Abstract
Purpose
To investigate the effects of long intergenic non-protein coding RNA 221 (LINC00221) on preeclampsia (PE) and its mechanism.
Methods
The expression of LINC00221 was detected in placental tissues from PE patients and normal pregnant women (non-PE). Next, the effects of LINC00221 silencing on trophoblast cells (HTR-8/SVneo and JEG-3) and co-cultured HUVECs or macrophages were evaluated. Afterwards, miR-542-3p was confirmed to bind to LINC00221 directly, and miR-542-3p mimics and inhibitors were transfected into trophoblast cells. Next, a rescue experiment was performed to examine the effect of LINC00221/miR-542-3p axis. Finally, the effect of LINC00221 was also verified in vivo in rat PE models.
Results
The expression of LINC00221 was higher in placental tissues of PE patients than those of non-PE. LINC00221 silencing significantly reduced MCP1 level and increased the VEGF level in trophoblast cells. LINC00221 knockdown in trophoblast cells remarkably enhanced VEGFR expression and the angiopoiesis of HUVECs, and decreased the migration and invasion of macrophages and reduced TNF-α level. Besides, LINC00221 knockdown decreased CHOP, p-IREα, p-PERK, and iNOS expression and increased Trx expression. Notably, LINC00221 negatively regulated miR-542-3p expression. MiR-542-3p overexpression had an effect to that of LINC00221 knockdown, while miR-542-3p inhibition had the opposite effect. Treatment with miR-542-3p inhibitors partially reversed the protective effect of LINC00221 silencing. PE rat model results were consistent with those of in vitro experiments.
Conclusions
Downregulation of LINC00221 might reduce dysfunction, inflammatory responses, endoplasmic reticulum stress, and oxidative stress, and thereby protect against PE by augmenting miR-542-3p.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10815-022-02593-y.
Keywords: Preeclampsia, LINC00221, Endothelial cell dysfunction, Endoplasmic reticulum stress, Oxidative stress
Introduction
As a common pregnancy complication, preeclampsia (PE) usually appears after 20 weeks of gestation and is characterized by clinical symptoms of hypertension and proteinuria [1]. PE influences 2–8% of pregnancies worldwide and leads to high risks for maternal and perinatal death [1]. Although progress has been made in treating its symptoms, the pathogenesis of PE remains unclear and is extremely complex [2]. Several factors, such as abnormal trophoblast cell invasion, endothelial cell damage, inflammatory responses, oxidative stress, abnormal immune regulation, genetic factors, and nutritional factors, are involved in the occurrence and development of PE [2]. Furthermore, extravillous trophoblasts play an important in placental development, and dysfunctional trophoblast cells are considered to be a major trigger of PE [3]. Therefore, it is important to understand the underlying mechanism of PE when searching for an effective treatment.
With the development of high-throughput sequencing and the continuous updating of computer algorithms, a large number of non-coding RNAs have been found to participate in a variety of human diseases [4]. Long non-coding RNAs (lncRNAs) are arbitrarily defined as non-coding RNA molecules containing > 200 nucleotides [5]. Some lncRNAs can interfere with the translation of miRNA molecules produced by specific genes and thereby participate in disease progression [6]. A growing number of investigators are exploring the functions of lncRNAs in distinct human disorders [7]. Several dysregulated lncRNAs have been suggested as potential biomarkers for use in diagnosing and treating PE, and usually function in PE development [8]. Previous studies reported the aberrant expression of lncRNAs in PE and identified several lncRNAs involved in PE development, such as lncRNA TCL6 [9], lncRNA BC030099 [10], lncRNA CCAT1 [11], and lncRNA HOXD-AS1 [12]. Several studies have demonstrated that LINC00221 is abnormally expressed in various cancers and regulates tumorigenesis via complex mechanisms [13–15]. Notably, a recent microarray analysis showed that LINC00221 was more highly expressed in an abnormally invasive placenta than in a normal placenta [16]. However, the precise role and mechanisms of LINC00221 in PE remain unexplored.
In the present study, LINC00221 levels were first detected in the placental tissues of PE patients and PE model rats. Next, the effects of abnormal LINC00221 expression on the function of trophoblast cells (HTR-8/SVneo cells and JEG-3 cells) were examined. In addition, the potential mechanism of interaction between LINC00221 and its target miRNA was further investigated.
