Collection of human embryonic tissues
Fresh villi tissues of placenta from 45 women with RM diseases were collected. The patients’ age was ranged from 23 to 36 years (29.1 ± 6.9 years) and 8–10 weeks of gestation. These placenta villi tissues were obtained in the Department of Obstetrics and Gynecology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, from February 2017 to June 2018. The patients accompanying with autoimmune, chromosomal, parental, hormonal, infectious and anatomic disorders, or complications of thyroid abnormalities, diabetes and hypertension, or infection with ureaplasma and chlamydia in cervical mucus, were excluded from this study [19]. Fetal chromosomal abnormalities in RM placenta villi were also excluded.
Additionally, 39 women aged 22–37 years (28.5 ± 7.6 years) with normal gestation were enrolled in health controls (HCs) group. These women were arranged to the HCs group undergoing abortion at the gestation of 6–10 weeks. All HCs group had previous pregnancies without preeclampsia or spontaneous abortion. Written informed consent is requested from the patients involved in current study, which has been approved by the Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai. The samples from RM patients or HCs were frozen immediately in liquid nitrogen for RNA extraction or fixed with 4% paraformaldehyde for immunostaining. In some cases, the villous explants were freshly cultured. And the primary trophoblasts were isolated before storage.
Isolation of primary cytotrophoblast cells
Primary trophoblasts were digested by 0.125% trypsin supplemented with DNase I (0.1 mg/mL, Sigma, St. Louis, MO), followed by discontinuous percoll gradient centrifugation, as reported in the previous study [22]. Briefly, the placenta villous samples were rinsed in iced-PBS. The placenta villous samples were digested in a medium of Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Invitrogen, CA, USA) containing DNase I plus trypsin at 37 °C for 20 min for three times. Then, the cytotrophoblasts were isolated by centrifugation at 5% to 65% gradient at steps of 5% and centrifuged at 1000g for 10 min. The layer between 35 and 45% aliquots containing the cytotrophoblasts was obtained. The collected primary trophoblasts had a purity of ~ 95%, as examined by FACS for vimentin-negative cells and cytokeratin 7-positive. Purified primary cytotrophoblasts were immediately plated in each well of a 6-well plate at 8.0 × 105 cells/mL, and were further cultured in DMEM/F12 medium containing 20% fetal bovine serum (FBS) and penicillin/streptomycin/gentamicin antibiotics (Invitrogen, CA, USA).
Cell culture
A human trophoblast cell line derived the first-trimester extravillous trophoblast (EVT), HTR-8/SVneo, was a generous gift provided by Professor. PK Lala (Ontario, Canada) [21]. HTR-8/SVneo cell line was routinely maintained in DMEM/F12 medium containing 10% of FBS and penicillin/streptomycin antibiotics (Invitrogen, CA, USA).
UCA1 expressing plasmid
To construct an overexpressing UCA1 plasmid, the RNA sequence of human lncRNAs UCA1 was cloned into the modified pcDNA3.1 vector with the primers as following: (1) Forward primer sequence, 5′-ATTGAATTTGACATTCTTCTGGACAATGAGT-3′; (2) Reverse primer sequence, 5′-ACGCGGATCCCTGACTCTTTAGGAAGATTTCT-3′. The pcDNA3.1-UCA1 expressing vector and its empty vector were purified with a commercial Plasmid kit (Qiagen, Hilden, Germany). Then 1.5 μg pcDNA3.1-UCA1 plasmids were transfected into HTR-8 cells when these cells reached 60–70% confluence in a 6-well plate using the jetPRIME® kit following the manufacturer’s instructions (PolyPlus Transfection, Illkirch, France). The pcDNA3.1 empty vector was served as a negative control. HTR-8 cells were treated for the subsequent experiments 24–72 h after transfection.
Knockdown of UCA1
UCA1 knockdown was performed using three specifics siRNAs that were purchased from GenePharma Inc (Shanghai, China). Briefly, 24 h prior to transfection, HTR-8 cells were cultured in a 6-well plate to reach approximately 40–50% confluence. Then, the trophoblasts were transfected with 100 nmol/L UCA1 siRNA by using Oligofectamine@ transfection kit (Invitrogen, CA, USA) in optiMEM medium (Invitrogen, CA, USA) following the manufacturer’s instructions. Random sequence siRNA has been routinely included as a negative control. All the transfected trophoblasts were further used for the subsequent migration and invasion assays 24 h after transfection.
