2.1. Clinical tissue samples
Placenta tissues were sampled from the department of Gynecology and Obstetrics, Nanjing Maternity and Child Health Care Hospital. Placentas from 40 participants were included in the study, including 20 pregnant women with FGR and 20 normal pregnant women. FGR is defined as a fetus that has failed to achieve its genetically determined growth potential and is defined as EFW less than the 10th centile with respect to the reference value for current pregnancy age. This study was approved by the Ethics Committee of the Nanjing Maternity and Child Health Hospital. Written informed consent was obtained from all patients before using. All placenta tissues frozen in liquid nitrogen were then stored at−80℃.
2.2. RNA isolation and quantitative reverse transcription PCR (qRT-PCR) assay
Total RNAs were extracted from cells and tissues by traditional methods. cDNA was synthesized using the reverse transcription kit (Vazyme, China) in a 20 μl reaction containing 1000ng of total RNA. qRT-PCR reactions were carried out triplicate with 384-well plates on ViiA7 real-time PCR System-Life Tech (Applied Biosystems, USA) with a standard absolute quantification thermal cycling program. The threshold cycle (Ct) values of each sample were used in the post-PCR data analysis. Comparative quantification was performed according to the 2−ΔCt method and GAPDH was used as internal reference of mRNA for normalization. The sequences of the primers were as follows: LINC00881, Forward primer 5'-AGGTGCTGTATTGGCTCTTGAC-3'; Reverse primer 5'-GCTTGCTGGCTTGTTTCTGC-3'；FSCN1, Forward primer 5'- CCTTCCGTACCCACACG-3'; Reverse primer 5'- CATTGGACGCCCTCAGT-3'. ACTB, Forward primer 5'- TCTCCCAAGTCCACACAGG-3'; Reverse primer 5'- GGCACGAAGGCTCATCA-3'. PRDX6, Forward primer 5'- GAGCTGTTCAAGGGCAAGA-3'; Reverse primer 5'-TCGCCAGTCACAAAGGC -3'. ANXA1, Forward primer 5'-AAATGCCTCACAGCTATCG-3'; Reverse primer 5'-CCTTATGGCGAGTTCCAA-3'. PRDX1, Forward primer 5'- GAAACAAGGAGGACTGGGA-3'; Reverse primer 5'- CCCTGAACGAGATGCCT-3'. CALR, Forward primer 5'- CCTGCCGTCTACTTCAAGGAG-3'; Reverse primer 5'- GAACTTGCCGGAACTGAGAAC-3'. GAPDH, Forward primer 5'- GAACGGGAAGCTCACTGG-3'; Reverse primer 5'- GCCTGCTTCACCACCTTCT-3'.
2.3. Cell culture and transfection
The HTR8/SVneo trophoblast cell is the storage cell line of our laboratory, which was derived from placental trophoblast. The HTR8/SVneo trophoblast cells were cultured in complete growth medium: DMEM basic(Gibco, USA), supplemented with 10% fetal bovine serum(10% FBS) (Gibco, USA) under the condition of 37 °C and 5% CO2. Three Small interfering RNAs (siRNAs) sequences of FSCN1 are as follows: NC, Sense5'-3' UUCUCCGAACGUGUCACGUTT; Antisence 5'-3'ACGUGACACGUUCGGAGAATT. hs-FSCN1-si1, Sense5'-3' GCAAGAAUGCCAGCUGCUATT; Antisence 5'-3' UAGCAGCUGGCAUUCUUGCTT. hs-FSCN1-si2, Sense5'-3' GCAAGUUUGUGACCUCCAATT; Antisence 5'-3' UUGGAGGUCACAAACUUGCTT. hs-FSCN1-si3, Sense5'-3' GCUCCAGCUAUGACGUCUUTT; Antisence 5'-3' AAGACGUCAUAGCUGGAGCTT. Overexpression plasmid of FSCN1, overexpression lentiviruses and negative control of LINC00881 were synthesized from HANBIO (Shanghai, China). Smart Silencer and negative control were synthesized from RIBOBIO (Guangzhou, China). For preparation, HTR8/SVneo trophoblast cells were seeded in six-well plates overnight until growing to 60-70%. Afterwards, the operation was carried out according to the manufacturer’s protocol of Lipofectamine 3000(Invitrogen, USA). Transfected cells were divided into LINC00881⁃NC, LINC00881⁃OE, LINC00881-SiNC, LINC00881-Si, LINC00881-OE+NC-FSCN1, LINC00881-OE+OV-FSCN1 NC, Si1, Si2, Si3, NC-FSCN1, OV-FSCN1.