Materials and methods
Clinical sample collection
This study enrolled 20 PE patients who were treated at The Third Affiliated Hospital of Zhengzhou University between 2020 and 2022. After obtaining written informed consent, the placental tissues of the patients were collected. Placental tissues were also obtained from 20 age and sex-matched normal pregnant women who served as control subjects. In addition, the clinical characteristics of the PE patients, including age, urine protein levels, systolic and diastolic blood pressures, gestational weeks, and neonatal weights, were recorded.
Cell culture and treatments
Trophoblastic HTR-8/SVneo cells, JEG-3 cells, HUVECs, and THP-1 cells were purchased and transferred into complete DMEM medium (Thermo Fisher, China) prior to use, and then cultured in a 5% carbon dioxide atmosphere at 37 °C. Macrophage formation by THP-1 cells was inducted by treatment with 25 ng/mL of phorbol 12-myristate 13-acetate (PMA, Sigma) for 48 h. First, the HTR-8/SVneo cells and JEG-3 cells were transiently transfected with siRNA #1, siRNA #2, and siRNA #3 to screen the siRNAs for an optimal LINC00221 silencing effect. For subsequent analysis, the downstream target miRNA of LINC00221, an LINC00221 overexpression vector, siRNA, an miR-542-3p inhibitor, the miR-542-3p mimic, siRNA + the miR-542-3p inhibitor, and their corresponding controls were transfected into both the HTR-8/SVneo cells and JEG-3 cells, respectively, by using Lipofectamine™ 2000 transfection reagent (Thermo Fisher, Waltham, MA, USA).
Co-culture of HUVECs or macrophages with trophoblast cells
Co-culture experiments were conducted using Transwell inserts (Millipore, Burlington, MA, USA) to confirm the effects of trophoblast cells on HUVECs and macrophages. HUVECs (1.5 × 105) were seeded into the upper chamber of a Transwell plate, while HTR-8/SVneo or JEG-3 cells (2 × 105) were added to the lower chamber. Following incubation for a specific time period, the HUVECs were collected and analyzed for VEGFR expression and angiogenesis. The co-culture of macrophages (2 × 105) with trophoblast cells was also performed using Transwell inserts. In those studies, the migration and invasion abilities of macrophages were detected simultaneously, and their supernatants were analyzed for TNF-α levels (Fig. 1).
Fig. 1.
Experiment design of this research
Animal experiments
Female pregnant rats (gestation period, 4–8 days) were purchased and housed in our laboratory. To establish the PE rat model, the rats were treated with 0.3 g/L (at dose of 50 mg/Kg/d) of N-nitro-L-arginine methyl ester (L-NAME) via intragastric administration. To explore the effects of LINC00221 on PE rats, the rats were randomly assigned to three separate groups (n = 6 rats per group), which were treated with equal volumes of saline (sham group), L-NAME + LINC00221 NC (model group), and L-NAME + LINC00221 shRNA (shRNA group), respectively. LINC00221 shRNA (20 μL containing 10 μg of shRNA) was intraperitoneally injected everyday before 16 dpp (days post-pregnant). At 20 days, samples of peripheral blood and placental tissue were obtained from rats in the different groups and used in subsequent experiments.
Fluorescence in situ hybridization (FISH) assay
The expression and localization of LINC00221 in HTR-8/SVneo cells and JEG-3 cells were evaluated by FISH. In brief, cells were fixed, permeabilized, and rehydrated. Next, the cells were incubated overnight at 42 °C with a biotin-labeled probe mixture targeting LINC00221, after which they were washed again with PBS solution and incubated with an anti-biotin monoclonal antibody, followed by incubation with a secondary antibody. The cell nuclei were stained with DAPI. Finally, the cells were mounted onto slides with Prolong Gold Antifade Reagent (Cell Signaling Technology, Danvers, MA, USA) and evaluated by fluorescence microscopy.
Quantitative RT-PCR
The total RNA from treated cells, serum, and tissues was extracted using TRIzol Reagent (Thermo Fisher). Samples of complementary DNA were prepared from the isolated RNA by using a QuantiTect Reverse Transcription Kit (QIAGEN, Germany). Quantitative PCR was performed using SYBR Premix Ex Taq TM II (Takara). β-Actin or U6 was used as an internal standard for quantitation of LINC00221 or miRNA, respectively. The sequences of the primers are shown in Table 1.
Table 1.
