Our previous population study suggested that folate reduction may be a mediator between gestational As exposure and FGR.50 In present study, we found no difference in whether the mothers of AGA and SGA infants received FA supplementation. There was a negative correlation between maternal urinary As concentration and plasma folate content in SGA subjects. Surprisingly, there was a weak correlation between maternal plasma folate and CYR61 contents in SGA subjects. Taken together, these results provide evidence that FA might be used for the clinical prevention of As-evoked FGR.

In conclusion, the current results suggest that gestational As exposure induced defective trophoblastic invasion and FGR through inhibiting trophoblast migration and invasion. The in vivo and in vitro experiments support a model in which CYR61 down-regulation was involved in As-induced inhibition of migration and invasion in trophoblast cells. Mechanistically, CYR61 down-regulation was attributed to reduction of Cyr61 m6A modification and Cyr61 mRNA stability. SAM depletion-mediated suppression of m6A methyltransferase activity partially contributed to As-induced reduction of Cyr61 m6A modification.

Limitations of Study

This study had several flaws. First, differential transcriptional expression of placenta is sex-dependent.93 However, we randomly selected placentas from different dams for RNA-seq analysis, without gender distinction. We need to consider the gender in placental RNA-seq to verify our study. Second, we reported that CYR61 down-regulation was partly due to reduction of Cyr61 m6A modification and Cyr61 mRNA stability. However, inorganic As accepts methyl groups from SAM, producing monomethylarsonic acid and dimethylarsonic acid. Hence, epitranscriptomics might be relevant in this study, but we did not rule out other modes of regulation (e.g., DNA methylation, miRNA). Finally, the case-control study in our research is a small sample size. We need to expand the population sample to verify our findings.

Acknowledgments

The authors thank all participants in the case-control study and the medical staff for collecting the recruited specimens at Wuxi Maternal and Child Health, China.

Funding was received from the National Natural Science Foundation of China, grant #81930093 (D.X.X) and #82173565 (C.Z.), and the Natural Science Foundation of Anhui province, #2208085MH205 (Y.J.F.). RNA-seq was supported by Genesky Biotechnologies.

Conceptualization: D.-X.X., W.W., and H.W. Methodology: Y.-P.S., J.-W.L., Z.-Z.C, Q.-Q.H, F.-X.X., and Y.H. Investigation: D.-Z.C, H.Z., and Y.-J.F. Visualization: D.-X.X. and Y.-P.S. Funding acquisition: D.-X.X., CZ. and Y.-J.F. Project administration: H.W. and D.-X.X. Supervision: D.-X.X. and W.W. Writing-original draft: D.-X.X. and Y.-P.S. In addition, D.-X.X. and Y.-P.S. accessed and verified the data and were responsible for the decision to submit the manuscript; Writing-review and editing: D.-X.X. and Y.-P.S. All authors approved the final manuscript.

All data needed to evaluate the conclusions in the paper were present in the paper and the Supplementary Materials (Table Sl-S8; Figure S1-S7). RNA-seq data were uploaded to the National Center for Biotechnology Information (GSE222092).

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