Clinical sample collection

Human placenta samples were collected from 24 preeclampsia patients and 24 normal pregnant women registered at the Department of Obstetrics, Henan Provincial People’s Hospital (Zhengzhou, Henan, China) after their delivery, and then quickly put into − 80 °C freezer. The clinical characteristics of normal pregnant women and preeclampsia patients were shown in Table 1. The diagnostic criteria of preeclampsia was described as follows: hypertension (systolic blood pressure not lower than 140 mmHg and/or diastolic blood pressure not lower than 90 mmHg) and newly-onset proteinuria (higher than 300 mg per day) (Li et al. 2020; Wang et al. 2019). Exclusion criteria included pre-delivery infection, multiple pregnancies, alcoholism, smoking and alcohol abuse, genetic abnormalities, fetus growth retardation, rupture of membrane, cholestasis of pregnancy, and history of cardiovascular diseases, renal diseases, autoimmune diseases, diabetes, chronic hypertension, or other metabolic diseases. Written informed consent was collected from all participants before the procedure. The research protocol was approved by the Ethical Committee of the Henan Provincial People’s Hospital (Zhengzhou, Henan, China).

Table 1 Clinical characteristics of normal and preeclamptic pregnanciesCell culture

Human placenta trophoblasts (HTR-8/SVneo, JEG-3, BeWo) and human embryonic kidney (HEK) 293 T cells were purchased from the American Type Culture Collection (ATCC, USA) and cultured in RPMI 1640 (Invitrogen, USA) with 10% fetal bovine serum (Invitrogen, USA) and 1% penicillin/streptomycin (Invitrogen, USA) at 37 °C in a humidified incubator with 5% CO2. In order to mimic the hypoxic condition in preeclampsia placenta, cells were cultured in a hypoxic workstation (AW200SG, Electrotek, UK) with 2% O2, 5% CO2 and 93% N2 at 37 °C. For the normoxic group, a mixture of 20% O2, 5% CO2 and 75% N2 was used instead. Culture medium was changed every two to three days. The treatment of HMGB1 was given as described in Son et al. (2016). Recombinant HMGB1 (ab167718, Abcam, USA) was added into the culture medium to a final concentration of 3 μg/mL, and cells were treated for 24 h.

Cell transfection

The sh-LINC00240 vector for silencing of LINC00240 expression, the sh-Nrf2 vector for silencing of Nrf2 expression and the negative control sh-NC, the miR-155 inhibitor for silencing of miR-155 expression and the inhibitor NC, the miR-155 mimics and mimics NC were all designed and synthesized by GenePharma (Shanghai, China). The above shRNAs, miRNA inhibitor and mimics, and their corresponding negative controls were transfected into cells using the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s guidelines. For overexpression of LINC00240 (OE-LINC00240), cells were transfected with lentivirus (LV)-LINC00240 (LV5-EF1a-GFP/Puro vector, GenePharma, China). The lentiviral vectors were transfected into cells for 24 h at a multiplicity of infection of 50, followed by puromycin antibiotic selection (5 µg/mL) for 4 days.

Dual luciferase reporter assay

Wild type or mutant 3′-untranslated region (UTR) of Nrf2 (Nrf2-WT/Nrf2-MUT) or LINC00240 (LINC00240-WT/LINC00240-MUT) was constructed into pmirGLO Dual-Luciferase miRNA Target Expression plasmid (Promega, USA). The reporter plasmids were co-transfected with miR-155 mimics or miR-155 inhibitor into 293 T cells using Lipofectamine 2000 (Thermo Fisher Scientific, USA). The firefly and renilla luciferase activities were sequentially measured after 48 h using Dual-Luciferase Reporter Assay System (Promega, USA) and the firefly luciferase results were normalized to renilla luciferase activities.