Specific primers for qPCR assay
| Gene | Primer sequence |
|---|---|
| LINC00221 | Sense primer: 5′-CGGGGACTTCCTTTATGTGG-3′ |
| Antisense primer: 5′-TATGCAAACAGCCTCTCGTG-3′ | |
| miR-542-3p | Sense primer: 5′-ACACTCCAGCTGGGTGTGACAGATTGATAACT-3′ |
| RT primer: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTTTCAGT-3′ | |
| GAPDH | Sense primer: 5′-TGTTCGTCATGGGTGTGAAC-3′ |
| Antisense primer: 5′-ATGGCATGGACTGTGGTCAT-3′ | |
| U6 | Sense primer: 5′-CTCGCTTCGGCAGCACA-3′ |
| Antisense primer: 5′-AACGCTTCACGAATTTGCGT-3′ |
LINC00221, long intergenic noncoding RNA 00,221; GAPDH, glyceraldehyde-phosphate dehydrogenase
Western blotting assay
The treated cells and tissues were lysed with cold lysis buffer (Beyotime, China) to extract their total proteins. Next, an aliquot of protein from each extract was separated on a PAGE gel and the protein bands were transferred onto PVDF membranes, which were subsequently blocked. The membranes were then incubated with primary antibodies against VEGF, CHOP, IREα, PERK, phosphorylated-IREα (p-IREα), p-PERK, thioredoxin (Trx), inducible nitric oxide synthase (iNOS), or GAPDH (1:1000, Abcam, Cambridge, MA, USA) at room temperature for 3 h. Following incubation with a secondary antibody (1:3000, Abcam), the expression levels of the target proteins were observed by enhanced chemiluminescence (Millipore, Burlington, MA, USA).
Luciferase activity assay
The targeting relationship between LINC00221 and miR-542-3p was verified by the luciferase reporter gene assay. The psiCHECK-2 promoter vector luciferase gene was modified to contain the mutant 3′UTR of LINC00221 (MUT) or wild-type 3′UTR of LINC00221 (WT) to yield the recombinant plasmids psiCHECK-2-LINC00221-WT and psiCHECK-2-LINC00221-MUT. Next, the psiCHECK-2-LINC00221-WT or psiCHECK-2-LINC00221-MUT, the miR-542-3p mimics or NC, and the plasmid pRL-TK with Renilla luciferase activity were co-transfected into 293 T cells for 24 h by using LipofectamineTM2000. The luciferase activity was then measured.
Enzyme-linked immunosorbent assay (ELISA)
The relative levels of tumor necrosis factor-α (TNF-α), VEGF, and MCP1 in cells were detected by ELISAs that were performed with corresponding ELISA KITs purchased from Solarbio Life Science Company (Beijing, China) All ELISAs were performed according to the manufacturer’s instructions.
In vitro angiogenesis assay
After co-incubation with treated trophoblast cells, the angiogenesis activity of HUVECs was evaluated by a tube formation assay. Briefly, a precooled 96-well plate was coated with Matrigel and then incubated at 37 °C for 45 min. Subsequently, treated HUVECs were seeded into the wells of the 96-well plate, after which the plate was incubated for 8 h and then photographed under a microscope (Olympus Corporation, Tokyo, Japan).
Cell immunofluorescence
The expression of the VEGR receptor (VEGFR) in treated HUVECs was also evaluated by cell immunofluorescence. In brief, cells were fixed in 4% formaldehyde solution for 12 min, blocked with 5% BSA solution, incubated with antibodies targeting VEGFR (Abcam; 1: 500) overnight at 4 °C, washed again with PBS solution, and then incubated in the dark with secondary antibodies for 1–2 h. Cell nuclei were stained with DAPI. The cells were then mounted onto slides using Prolong Gold Antifade Reagent (CST), and their fluorescence intensities were evaluated under a fluorescence microscope.
Transwell assay
Macrophages induced from THP-1 cells were co-cultured with trophoblast cells as described above. The migration and invasion abilities of the macrophages were detected. Briefly, macrophages suspended in DMEM without serum were seeded into the upper chamber of a Transwell plate and HTR-8/SVneo or JEG-3 cells suspended in complete DMEM culture medium with serum were added to the lower compartment as a chemotactic factor. The plate was then incubated at 37 °C. Cells in the lower compartment were stained with crystal violet, and the numbers of migrated and invasive cells were counted under a light microscope.
