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GADD45A axis
Author’s Accepted Manuscript Long non-coding RNA DLX6-AS1 is upregulated in preeclampsia and modulates migration and invasion of trophoblasts through the miR376c/GADD45A axis Yan Tan, Di Xiao, Yanbin Xu, Chenhong Wang www.elsevier.com/locate/yexcr
PII: DOI: Reference:
S0014-4827(18)30579-2 https://doi.org/10.1016/j.yexcr.2018.07.039 YEXCR11143
To appear in: Experimental Cell Research Received date: 14 March 2018 Revised date: 23 July 2018 Accepted date: 24 July 2018 Cite this article as: Yan Tan, Di Xiao, Yanbin Xu and Chenhong Wang, Long non-coding RNA DLX6-AS1 is upregulated in preeclampsia and modulates migration and invasion of trophoblasts through the miR-376c/GADD45A axis, Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2018.07.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Long non-coding RNA DLX6-AS1 is upregulated in preeclampsia and modulates migration and invasion of trophoblasts through the miR-376c/GADD45A axis Yan Tan1, Di Xiao2, Yanbin Xu1, Chenhong Wang1,3* 1
Department of Gynecology and Obstetrics, Affiliated Shenzhen Maternity & Child Healthcare
Hospital, Southern Medical University (Shenzhen Maternity & Child Healthcare Hospital), Shenzhen 518028, Guangdong, China 2
Department of Gynecology and Obstetrics, The Third Affiliated Hospital, Southern Medical
University, Guangzhou 510150, Guangdong, China 3
Department of Gynecology and Obstetrics, Shenzhen Hospital, Southern Medical University,
Shenzhen 518000, Guangdong, China *Correspondence to: Chenhong Wang, Present address: Department of Gynecology and Obstetrics, Shenzhen Hospital of Southern Medical University, No.1333, Xinhu Road, Baoan district, Shenzhen 518000, Guangdong Province, China. Tel./ Fax: +86-0755-23360520. [email protected] Abstract Long non-coding RNA (lncRNA) are key regulatory molecules that are implicated in diverse biological processes and human diseases, including preeclampsia. However, their expression and functions in the progression of preeclampsia remains largely unclear. In this study, lncRNA DLX6-AS1 was confirmed to be significantly upregulated in the placentas of patients with preeclampsia, compared with normal controls. Overexpression of DLX6-AS1 dramatically decreased proliferation, migration and invasion in trophoblast JEG3 and HTR-8/SVneo cells. Luciferase activity and RNA pull-down assays showed that DLX6-AS1 interacted with miR-376c, and that overexpression of DLX6-AS1 significantly reduced expression of miR-376c
in HTR-8/SVneo cells. Also, miR-376c targeted and downregulated GADD45A in HTR-8/SVneo cells. Overexpression of GADD45A effectively attenuated a miR-376c-induced increase in the proliferation, migration and invasion of HTR-8/SVneo cells. Further research showed that DLX6-AS1-induced inhibition of trophoblast proliferation, migration and invasion was effectively neutralized by GADD45A knockdown and miR-376c overexpression. Taken together, these findings suggest that DLX6-AS1 may contribute to preeclampsia by impairing proliferative, migratory and invasive abilities of trophoblasts via the miR-376c/GADD45A axis. Keywords Preeclampsia, DLX6-AS1, trophoblast, GADD45A, miR-376c
Introduction Preeclampsia (PE), a pregnancy-specific syndrome, remains a leading cause of maternal and neonatal morbidity and mortality[1]. This multi-systemic disorder clinically manifests as new-onset hypertension (≥ 140/90 mmHg) and overt proteinuria (≥ 300 mg/24 h) after 20 weeks of gestation[2]. Trophoblastic dysfunction, such as inadequate migration and invasion, are well-known to be correlated with PE[2, 3]. However, the molecular mechanism implicated in the pathogenesis of PE and the regulation of trophoblast behavior are still largely unknown. Long non-coding RNAs (lncRNAs) are a class of RNA transcripts longer than 200 nucleotides in length which play multiple and crucial roles in many fundamental biochemical and disease processes[4, 5]. LncRNAs help regulate gene expression through chromosome dosage compensation, genomic imprinting, and transcriptional and epigenetic regulation[1, 5, 6]. Numerous lncRNAs are differentially expressed in the placentas from healthy subjects and in patients with PE, suggesting that aberrant expression of lncRNA may contribute to PE[4, 7].