RNA binding protein immunoprecipitation (RIP) assay

To demonstrate that LINC00240 could bind to miR-155, RIP assay was performed as described in Meier et al. (2013). Briefly, 293 T cells transfected with miR-155 mimics or mimics NC were lysed in polysome lysis buffer and stored at − 80 °C. Sepharose beads were coated with anti-Ago2 antibody (1:500, Sigma-Aldrich, USA) or control IgG antibody (1:500, Sigma-Aldrich, USA). After thawing, cell lysates were mixed at 1:10 with a cocktail buffer containing RNAseOUT (Invitrogen, USA) and then transferred into tubes containing beads coated with Ago2 or control IgG antibody. After incubation at 4 °C, beads were collected by centrifugation and washed in NT2 buffer. The proteins were eluted using glycine and then degraded by proteinase K (Sigma-Aldrich, USA). The immunoprecipitated RNAs were isolated using TRIzol reagent (Invitrogen, USA) for further measurement using quantitative PCR.

Measurement of cell viability by CCK-8

An equal amount of cells (5 × 103 cells per well) were transferred into wells of a 96-well plate with culture medium and incubated for indicated time. Cell Counting Kit-8 (CCK-8, #96992, Sigma-Aldrich, USA) was added into the cell culture medium at 1:10. After incubation at 37 °C for 2 h, absorbance was measured at 450 nm using a plate reader (Thermo Fisher Scientific, USA).

Measurement of cell proliferation by colony formation assay

Cultured cells were firstly trypsinized and suspended in culture medium. Cells were diluted and planted in a 6-well plate at a density of 100 cells per well. After incubation for 2 weeks, colonies were fixed with 3.7% methanol and stained with 0.5% crystal violet at room temperature for 2 h. The number of colonies was counted under a microscope (Zeiss, Germany).

Measurement of cell migration by wound healing assay

Cells were firstly seeded in 6-well culture plates (6 × 105 cells per well) with culture medium. After cells reached 90% confluence, the plate was scratched across the center of the well using a 10 µL pipette tip. After the scratching, wells were gently washed with PBS to remove detached cells and image of the wound was taken under an inverted microscope (Zeiss, Germany). Culture medium was then added and cells were cultured for another 24 h. After washing with PBS, images of the wound were taken under microscope. Widths of the wound were measured using Image J software, and the migration ability of the cells was evaluated by the following formula: (W0 h − W24 h)/W0 h × 100%.

Measurement of cell invasion by Transwell assay

Transwell assay was performed to study the cell invasion capacity using Transwell permeable supports (8 μm, Corning, USA). After indicated treatments, cells were harvested and diluted in serum-free medium. Cells (5 × 104) were seeded in the upper chamber which was pre-coated with matrigel, whereas fresh medium containing 10% FBS was added to the bottom chamber. After further incubation for 48 h, cotton swab was used to remove the cells on the upper side of the filter. The invaded cells were fixed in 4% paraformaldehyde, and then stained with 1% crystal violet and counted under the microscope (Zeiss, Germany).

Co-culture system and detection of M2 macrophage using flow cytometry

Human placenta trophoblast cell lines (HTR-8/SVneo, JEG-3), with or without transfection, were cultured in normoxic or hypoxic condition. After 48 h, supernatants of those cultured cells were taken and used to treat M0 macrophages for 24 h. After the treatment, flow cytometry was used to assess the phenotype of M2 macrophages (both positive in CD68 and Arg-1) as described in Lindau et al. (2018). Macrophages were resuspended in PBS and incubated with antibodies against CD68 (12-0689-42, Thermo Fisher Scientific) and Arg-1 (17-3697-82, Thermo Fisher Scientific) at 4 °C for 30 min in dark. After washing with PBS, cells were resuspended in PBS and analyzed using FACSCanto II (BD Biosciences, USA) with gating at CD68+Arg-1+. Results were obtained using FACSDiva 6.0 software (BD Biosciences, USA).

Measurement of cell pyroptosis using flow cytometry

FAM-FLICA in vitro Caspase-1 Detection Kit (ImmunoChemistry, USA) was used to measure levels of caspase-1 following the manufacturer’s instruction. Briefly, each sample was added 5 μL FLICA and 2 μg/mL PI to a final volume of 300 μL. After incubation at 37 °C in dark for 1 h, cells were washed twice with wash buffer and analyzed with flow cytometer (BD Biosciences, USA).