Immunohistochemistry (IHC)
Samples of placental tissue were treated with 4% paraformaldehyde and embedded in paraffin. After being de-waxed and hydrated, the placental tissue slides were exposed to citrate buffer. Next, the slides were pre-treated at 80 °C and blocked by endogenous peroxide, after which the slides were reacted with a CK7, α-SMA, or MCP1 antibody and then with a secondary antibody. Finally, the slides were mounted and analyzed under a light microscope (Nikon).
Statistical analysis
Quantitative data were analyzed using SPSS Statistics for Windows, Version 20.0 software (IBM Corp., Armonk, NY, USA). Differences among various groups were evaluated by the t test or ANOVA. A P-value < 0.05 indicated statistical significance.
Results
Clinical characteristics and LINC00221 levels in PE patients
As shown in Table 2, in addition to age, the urine protein levels and systolic and diastolic blood pressures were significantly higher, while the gestational weeks and neonatal weights were obviously lower in PE patients than in normal pregnant women. Notably, qPCR results showed that LINC00221 was much more highly expressed in the placental tissues of PE patients when compared with the placental tissues of normal pregnant women (Fig. 2A). FISH assays confirmed the expression of LINC00221 in both HTR-8/SVneo and JEG-3 cells (Fig. 2B).
Table 2.
Comparison of clinical characteristics between preeclampsia (PE) patients and normal pregnant women involved in this study
| Clinical characteristics | Control (n = 20) | PE | P-value | ||
|---|---|---|---|---|---|
| Early onset (n = 12) | Late onset (n = 8) | Total (n = 20) | |||
| Urine protein | 0.11 ± 0.02 | 4.76 ± 0.62 | 4.54 ± 0.58 | 5.09 ± 0.57 | P < 0.01 |
| Age | 31.3 ± 2.05 | 29.85 ± 4.13 | 29.92 ± 4.46 | 29.75 ± 3.88 | P > 0.05 |
| Systolic blood pressure | 119.75 ± 7.71 | 167.26 ± 8.3 | 165.72 ± 7.89 | 169.57 ± 8.89 | P < 0.01 |
| Diastolic blood pressure | 75.27 ± 3.37 | 109.75 ± 7.02 | 107.42 ± 7.72 | 113.25 ± 4.1 | P < 0.01 |
| Gestational age | 36.94 ± 0.47 | 30.88 ± 1.31 | 31.09 ± 1.22 | 30.56 ± 1.46 | P < 0.01 |
| Neonatal weight | 3223.4 ± 146.83 | 2247.44 ± 236.59 | 2323.77 ± 240.07 | 2132.94 ± 190.77 | P < 0.01 |
Fig. 2.
LINC00221 was upregulated in the placental tissues of preeclampsia (PE) patients. A The levels of LINC00221 in the placental tissues of PE patients and normal pregnant women as determined by qRT-PCR; B the location of LINC00221 (red) in HTR-8/SVneo cells and JEG-3 cells as determined by fluorescence in situ hybridization assays. **P < 0.01
LINC00221 silencing reduced MCP1 levels, increased VEGF levels, and reduced endoplasmic reticulum stress and oxidative stress in trophoblast cells
To screen for LINC00221 specific siRNAs, both HTR-8/SVneo and JEG-3 cells were transiently transfected with siRNA #1, siRNA #2, and siRNA #3, and the results showed that siRNA #1 exhibited the optimal silencing effect on LINC00221 expression (Fig. 3A); thus, siRNA #1 was used in subsequent experiments. ELISA results revealed that LINC00221 silencing significantly reduced cellular MCP1 levels and increased cellular VEGF levels (Fig. 3B, C). Furthermore, endoplasmic reticulum stress-related molecules, such as CHOP, IREα, and PERK, as well as oxidative stress-related proteins (Trx and iNOS) were detected in trophoblast cells by western blotting. Those results showed that LINC00221 silencing decreased CHOP, p-IREα, p-PERK, and iNOS expression but increased Trx expression in both HTR-8/SVneo and JEG-3 cells (Fig. 3D).
Fig. 3.