2.4. Cell proliferation, Cell migration and invasion assay
The Cell Counting Kit-8 (CCK8) assay (Dojindo, Japan) was applied to test the cell proliferation as manufacturer’s protocol. Briefly, the transfected cells were plated in 96‑well plates at a density of 3×103 cells per well and cultured at 37 °C with 5% CO2 for 0, 24, 48 and 72 h. 10μl per well of CCK‑8 was added to the cells and the 96‑well plates were incubated for another 2h. The absorbance was measured by a multifunctional microplate reader at 450 nm. Cell migration assays were performed using the Transwell system (Corning, USA). Cells (3×104) were added to the upper well in 200μl of culture medium without FBS. Next, 600 µl of medium (with 10% FBS) was added to the lower chamber. The protocol for the cell invasion assay was the same as the procedure for the migration assay, except that the upper side of the polycarbonate membrane was coated with a thin layer of Matrigel (BD Biosciences, USA). 60 μl Matrigel, 1:7 diluted in serum-free DMEM, was coated onto the upper chamber and incubated at 37°C for 2 h. The cells that migrated (after 48h) and the cells that invaded (after 96h) through the membrane were fixed in methanol and stained with 0.5% crystal violet (Beyotime, China). The cells that did not migrate through the pores were removed from the upper surface of the membrane by scraping with a cotton swab. The mean number of cells was calculated from counts in five random fields under a microscope.
2.5. Cell apoptosis and cycle assay
Cell apoptosis rate was evaluated using the AnnexinV-PE/7AAD Apoptosis Detection kit (Vazyme, China) according to the instructions from the manufacturer. Cells were collected, washed with PBS, and resuspended in 500 binding buffer. Then 5μl Annexin V-PE and 5μl 7-AAD staining solution were added to the buffer and incubated at room temperature for 15 min in the dark. Cells were analyzed by flowcytometry (Beckman Coulter, USA) within 1 h. Cell cycle analysis was evaluated using the cell cycle analysis kit (Beyotime, China) according to the instructions from the manufacturer. The transfected cells were collected, washed with cold PBS, and fixed with 70% ethanol at 4 ℃ for more than 12 h. The fixed cells were washed with cold PBS (Gibco, USA) and then stained with propidium iodide for 60 min at 37°C in the dark. Cells were analyzed by flowcytometry within 24 h. The percentage of cells in the G1 phase, the Sphase, and the G2 phase was analyzed.
2.6. RNA pull‑down and mass spectrometry assay
The synthesis of LINC00881 was carried out with MAXIscript® Kit (Thermo Fisher Scientific, Inc.) according to manufacturer’s instructions. The interaction between LINC00881 and proteins were examined using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Inc.) following the instructions of the manufacturer. The RNA-binding protein complexes were washed, eluted, and could be analyzed by Mass spectrometry (MS) (5600-plus, AB SCIEX, USA) and/or western blot (WB) analysis.
2.7. RNA immunoprecipitation (RIP) assay
The RIP assay was conducted by the RNA-Binding Protein Immunoprecipitation Kit (EMD Millipore, USA) according to the instructions of manufacturer. Briefly, the cells were lysed and the resulted lysis solutions were subsequently incubated with antibody against FSCN1 or isotype control IgG. The RNA-protein complexes were immunoprecipitated with protein A agarose beads, and the RNA was extracted and purified. LINC00881 could be quantified by qPCR.
2.8. Western blot analysis
Total protein was extracted using the RIPA lysis buffer phosphatase inhibitor (Beyotime, China), and the concentration was measured with the BCA assay (Beyotime, China). Proteins (40μg) were separated with SDS‐PAGE and then transferred to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% skim milk (Biofroxx, Germany) for 2 h and incubated with primary antibody overnight at 4℃, including cyclin L1(Santa Cruz, USA), monoclonal anti-Fascin (ab126772, Abcam, Britain) and monoclonal anti-GAPDH (ab8245, Abcam, Britain). The membranes were then washed with TBST (three times, 10 minutes/time), incubated with the HRP-conjugated goat anti-rabbit IgG (7074P2,Cell Signaling Technology (CST), USA) and HRP-conjugated horse anti-mouse IgG (7076P2,Cell Signaling Technology (CST), USA). Proteins were visualized with the ECL system (Millipore, USA) using the automatic chemiluminescence image analysis system (Tanon, China). Bands on Western blot gels were quantified using ImageJ. These values were then normalized to GAPDH level.
2.9. Statistical analysis
All data were presented as mean ± sd. Student’s two-tailed t-test was used to analyze the differences between groups. Values were considered statistically significant at P < 0.05.