Assessment of oxidative stress

Reactive oxidative stress (ROS) Assay Kit (ab186027, Abcam, USA) and Lipid Peroxidation (MDA) Assay Kit (ab118970, Abcam, USA) were used to evaluate the oxidative stress levels as per the manufacturer’s instruction. For ROS assay, cultured cells were stained with ROS Red working solution and fluorescence intensity was measured at 520/605 nm (Ex/Em). For lipid peroxidation assay, 600 μL of thiobarbituric acid solution was added to 200 μL samples or standards. After incubation at 95 °C for 1 h, samples and standards were cooled to room temperature in ice bath. 200 μL of the reaction mixture from each sample or standard was moved into a 96-well plate and optical density (OD) value was immediately measured at 532 nm using a plate reader (Thermo Fisher Scientific, USA).

ELISA assay

ELISA Kit for HMGB1 (ABIN6574155, Antibodies-online, Germany) was used to measure HMGB1 level in cell supernatant following the manufacturer’s instruction. Briefly, samples and standards were added into wells of a 96-well plate pre-coated with anti-HMGB1 antibody and incubated at 37 °C for 2 h. After discarding the supernatant and washing the wells with wash buffer, 100 μL of Detection Reagent A was added into the wells and the plate was incubated at 37 °C for 1 h. After washing, 100 μL of Detection Reagent B was added and the plate was incubated at 37 °C for 30 min. After complete washing of the wells, 90 μL of Substrate Solution was added and the plate was incubated at 37 °C for 15 min. 50 μL of Stop Solution was then added and OD value was measured at 450 nm using a plate reader (Thermo Fisher Scientific, USA).

Animal model

Female Sprague–Dawley rats weighing from 200 to 250 g were obtained from the Shanghai SLAC Laboratory Animal Center (Shanghai, China) and kept in temperature and humidity-controlled environment with 12:12 h light–dark cycle. Animals were given free access to water and food and cages were changed three times per week. After acclimation for one week, the animals were mated overnight with healthy male rats. After confirming the pregnancy by presence of sperms in vagina (defined as embryonic day 0) (Li et al. 2018a; Xu et al. 2019, 2021), animals were randomly put into four groups: control group, lipopolysaccharide (LPS) group, LPS + OE-NC group and LPS + OE-LINC00240 group (n = 12 per group). For LPS group, 1.0 μg/kg LPS (Sigma-Aldrich, USA) was injected through tail vein on the 5th embryonic day to generate preeclampsia model. For control group, normal saline was injected instead of LPS on the same day. For LPS + OE-NC or LPS + OE-LINC00240 group, 1.0 μg/kg LPS was also injected through tail vein on the 5th embryonic day, and lentivirus vector carrying human LINC00240 by PCR or control vector was injected on the 6th and 15th embryonic day to generate LINC00240-overexpressed preeclampsia rat model. Animals were monitored for blood pressure and urine protein levels on embryonic day 0, 3, 6, and every 3 days thereafter. On embryonic day 20, animals were euthanized by cervical vertebra dislocation. Fetus, placenta, liver and kidney tissues were collected, weighed, and frozen at − 80 °C until further analyses. All the animal experiment procedures were approved by the Animal Ethics Committee of Henan Provincial People’s Hospital (Zhengzhou, Henan, China). All the animal experiments were also performed in Henan Provincial People’s Hospital.

Immunohistochemistry assay

Rat placenta samples were collected and fixed in 4% paraformaldehyde for 24 h. After fixation, samples were dehydrated, embedded in paraffin, and stored at room temperature for further analyses. Before staining, the paraffin-embedded placenta samples were sectioned, and slices were de-paraffinized and hydrated in de-ionized water. After antigen-retrieval, slices were stained with anti-cytokeratin 7 (CK7) antibody (1:1000, ab199718, Abcam, USA) at 4 °C overnight, followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000, ab205718, Abcam, USA) for 1 h. Slices were then counterstained by hematoxylin for 30 min. After washing, slices were visualized by 3,3′-diaminobenzidine, and staining results were examined under a light microscope (Zeiss, Germany).