Effects of LINC00221 silencing on trophoblast cells, co-cultured HUVECs, and co-cultured macrophages. A The LINC00221 levels in HTR-8/SVneo cells and JEG-3 cells transfected with siRNA #1, siRNA #2, and siRNA #3 was determined by qRT-PCR; B, C the levels of monocyte chemotactic protein 1 (MCP1) (B) and vascular endothelial growth factor (VEGF) (C) in HTR-8/SVneo cells and JEG-3 cells transfected with siRNA #1 was determined by enzyme-linked immunosorbent assays (ELISAs); D the expression of endoplasmic reticulum stress-related molecules, including CHOP, IREα, and PERK, as well as oxidative stress-related proteins (Trx and iNOS) in trophoblast cells was determined by western blotting; E the angiogenic activity of HUVECs co-incubated with trophoblast cells as determined using an in vitro angiogenesis assay; F expression of the VEGF receptor in HUVECs co-incubated with trophoblast cells was determined by western blotting; G expression of the VEGF receptor in HUVECs co-incubated with trophoblast cells was determined by cell immunofluorescence; H, I the migration and invasion abilities of macrophages co-incubated with trophoblast cells were determined by Transwell assays; J the levels of tumor necrosis factor-α (TNF-α) in the supernatants of co-cultured trophoblast cells and macrophages were determined by ELISA. ***P < 0.001, vs. NC
LINC00221 silencing in trophoblast cells promoted VEGFR expression and angiogenesis by co-cultured HUVECs, and inhibited migration/invasion and TNF-α production by macrophages
To confirm the effect of trophoblast cells on HUVECs, HTR-8/SVneo or JEG-3 cells were co-cultured with HUVECs. The results showed that when compared with a Blank or NC group, VEGFR expression and the angiogenic ability of HUVECs were remarkably enhanced in the siRNA#1 group (Fig. 3E, F,G).
Because the MCP-1 levels in trophoblast cells were reduced by siRNA#1, we speculated whether the macrophage recruitment ability of trophoblast cells had changed. During co-culture of trophoblast cells and macrophages, LINC00221 silencing in trophoblast cells obviously decreased the numbers of migrated and invaded macrophages (Fig. 3H, I), and also reduced TNF-α levels (Fig. 3J).
Association between LINC00221 and miR-542-3p
Based on GEO datasets, PE-related miRNAs were screened and identified in the placental tissues of PE patients and normal pregnant women. Q-PCR results showed that the levels of miR-518-3p, miR-542-3p, miR-3652, miR-4316, miR-548o, miR-3907, miR-320c, and miR-516a-3p were significantly lower, while miR-152 and miR-544b levels were higher in the placental tissues of PE patients than in the placental tissues of normal pregnant women (Fig. 4A). A bioinformatics analysis revealed that miR-542-3p was a potential target gene of LINC00221. Luciferase receptor assays showed that after co-transfection with psiCHECK-2-LINC00221-WT and miR-542-3p mimics, the luciferase activity was reduced when compared to co-transfection with the mimic control, while no significant difference was found after psiCHECK-2-LINC00221-MUT treatment, suggesting that miR-542-3p binds to LINC00221 (Fig. 4B). In addition, the levels of miR-542-3p were obviously decreased in HTR-8/SVneo or JEG-3 cells transfected with the LINC00221 overexpression vector, and increased in cells transfected with siRNA #1 (Fig. 4C).
Fig. 4.
LINC00221 negatively regulated miR-542-3p expression. A The levels of differentially expressed miRNAs in the placental tissues of PE patients and normal pregnant women were determined by qRT-PCR; B the targeted regulation of miR-542-3p by LINC00221 was proven by a luciferase reporter assay; C the miR-542-3p levels in HTR-8/SVneo cells and JEG-3 cells transfected with the LINC00221 overexpression vector or siRNA were determined by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001
Effect of abnormal miR-542-3p expression on trophoblast cells, co-cultured HUVECs, and co-cultured macrophages
MiR-542-3p regulated MCP1 and VEGF release, endoplasmic reticulum stress, and oxidative stress in trophoblast cells
Both HTR-8/SVneo and JEG-3 cells were exposed to miR-542-3p mimics or the inhibitor, respectively. When compared with untreated cells, the miR-542-3p levels were remarkably upregulated in cells treated with miR-542-3p mimics and downregulated in cells treated with the miR-542-3p inhibitor (Fig. 5A). Additionally, treatment with miR-542-3p mimics significantly reduced cellular MCP1 levels and increased VEGF levels, while cells treated with the miR-542-3p inhibitor showed the opposite changes in expression when compared to untreated cells (Fig. 5B, C). Furthermore, western blot assay showed that miR-542-3p mimics decreased the expression of CHOP, p-IREα, p-PERK, and iNOS, but increased Trx expression in both HTR-8/SVneo and JEG-3 cells, and treatment with the miR-542-3p inhibitor produced the opposite changes in expression (Fig. 5D).
