Human trophoblast cells (JEG3 and HTR-8) and human embryonic kidney 293 T (HEK-293 T) were selected for this study. Both JEG3 and HTR-8 showed epithelial-like phenotypes under a light microscope (Supplementary file 1). JEG3 cell line was obtained from Huatuo Biotechnology Co., Ltd. (Shenzhen, China) while HTR-8 and HEK-293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). JEG3 cells were incubated in EMEM Medium containing 10% FBS (Gibco). HTR-8 cells were maintained in RPMI-1640 Medium containing 5% FBS while HEK-293 T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS. All mediums were kept under the condition of 5% CO2 at 37 °C. Cell cultures were regularly checked by polymerase chain reaction (PCR) for mycoplasma contamination, as previously described . Additionally, the authentication of JEG3 and HTR-8 cells, as well as cross-contamination, was checked by STR profiling, and the corresponding STR reports were provided in Supplementary files 2 and 3.
Genechem (Shanghai, China) synthesized the short hairpin RNAs (shRNAs) targeting JUN or SLC35C1, as well as sh-NC. In addition, pcDNA3.1-JUN and the empty vector were also obtained from Genechem while miR-218-1-3p mimics/inhibitor and negative control were procured from RiboBio (Shanghai, China). Transfection of the above-mentioned plasmids was completed with Lipofectamine 3000 (Invitrogen). The transfection efficiency was ranged from 75 to 85%.
Quantitative real-time PCR (RT-qPCR) analysis
Firstly, total RNA was obtained with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Then M-MLV reverse transcriptase (Promega, Madison, USA) was employed to convert total RNA into cDNA. Through using SYBR Green Real-Time PCR Kit (Takara), RT-qPCR was performed. All target genes were measured with 2−ΔΔCt method and GAPDH or U6 was considered as the internal control. The Ct values obtained from real-time PCR analyses for indicated genes under different conditions were presented in Supplementary file 4.
Cell counting kit-8 (CCK-8) assay
At first, cells were incubated into 96-well plates (5 × 103 cells/well). After the addition of CCK-8 solution to each well, the optical density at 450 nm at indicated time points (0, 24, 48, 72 h) was measured with a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) after additional 2-h incubation to reflect changes in cell viability.
In detail, the upper chambers of Transwell inserts (Corning Incorporated, Corning, NY) with serum-free medium were used to accommodate trophoblast cells (5 × 104 cells). Transwell inserts covered by Matrigel was used for invasion assay. Then, the basal chamber was supplemented with culture medium containing 10% FBS. After being dyed by crystal violet, cells were counted 24 h later.
Wound healing assay
JEG3 and HTR-8 cells were plated in six-well plates with 5 × 105 cells/well and grown in complete culture media. After reaching 80% confluence, cells were starved for 24 h. Then, the surface of each well was lightly wounded using a sterile micropipette tip, and the cells were treated with complete culture media again. An inverted optical microscope (× 200) (Nikon, Japan) was used to observe and monitor the wound width at 0, 6, 12, 18, 24 h. The closure rate was defined as per the formula: closure rate = (wound width at 0 h – wound width at indicated time)/wound width at 0 h.
Chromatin immunoprecipitation (ChIP)
EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore, Burlington, MA, USA) was used for ChIP assay. Briefly, the crosslinked chromatin DNA was sonicated to obtain chromatin fragments which were precipitated with anti-c-Jun (ab32137, 1/50 dilution, Abcam, Cambridge, MA, USA) antibody and immunoglobulin G (IgG) antibody (#3900, 1/50 dilution, Cell signaling Technology, Boston, MA, USA). The immunoprecipitated DNA was subjected to PCR analysis.
Luciferase reporter assay
The MIR218–1 promoter region containing the binding sites (wild type or mutant type) of JUN was constructed into the pGL3 vector (Promega, Madison, WI) and co-transfected along with pcDNA3.1-JUN or the empty vector into JEG3 and HTR-8 cells. Similarly, SLC35C1 3’UTR with wild type or mutant type miR-218-1-3p binding sites were sub-cloned into pmirGLO vectors and then the constructs were co-transfected with NC mimics or miR-218-1-3p mimics into HEK-293 T cells. After 48 h, the Dual-Luciferase Reporter Gene Assay Kit (Yeasen, Shanghai, China) was applied to measure luciferase activity.
Western blot analysis
First of all, proteins in cells were extracted by RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) and then concentrated via bicinchoninic acid (BCA) kit (Thermo Fisher Scientific). Next, total of 20 μg proteins in RIPA buffer were added into each well of the gel, and were then separated by 12% SDS-PAGE. After that, proteins were transferred onto PVDF membranes (Millipore, Billerica, MA, USA), and the membranes were then blocking with 5% non-fat milk. Afterwards, membranes were cropped as needed and then incubated by primary antibodies at 4 °C overnight, followed by PBS washing for 3 times and 1-h incubation with secondary antibody at dark room. The antibodies against IL-6 (ab233706, 1/1000 dilution, Abcam), IL-1β (ab216995, 1/1000 dilution, Abcam), TNF-α (ab66579, 1/1000 dilution, Abcam), CXCL-8 (ab154390, 1/2000 dilution, Abcam), c-Jun (ab32137, 1/2000 dilution, Abcam), SLC35C1 (ab60336, 1.25 μg/mL, Abcam) and GAPDH were all bought from Abcam. Lipopolysaccharide (LPS)-treated HUVEC whole cell lysate (LPS-HUVEC-WCL), LPS-treated THP-1 whole cell lysate (LPS-THP-1-WCL), HeLa whole cell lysate (HeLa-WCL; ab150035) and human Jurkat cell lysate (Jurkat-CL) were used as positive controls for antibodies against IL-6, IL-1β/TNF-α, CXCL-8/c-JUN and SLC35C1, respectively. Protein quantification was conducted by Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerican MA, USA). The original blots with clear edges and marker band sizes were provided in Supplementary file 5.
SPSS version 16.0 (IBM, Chicago, IL) was used to analyze data. The data are displayed as the mean ± SD. All experiments were conducted in triplicate. Unpaired two-tailed Student’s t-test or ANOVA was used to analyze the differences between two or more groups. P value less than 0.05 was thought to be statistically significant.