H&E staining assay

Paraffin-embedded rat kidney and liver samples were sectioned, followed by three changes of xylene and hydration in 100%, 95%, 80% ethanol. The slices were stained with hematoxylin for 3 min and rinsed three times with de-ionized water for 30 s, followed by differentiation in 1% acid ethanol. The slices were then stained with 0.5% eosin for 30 s and dehydrated in ethanol, followed by three changes of xylene. A drop of neutral balsam mounting medium was added. The slices were naturally dried overnight and observed under a microscope (Zeiss, Germany).

Western blot assay

Cells were firstly lysed in RIPA buffer (Sigma-Aldrich, USA) and protein concentrations of each homogenate were measured using a commercial BCA protein assay Kit (Thermo Fisher Scientific, USA). 30 μg of proteins were separated on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then blocked in 5% skimmed milk in TBST, and stained overnight at 4 °C with primary antibodies against Nrf2 (1:1000, ab137550, Abcam), HO-1 (1:2000, ab13243, Abcam), eNOS (1:5000, ab76198, Abcam), NQO1 (1:10,000, ab80588, Abcam), caspase-1 (1:1000, ab238979, Abcam), NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, ab180799, Abcam), HMGB1 (1:10,000, ab79823, Abcam), or β-actin (1:5000, ab115777, Abcam). Samples were then stained with goat anti-rabbit or goat anti-mouse IgG secondary antibody (1:10,000, ab205718/ab205719, Abcam) at room temperature for 2 h. Bands were developed using chemiluminescence substance (Thermo Fisher Scientific, USA). The proteins were quantified using Quantity One software (Bio-Rad, USA).

Quantitative PCR (qPCR) assay

Total RNA from cells or tissue samples was extracted using TRIzol reagent (Invitrogen, USA). Reverse transcription was performed using 1 μg total RNA from each sample using PrimeScript RT reagent Kit (for mRNAs, Takara, China) or TaqMan MicroRNA Reverse Transcription Kit (for miR-155, Thermo Fisher Scientific, USA). qPCR was performed using SYBR Premix EX Taq Kit (Takara, China) on Applied Biosystems 7500 Real Time PCR system (Thermo Fisher Scientific, USA). β-actin and U6 small nuclear RNA (U6 snRNA) were used as internal references for mRNA and miRNA, respectively. Relative expression level was calculated by the 2−ΔΔCt method. Specific primers for qPCR were obtained from Sangon Biotech (China) as listed in Table 2.

Table 2 Primers used for qRT-PCRCell–cell fusion

The cell–cell fusion of BeWo cells was detected and fusion index was calculated as described in Zhang et al. (2020) with some modifications. Briefly, forskolin was used to induce the syncytialization of BeWo cells. After being cultured for 24 h on poly-l-lysine-coated cover glass, BeWo cells with indicated treatments were treated with 30 μM forskolin (Cell Signaling Technology, USA) for 48 h. After the treatment, the culture medium was discarded and 4% paraformaldehyde was added to fix the cells by incubation for 20 min at room temperature. 0.5% Triton X-100 in PBS was used to permeabilize the cells for 3–5 min at room temperature. Blocking buffer was added to block the cells for 1 h at room temperature. Specific primary antibody anti-E-cadherin (Cell Signaling Technology, USA) was added in the blocking buffer and incubated with cells overnight at 4 °C. Primary antibody was washed out using PBS for 3 times, followed by incubation of Alexa fluor-conjugated secondary antibody for 2 h at room temperature. Cells were washed three times again by PBS and then treated with DAPI-containing mounting media (Thermo Fisher Scientific, USA). The cells were captured using fluorescence microscope (Zeiss, Germany).

Statistical analysis

All the experiments were repeated at least three times. Data were analyzed with GraphPad Prism 6.0 and results were expressed as mean ± standard deviation. Statistical evaluation was performed using Student’s t test between two groups or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparison. Spearman correlation analysis was performed to analyze the correlation between LINC00240, miR-155, Nrf2, Arg-1, and inducible nitric oxide synthase (iNOS) in placenta samples. P values less than 0.05 were considered as statistically significant for all analyses.