Fig. 5.
Effect of abnormal miR-542-3p expression on trophoblast cells, co-cultured HUVECs, and co-cultured macrophages. A The miR-542-3p levels in HTR-8/SVneo cells and JEG-3 cells treated with mimics or the inhibitor were determined by qRT-PCR; B, C the levels of monocyte chemotactic protein 1 (MCP1) and vascular endothelial growth factor (VEGF) in HTR-8/SVneo cells and JEG-3 cells transfected with mimics or the inhibitor were determined by enzyme-linked immunosorbent assays (ELISAs); D the expression of endoplasmic reticulum stress-related molecules, including CHOP, IREα, and PERK, as well as oxidative stress-related proteins (Trx and eNOS) in trophoblast cells treated with mimics or the inhibitor was determined by western blotting; E the angiogenic ability of HUVECs co-incubated with trophoblast cells was determined by in vitro angiogenesis assays; F expression of the VEGF receptor in HUVECs co-incubated with trophoblast cells was determined by western blotting; G expression of the VEGF receptor in HUVECs co-incubated with trophoblast cells was determined by cell immunofluorescence; D, H, I the migration and invasion abilities of macrophages co-incubated with trophoblast cells was determined by Transwell assays; J the levels of tumor necrosis factor-α (TNF-α) in the supernatants of co-cultured trophoblast cells and macrophages was determined by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001, vs. NC
MiR-542-3p in trophoblast cells regulated VEGFR expression and the angiogenic ability of co-cultured HUVECs, as well as the migration and invasion abilities, and TNF-α production by macrophages.
After co-incubation of trophoblast cells and HUVECs, VEGFR expression and the angiogenic ability of HUVECs were remarkably enhanced in the miR-542-3p mimics group, while treatment with the miR-542-3p inhibitor produced the opposite effects on HUVECs (Fig. 5E, F, G).
Moreover, during co-culture with miR-542-3p overexpressing trophoblast cells, the migration and invasion abilities of macrophages were obviously reduced (Fig. 5H, I), and the TNF-α levels (Fig. 5J) in both HTR-8/SVneo and JEG-3 cells were also reduced, while the miR-542-3p inhibitor group exhibited the opposite effects.
MiR-542-3p reversed the effect of LINC00221 on trophoblast cells, co-cultured HUVECs, and co-cultured macrophages
MiR-542-3p reversed the effect of LINC00221 on trophoblast cells
To further explore the relationship between LINC00221 and miR-542-3p, rescue experiments were performed with both HTR-8/SVneo and JEG-3 cells by treating the cells with LINC00221 siRNA and/or the miR-542-3p inhibitor, respectively. We found that compared to the NC group, LINC00221 silencing significantly reduced LINC00221 expression and increased miR-542-3p levels, while addition of the miR-542-3p inhibitor had no effect on LINC00221 expression, but decreased miR-542-3p levels (Fig. 6A, B). When compared with the siRNA group, the miR-542-3p inhibitor significantly increased cellular MCP1 levels and decreased cellular VEGF levels (Fig. 6C). Furthermore, addition of the miR-542-3p inhibitor reversed the expression of endoplasmic reticulum stress-related molecules and oxidative stress-related proteins induced by LINC00221 silencing (Fig. 6D).
Fig. 6.
MiR-542-3p suppression reversed the effect of LINC00221 knockdown on trophoblast cells and co-cultured HUVECs. A The expression of LINC00221 in HTR-8/SVneo cells and JEG-3 cells treated with siRNA or siRNA + inhibitor was determined by qRT-PCR; B the miR-542-3p levels in HTR-8/SVneo cells and JEG-3 cells treated with siRNA or siRNA + inhibitor was determined by qRT-PCR; C the levels of monocyte chemotactic protein 1 (MCP1) and vascular endothelial growth factor (VEGF) in HTR-8/SVneo cells and JEG-3 cells transfected with siRNA or siRNA + inhibitor was determined by enzyme-linked immunosorbent assays (ELISAs); D the expression of endoplasmic reticulum stress-related molecules, including CHOP, IREα, and PERK, as well as oxidative stress-related proteins (Trx and iNOS) in trophoblast cells treated with siRNA or siRNA + inhibitor was determined by western blotting; E the angiogenic ability of HUVECs co-incubated with trophoblast cells was determined by in vitro angiogenesis assays; F expression of the VEGF receptor in HUVECs co-incubated with trophoblast cells was determined by western blotting; G expression of the VEGF receptor in HUVECs co-incubated with trophoblast cells was determined by cell immunofluorescence. **P < 0.01, ***P < 0.001, vs. NC. ###P < 0.001, vs. siRNA + miRNA NC
MiR-542-3p reversed the effect of LINC00221 on co-cultured HUVECs and co-cultured macrophages
After co-culture with trophoblast cells treated with LINC00221 siRNA and the miR-542-3p inhibitor, the VEGFR expression and angiogenic ability of HUVECs were reduced when compared with the siRNA group (Fig. 6E, F, G).
