Background
Spontaneous abortion is defined as the loss of a pregnancy without outside intervention before 20 weeks’ gestation [
1]. It is the commonest complication of pregnancy with an incidence estimated at 10–15% [
2,
3]. For clinical purposes, spontaneous abortion could be divided into several recognized types including threatened abortion, inevitable abortion, incomplete abortion, complete abortion, missed abortion and septic abortion [
4]. There are several risk factors prior to pregnancy associated with spontaneous abortion, which are complex and modifiable, including menarche age, family genetic diseases and maternal congenital defects [
5]. Moreover, more women are vulnerable to mental health problems after the occurrence of spontaneous abortion, especially for immigrants, childless women and women in a low socioeconomic status [
6]. Meanwhile, we have to recognize that dysfunction of trophoblast cells is responsible for the abortion [
7], and inflammatory response is involved in abortion [
8].
Triclosan (TCS) is an antimicrobial which is frequently applied in toothpaste, mouthwash, hand sanitizer, and surgical soaps [
9]. TCS has been sustained as an essential effector in human diseases in accumulating studies. For instance, TCS have been proposed to induce human vascular endothelial cell injury via modulation on PI3K/Akt/mTOR [
10]. TCS exacerbates the development of steatohepatitis and fibrosis, coupled with an increment in levels of hepatic lipid droplets and oxidative stress [
11]. Liu et al. have demonstrated that enough concentrations of TCS are capable of inhibiting the viability and growth of breast cancer cells (MCF-7 and SKBr-3) in culture [
12]. Meanwhile, Wang et al. have revealed that patients who underwent spontaneous abortion had much higher urinary TCS (over 10 folds) than normal pregnancy, and TCS exposure in mice may lead to spontaneous abortion in mid-gestation [
13]. However, the way in which TCS modulated the process of spontaneous abortion needs to be investigated.
MicroRNAs (miRNAs) are short non-coding RNAs which play an important role in regulating gene expression and the implication of aberrantly expressed miRNAs in tumorigenesis and progression of malignancies has been unveiled in previous papers [
14,
15]. For instance, miR-93 influences cell proliferation and apoptosis in recurrent spontaneous abortion via targeting BCL2L2 [
16] and miR-181b-5p has been suggested to exert suppressive impacts on trophoblast cell migration and invasion via directly targeting S1PR1 [
17]. Moreover, the association between TCS and miRNAs in human diseases has also been mentioned in previous research. For instance, Ha et al. have presented that miR-6321 induced by TCS is implicated in testicular steroidogenesis, and specifically, miR-6321/Map 3 k1-mediated JNK/c-Jun/Nur77 cascade contributes to TCS-induced inhibition on testicular steroidogenesis [
18]. However, miRNAs influenced by TCS in trophoblast cells require further exploration.
In this paper, the main aim was to uncover the role of TCS in spontaneous abortion in vitro and explore the underlying downstream regulatory mechanism of TCS.
Methods
Cell culture
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% CO
2 at 37 °C. Cell cultures were regularly checked by polymerase chain reaction (PCR) for mycoplasma contamination, as previously described [
19]. 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.
Plasmid transfection
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.
Transwell assay
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.
Statistical analysis
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.
Discussion
Spontaneous abortion is a serious problem troubling women at the reproductive age [
1]. The trophoblast has been reported as a crucial effector in implantation and placentation [
23]. Wang et al. have reported the association between TCS and spontaneous abortion in human cases and mice model [
13]. Aiming to explore the underlying regulatory mechanism, we firstly utilized TCS to create in vitro model simulating spontaneous abortion in trophoblast cells and discovered that TCS suppressed cell proliferation, migration and invasion while inducing the inflammatory response of trophoblast cells in vitro.
SLIT2 has been identified as an important regulator in trophoblast differentiation and invasion during pregnancy [
20]. It has been reported that Benzophenone-3 (BP-3) treatment can mediate the expression of SLIT2 and miR-218 [
24], a gene derived from introns of SLIT2 [
21]. Moreover, miR-218 has also been reported to play a central part in regulating trophoblast functions. For instance, hypoxia-induced miR-218 exerts suppressive impacts on trophoblast invasion via targeting LASP1 [
25]. Additionally, miR-218 weakens the migratory and invasive abilities of HTR-8 cells via targeting SOX4 [
26]. In our study, we discovered that TCS induced the up-regulation of miR-218-1-3p in human trophoblast cells. It was suggested in functional assays that overexpression of miR-218-1-3p impeded the proliferation, migration and invasion while stimulating the inflammatory response of trophoblast cells.
The involvement of c-Jun in multiple human diseases has also been unveiled in previous papers. For example, Peng et al. have illustrated that c-Jun, mediated by GnRH, influences trophoblast cell invasion [
27]. C-Jun also displays peak expression in human placenta at early gestation, which might be associated with cytotrophoblastic proliferation [
28]. As a transcription factor, c-Jun also influences gene transcription. For example, c-Jun restrains the transcriptional activity of NF-E2 via inactive c-Jun/NF-E2p18 heterocomplexes [
29]. Additionally, TCS has been mentioned to modulate c-Jun production [
22]. Through our investigation, we discovered that TCS at a high concentration also contributed to an augment in c-Jun level. Moreover, c-Jun activated the transcription of MIR218–1 to enhance the expression of miR-218-1-3p.
SLC35C1 has been uncovered to abrogate the progression of colon cancer via inactivation of Wnt pathway [
30]. Our experimental results confirmed that SLC35C1 was a target gene of miR-218-1-3p, and the negative correlation between SLC35C1 and miR-218-1-3p was ascertained. In addition, SLC35C1 silence hindered the proliferation, migration and invasion of trophoblast cells while promoting cellular inflammatory response. Rescue assays also validated that TCS modulated the proliferation, migration, invasion and inflammatory response of trophoblast cells in vitro via miR-218-1-3p/SLC35C1 axis.
To be summarized, miR-218-1-3p was testified to be highly expressed in trophoblast cells after TCS treatment and up-regulation of miR-218-1-3p impeded the proliferation, migration and invasion of trophoblast cells while stimulating cellular inflammatory response in vitro. Moreover, TCS-induced c-Jun could activate the transcription of MIR218–1, thereby enhancing miR-218-1-3p expression. After confirming SLC35C1 was targeted by miR-218-1-3p, we also confirmed the influences of miR-218-1-3p/SLC35C1 on TCS-medicated proliferation, migration, invasion and inflammation of trophoblast cells. In a word, our study unveiled the TCS/miR-218-1-3p/SLC35C1 axis in the modulation of trophoblast cells and might provide novel insights for preventing spontaneous abortion in vitro.
However, there are still some limitations in our present study. For example, whether other potential regulatory mechanisms in the downstream of TCS participate in the process of spontaneous abortion also needs to be confirmed. This study used “in vitro” methodology to evaluate the mechanism of action of TCS in the trophoblast. The association of TCS with abortion in humans needs further studies.
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