Bioscience Reports (2019) 39 BSR20182022 https://doi.org/10.1042/BSR20182022 Research Article HOXD8/DIAPH2-AS1 epigenetically regulates PAX3 and impairs HTR-8/SVneo cell function under hypoxia Yaling Feng1,*, Jianxia Wang2,*,YueHe2, Heng Zhang3, Minhui Jiang1, Dandan Cao1 and Aiping Wang2 1Department of Perinatal Health Care, Wuxi Matemal and Child Health Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu Province 214002, PR China; 2Department of Women Health Care, Wuxi Matemal and Child Health Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu Province 214002, PR China; 3Department of Child Health Care, Wuxi Matemal and Child Health Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu Province 214002, PR China Correspondence: YalingFeng([email protected]) The present study aimed to unravel the molecular basis underlying PAX3 down-regulation, known to be involved in pre-eclampsia (PE) occurrence and development. Data obtained from databases suggested that Pax3 methylation levels in the promoter region are high in the placentas of PE patients. However, the expression of methylation-adjusting en- zymes, including DNMT1, LSD1, and EZH2, did not change. Since lncRNAs enhance the function of methylation-related enzymes independently of expression, we selected three lncRNAs, RP11-269F21.2, DIAPH2-AS1, and RP11-445K13.2, predicted to interact with methylation-adjusting enzymes. Two transcription factors, HOXD8 and Lhx3, predicted to regulate the expression of lncRNAs, were also selected. Using RNA interference technol- ogy, HOXD8 and Lhx3 were found to positively regulate DIAPH2-AS1 and RP11-445K13.2 in HTR-8/SVneo cells. Chromatin immunoprecipitation assays determined that DIAPH2-AS1 recruited LSD1 to histone 3, increasing DNMT1 stability at H3. The HOXD8/DIAPH2-AS1 network regulated HTR-8/SVneo cell function under hypoxia by epigenetically regulating PAX3. This regulatory network may thus be responsible for PAX3 down-regulation in the placentas of PE patients. Introduction Pre-eclampsia (PE) is a common obstetrical disorder with characteristic symptoms including hyperten- sion and proteinuria. It has emerged as an important cause of maternal and perinatal morbidity and mor- tality for which there is currently no effective treatment except the termination of pregnancy [1]. The pathogenesis of PE is likely to be related to the impaired development of the placenta, which consequently causes a series of pathological processes, such as endothelial dysfunction, systemic inflammation, and ox- idative stress [2]. Our previous study identified that abnormal down-regulation of PAX3 expression plays a critical role in the occurrence and development of PE. Trophoblasts with a low PAX3 expression show * These authors contributed inhibited proliferative and invasive capacities [3], whereas the invasion of the foetally derived extravillous equally to this work. trophoblasts (EVTs) toward the myometrium layer of the uterine wall is a key step in placenta development Received: 05 November 2018 [4,5]. An impaired invasion of the trophoblasts is likely to cause defective arterial remodeling, resulting Revised: 18 December 2018 in inadequate blood flow in the placenta [6]. Accepted: 04 January 2019 The expression of most genes is generally regulated by complex molecular mechanisms involving many factors, including transcription factors, long non-coding RNAs, and enzymes regulating the methylation Accepted Manuscript Online: 09 January 2019 of genes and histones. Among these, long non-coding RNAs (lncRNAs), a new member of the non-coding Version of Record published: RNA family (ncRNA), can function as signals, decoys, guides, or scaffolds to participate in intertwined 25 January 2019 gene regulatory networks at various levels [7]. LncRNAs harbor many miRNA response elements and c 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 1 License 4.0 (CC BY). Bioscience Reports (2019) 39 BSR20182022 https://doi.org/10.1042/BSR20182022 Table 1 Clinical information of healthy controls and PE patients Control (N=10) PE (N=10) P-value + + Age (year) 30.5 − 4.37 31.3 − 4.84 0.063 + + Gestational age (week) 39.6 − 5.42 37.2 − 4.93 0.041 + + SBP (mmHg) 116.7 − 20.92 171.5 − 29.41 <0.01 + + DBP (mmHg) 77.6 − 13.78 103.8 − 19.6 0.025 + Proteinuria (g/24 h) N/A 3.27−0.59 N/A Abbreviations: DBP, diastolic pressure; SBP, systolic pressure. therefore can communicate and regulate other RNA transcripts by competing specifically for shared miRNAs. More- over, mounting evidence have shown that some lncRNAs can coordinate histone modifications by binding to vari- ous histone modification