Moreover, co-inhibition of LINC00221 and miR-542-3p in trophoblast cells promoted the migration and invasion of co-cultured macrophages (Fig. 7A, B), and increased their TNF-α levels (Fig. 7C), when compared with HTR-8/SVneo and JEG-3 cells with only LINC00221 inhibition.
Fig. 7.
MiR-542-3p inhibitor reversed the effect of LINC00221 siRNA on co-cultured macrophages’ migration and invasion. A, B The migration and invasion abilities of macrophages co-incubated with trophoblast cells was determined by Transwell assays; C the levels of tumor necrosis factor-α (TNF-α) in the supernatants of co-cultured trophoblast cells and macrophages as determined by ELISA. ***P < 0.001, vs NC. #P < 0.05, ###P < 0.001, vs. siRNA + miRNA NC
Effect of LINC00221 silencing on the PE model rats
The effects of LINC00221 silencing on PE were also investigated via in vivo experiments. A rat model of PE was induced by treatment with L-NAME and LINC00221 shRNA. Results showed that when compared with the sham group, LINC00221 was highly expressed and miR-542-3p was only slightly expressed in the placental tissues of the PE model rats; and those effects could be partially reversed by LINC00221 silencing (Fig. 8A). ELISA results revealed that serum TNF-α levels were upregulated in the model group when compared with the sham group, while LINC00221 silencing reduced the TNF-α levels (Fig. 8B). In addition, IHC results revealed that MCP1 and α-SMA expression were increased and CK7 expression was decreased in the model group when compared with the sham group, and those protein levels could be partially recovered by LINC00221 silencing (Fig. 8C). Moreover, western blotting studies showed that when compared with the sham group, the levels of CHOP, p-IREα, p-PERK, and iNOS expression were increased and Trx expression was decreased in the model group, while LINC00221 silencing produced the opposite changes in expression (Fig. 8D).
Fig. 8.
Effect of LINC00221 silencing on the PE model rats. A LINC00221 and miR-542-3p expression in normal rats, PE model rats, and shRNA treatment PE rats as determined by qRT-PCR; B the levels of tumor necrosis factor-α (TNF-α) in normal rats, PE model rats, and shRNA-treated PE rats as determined by ELISA; C MCP1, α-SMA, and CK7 expression in normal rats, PE model rats, and shRNA-treated PE rats as determined by immunohistochemistry assay; D The expression of endoplasmic reticulum stress-related molecules, including CHOP, IREα, and PERK, as well as oxidative stress-related proteins (Trx and iNOS) in the normal rats, PE model rats, and shRNA-treated PE rats as determined by western blotting. ***P < 0.001, vs. Sham. ###P < 0.001, vs. model
Discussion
Preeclampsia (PE) is one of the most serious pregnancy-related complications, and termination of a pregnancy to deliver the placenta is the only way to completely cure PE. Thus, early clinical diagnosis and intervention for placental lesions are potential approaches for improving pregnancy outcomes. A recent study reported that LINC00221 was overexpressed in an abnormally invasive placenta. Thus, we speculated whether LINC00221 might be involved in the occurrence and progression of PE. In the present study, we found that LINC00221 levels were upregulated in the placental tissues of PE patients when compared with the placental tissues of normal pregnant women. Moreover, LINC00221 silencing significantly inhibited MCP1 expression and promoted VEGF expression in trophoblast cells. Notably, trophoblast cells with LINC00221 silencing promoted the angiogenic ability of HUVECs, and also inhibited the migration and invasion of macrophages.