Cell growth assays
For the cell growth studies, 2 × 103 HTR-8 trophoblast cells were treated with control siRNA, siUCA1#3, empty vector, or UCA1 overexpression vector in each well of a 96-well plate. Cell viability at indicated time points was evaluated with a commercial available Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Shanghai, China). The absorbance counts were measured at 450 nm by Spectra Max 190 microplate reader (BIO-RAD; Hercules, CA, USA).
Immunohistochemistry
Immunohistochemical assay was performed according to a previous protocol [23]. Briefly, villous tissue slides were blocked with 5% FBS, followed by the incubation at room temperature at least for 30 min. Then, slides were incubated with anti-MMP9 antibodies (Cat#13667, CST, Massachusetts, USA) at 4 °C for 12 h, followed by the treatment with a HRP/DAB IHC Kit (Abcam, MA, USA). Nuclei of cells were counterstained with Meyer’s hematoxylin (Sigma, MO, USA). The sections were evaluated for the expression of MMP9 using a Leica microscope (Leica, IL, USA).
Quantification of MMP2 and MMP9
To evaluate the level of MMP2 and MMP9 expression in response to UCA1 knockdown, human HTR-8 cell line was plated and cultured in the 6-well plate, followed by the transfection with control siRNA (siCtrl) or siUCA1 and further cultured for 24 h. The supernatants were collected after centrifugation. The level of MMP2 and MMP9 expression in supernatants were determined by ELISA analysis following the manufacturer’s instructions (R&D Systems, MN, USA).
Wound healing assay
The wound healing analysis was conducted according to a previous method [24]. In brief, 5 × 104 HTR-8 cells were plated into individual well of a 6-well plate and cultured for 24 h. Then, the trophoblasts were treated with control siRNA, siUCA1 oligos, empty vector, or UCA1 overexpression vector. When the cells reached approximately 85% confluence, the trophoblasts were incubated with Mitomycin C (10 µg/mL, Tocris Bioscience, Bristol, UK) in DMEM/F12 medium for 2 h to eliminate cell proliferative ability. A sterile 200-μL tip was applied to make a scrape across the middle area of each well, followed by washing HTR-8 cells with iced-PBS for three times to remove floating cells or debris. Cells were then cultured in DMEM/F12 medium with 1% FBS for another 16 h. The distance between the edges of the scrape in each well were photographed with a Leica microscope and evaluated in collected photomicrographs. Cell migration was evaluated by comparison of the width of wound closure relative to the initial wound area at 0 h and 16 h.
Invasion assay
We evaluated trophoblast invasive ability with a Transwell Matrigel invasion method as reported previously [22]. Briefly, 50 µL of Matrigel (dilution 1:4; Corning, New York, USA) was used to coat with the inserts in a 24-well plate (pore size, 8 µm; Corning, New York, USA) on ice. HTR-8 cells or primary trophoblasts freshly isolated from the patients with RM disorders were subjected to the transfection with siCtrl, siUCA1, control vector, and the UCA1 overexpression plasmid and cultured for 48 h. All these transfected cells were plated onto the upper chamber of the transwell insert at 8.0 × 104 cells/200 µL DMEM/F12 medium with 1% FBS. Then, 800 µL of DMEM/F12 medium with 15% FBS were added into the lower chambers. These cells were continually cultured at 37 °C for another 48 h. The inserts were then extensively rinsed with iced-PBS for three–five times, followed by staining with crystal violet (Sigma, MO, USA), and were evaluated under a Leica microscope. The total cell numbers of trophoblasts that had migrated into the low surface of inserts were carefully scored using a microscope. The assay was conducted duplicately and the three repeated experiments were performed independently.
Statistical analysis
All data are represented as mean ± SD unless otherwise indicated. The software of SPSS 13.0 (IBM, IL, USA) was used for all the statistical analyses. Independent two-tailed Student’s t-test was performed for the comparison of two groups. In some cases, one-way ANOVA with post hoc Tukey’s test was applied for the comparison within multiple groups. Correlations analyses were conducted using Spearman’s rank correlation test. In all the experiments/results, P < 0.05 was set as the statistical significance.
Detailed methods are available in the section of Additional file 1.