enzymes, such as lysine-specific demethylase 1 (LSD1) and enhancer of zeste homolog 2 (EZH2). LSD1 demethylates mono- and di-methylated residues of lysine-4 on histone H3 (H3K4me1, H3K4me2, and H3K9me1) and results in transcriptional repression, but LSD1 also activates transcription through the demethyla- tion of H3K9me2 [8]. In addition to histone 3, LSD1 also demethylates and thus stabilizes DNA methyl-transferase 1 (DNMT1). Targeted deletion of the gene encoding LSD1 in embryonic stem cells correlates with decreased DNMT1, resulting in progressive loss of DNA methylation [9]. These data indicate that LSD1 also regulates DNA methyla- tion [9]. As such, LSD1 is likely to mediate lncRNAs, epigenetically regulating gene expression by influencing DNA methylation. Previous studies have used high-throughput genomic and epigenomic technologies to investigate the difference in gene expression at various transcriptional levels between patients with PE and healthy participants. The data from the GSE44667 database showed high methylation levels of the PAX3 gene in the placenta of patients with PE [10]. This result is consistent with the low expression of PAX3 in the same patients, which was identified in our previous study [3]. Therefore, this evidence suggest that high methylation levels of PAX3 gene are an important cause of low expression. The present study aimed to elucidate the mechanism underlying the abnormally high methylation levels of PAX3 gene in the placenta of patients with PE. Materials and methods Sample collection Placentas (PE, n=10 and normal, n=10) were collected from women who had undergone Caesarean deliveries be- tween January 2015 and December 2015 at the Department of Gynaecology and Obstetrics in the Wuxi Maternity and Children Health Hospital Affiliated Nanjing Medical University (Wuxi, Jiangsu, China). All protocols used in the present study were approved by the Research Medical Ethics Committee of Wuxi Maternity and Children Health Hospital and the study abides by the Declaration of Helsinki principles. All women were informed of the research nature of our study and signed informed consent forms. The clinical data from all patients and normal controls are recorded in Table 1. Cell culture and treatments HTR-8/SVneo cells were obtained from ATCC (American Type Culture Collection, Manassas, VA) and maintained in RPMI-1640 medium (Life Technologies, Carlsbad, CA, U.S.A.) supplemented with 10% foetal bovine serum (In- vitrogen, Carlsbad, CA, U.S.A.), and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). For regular main- 2 ◦ tenance,thecellsweregrownin75-cm plastic flasks at 37 Cina5%CO2-95% air atmosphere with media changes every 2–3 days. For hypoxia, the cells were cultured in an AW200SG hypoxic workstation (Electrotek, U.K.) using a ◦ continuous flow of a humidified mixture of 1% O2,5%CO2, and 94% N2 at 37 C. Experiments were performed while the cells were under either hypoxic (2% O2) or normoxic (20% O2, as a control) conditions. Cell transfection HTR8/SVneo cells were transfected with various siRNAs designed by GenePharma (Shanghai, China) to knockdown the targeted genes. Table 2 shows the siRNA sequences. HTR8/SVneo cells were seeded onto six-well plates with a density of 1 × 105 cells/well. The cells were transfected with these siRNAs using Lipofectamine RNAi Max (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.), according to the manufacturer’s instructions. For the ectopic ex- pression of Pax3 in HTR-8/SVneo cells under hypoxia, transfection of pEGFP-C1-Pax3 vector (Genephama Biotech) 2 c 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2019) 39 BSR20182022 https://doi.org/10.1042/BSR20182022 Table 2 The sequences of siRNAs Name Sense (5-3) Antisense (5-3) HOXD8-SiRNA-702 UAGUAAGUGGGAUUGAUGGTT CCAUCAAUCCCACUUACUATT HOXD8-SiRNA-1210 UCUUCCUCUUCGUCUACCATT UGGUAGACGAAGAGGAAGATT HOXD8-SiRNA-1534 AGCAUAUGGUGAUUAUUAGTT CUAAUAAUCACCAUAUGCUTT LHX3-SiRNA-823 UCAUGUUGCGGAAAUACUGTT CAGUAUUUCCGCAACAUGATT LHX3-SiRNA-1625 UUUCAUGUCUAGAAAUAGCTT GCUAUUUCUAGACAUGAAATT LHX3-SiRNA-2312 AAUAGGUAGCUCGAGAUUCTT GAAUCUCGAGCUACCUAUUTT DIAPH2-AS1-SiRNA-196 AGUUGUUAUGUCUUCUUAGTT CUAAGAAGACAUAACAACUTT DIAPH2-AS1-SiRNA-341 AGGAUAAUCGCUUGAAUCCTT GGAUUCAAGCGAUUAUCCUTT DIAPH2-AS1-SiRNA-867 UAGGUUAAUUCCAUUUAUGTT CAUAAAUGGAAUUAACCUATT RP11-445K13.2-SiRNA-202 AUAAAGCAAUGUCGUUAUGTT CAUAACGACAUUGCUUUAUTT RP11-445K13.2-SiRNA-252 UCUCAAAGAGAAUCAGUAGTT CUACUGAUUCUCUUUGAGATT RP11-445K13.2-SiRNA-338 UUUGUAACUGGCCAAUUUCTT GAAAUUGGCCAGUUACAAATT Negative control FAM UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT Table 3 Primer sequences in PCR assay Length