It is well-known that the pathogenesis of PE is associated with various epigenetic and genetic factors. Especially, endothelial cell dysfunction is involved in abnormal placentation and considered to be a risk factor for the occurrence of PE [17]. VEGF was reported to promote decidual angiogenesis and function by binding to the VEGFR during early pregnancy [18]. A VEGF deficit was shown to participate in endothelial cell dysfunction in PE [19]. Our current study showed that a downregulation of LINC00221 levels increased the VEGF content in HTR-8/SVneo and JEG-3 cells, enhanced expression of the VEGF receptor, and also increased the angiogenic ability of co-cultured HUVECs. These findings suggest that LINC00221 silencing might improve endothelial cell function by regulating VEFG/VEGFR. The interaction between trophoblast cells and immune cells, such as macrophages, is also critical. During normal pregnancy, macrophages accumulate in the decidua to protect the fetus from infection. However, in a PE placenta, trophoblast cells produce excessive amounts of chemokines and recruit macrophages that induce inflammation and cell damage [20, 21]. In this study, we found that LINC00221 silencing significantly reduced the levels of MCP1 in trophoblast cells, and co-culture with si-LINC00221 trophoblast cells inhibited the migration and invasion of macrophages and reduced their levels of TNF-α. Thus, we speculated that LINC00221 silencing might prevent an inflammatory response by decreasing the recruitment of macrophages.
Endoplasmic reticulum stress and oxidative stress are two other risk factors for PE. A previous study proved that endoplasmic reticulum stress can be caused by activation of an unfolded protein response (UPR), and is involved in the occurrence of PE by inducing trophoblast cell apoptosis [22]. This study showed that LINC00221 silencing decreased the expression of CHOP, p-IREα, and p-PERK both in vitro and in vivo, indicating that the effects of LINC00221 in PE might be related to endoplasmic reticulum stress. As for oxidative stress, during normal placentation, ROS are generated and function to regulate cell signaling. However, excessive ROS production induces oxidative stress, further triggers inflammation, and promotes PE progression [23, 24]. In addition, oxidative stress has also been shown play a regulatory role during endothelial cell dysfunction, and thereby be responsible for the cardiovascular complications of PE [25, 26]. In this study, one oxidative stress biomarker (iNOS) was inhibited and another oxidative stress biomarker (Trx) was promoted by LINC00221 silencing, suggesting that LINC00221 might also be involved in oxidative stress and affect PE progression.
Subsequently, the downstream mechanism of LINC00221 in PE was explored in this study. Our data showed that LINC00221 could bind to miR-542-3p and negatively regulate miR-542-3p expression. Previous studies on the differential expression profiles of miRNAs have reported that miR-542-3p is significantly downregulated in the placentas of PE patients [27, 28]. Our study found that miR-542-3p might play a protective role in PE by suppressing endoplasmic reticulum stress and oxidative stress in trophoblast cells, enhancing HUVEC angiogenesis, and inhibiting macrophage recruitment. These findings suggest that a downregulation of LINC00221 expression might exert a protective effect against PE by upregulating miR-542-3p.
In this study, on the one hand, a downregulation of LINC00221 caused an upregulation of miR-542-3p, and placental trophoblasts were able to activate the ER stress pathway. On the other hand, a downregulation of trophoblast LINC00221 impaired the angiopoiesis activity of HUVECs and stimulated the recruitment of macrophages, thereby further amplifying the inflammatory response. In conclusion, our data showed that LINC00221 was overexpressed in the placental tissues of PE patients, and a downregulation of LINC00221 might regulate endothelial cell dysfunction, the inflammatory response, endoplasmic reticulum stress, and oxidative stress, and thereby help to protect against PE by augmenting miR-542-3p (Fig. 9).
Fig. 9.
The mechanism by which LINC00221 contributes to preeclampsia
Supplementary Information
(PNG 1912 kb)
Funding
This study is supported by the Joint Construction Project of Medical Science and Technology Key Project in Henan Province (LHGJ20190446).
Declarations
Ethical statement
For the placental tissue samples used in this study as well as clinical characteristics, prior patient consent were obtained and all experiments were conducted with the approval of the Ethics Committee of the First Affiliated Hospital of Xinxiang Medical University.
Competing interests
The authors declare no competing interests.
Footnotes
Highlights
1. LINC00221 expresses highly in the placental tissues of PE patients.
2. LINC00221 siRNA promotes angiopoiesis in co-cultured HUVECs.
3. LINC00221 promotes recruitment of macrophages in co-cultured macrophages.
4. LINC00221/miR-542-3p mediates the functions of trophoblast cells in vitro.
Publisher’s note
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