Some lncRNAs, such as MALAT-1, RPAIN and Uc.187 has been proved to be involved in regulating trophoblast behavior[6, 8, 9]. For instance, high expression of lncRNA SPRY4-IT1 in placentas from women with PE correlates with poor prognosis and contributes to PE by impairing trophoblast migration, invasion, and epithelial-mesenchymal transition[3, 10]. Therefore, increased understanding of the lncRNAs aberrantly expressed in PE patients are beneficial for understanding the pathogenesis and progression of PE, thereby proposing more therapeutic interventions. In the present study, lncRNA DLX6-AS1 was found to be upregulated in placentas from patients with PE, compared to those from women with normal pregnancies. Moreover, overexpression of DLX6-AS1 markedly repressed proliferation, migration and invasion of trophoblasts. Further investigation suggested that the inhibition effect of DLX6-AS1 on trophoblasts was related to the miR-376c/GADD45A axis. These findings indicate that DLX6-AS1 contributes to PE by regulating trophoblastic proliferation, migration and invasion via the miR-376c/GADD45A axis.
Materials and Methods Ethics statement and tissue samples Collection and experimentation of placental tissues in this study was approved by the Ethics Committee of the Affiliated Shenzhen Maternity & Child Healthcare Hospital of Southern Medical University (Guangdong, China). Subjects enrolled 24 patients with preeclampsia and 20 control patients who received a Caesarean section. Informed consent from all subjects was obtained before enrollment. Preeclampsia was diagnosed as systolic pressure ≥140 mm Hg and/or diastolic pressure ≥90 mm Hg, accompanied by proteinuria (urine protein ≥ 300 mg/24 h).
Control patients were those pregnant women who underwent caesarean section because of abnormal fetal position with delivery at < 37 weeks gestation. No subject was a smoker or had a history of any chronic diseases. Placental tissues were collected immediately after parturition and stored frozen in liquid nitrogen until use. Cell culture and transfection The human trophoblast cell lines JEG3 and HTR-8/SVneo were provided by American Type Culture Collection (Manassas, VA, USA) and cultivated in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and 100 U/mL of penicillin/streptomycin (Gibco) in a humidified atmosphere of 5% CO2 at 37 °C. Cells were plated in 24-well plates (2 × 104 cells/well), grown to 70% confluence, and then transfected with LipofectamineTM3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Overexpression
of
DLX6-AS1
was
achieved
using
pcDNA3.1-DLX6-AS1
plasmid
(GenePharma Co. Ltd., Shanghai, China) transfection, with an empty vector as a control. The pcDNA3.1-GADD45A and empty vectors, the miR-376c mimic and miR-NC mimic, and the GADD45A siRNA and scramble siRNA also were synthesized and provided by GenePharma. Cells were harvested 48 h after transfection for further studies. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted with Trizol reagent (Invitrogen) from placental tissue or trophoblasts, and cDNA was generated by using a Reverse Transcription Kit (Invitrogen) following the manufacturer’s instructions. RT-PCR was performed by using Syber Green PCR Mastermix (Applied Biosystems, Foster City, CA, USA). The specific primers were as follows: DLX6-AS1, forward 5′-CCA AAT GCT ACC ATC CAG CC-3′ and reverse 5′-TCT GGC TTC CCT TAA CCA AA-3′; GADD45A, forward 5′- GAG CAG AAG ACC GAA AGC GAC-3′ and
reverse 5′-GAA TGT GGA TTC GTC ACC AGC-3′; GAPDH, forward 5′-TTC GAC AGT CAG CCG CAT CTT-3′ and reverse 5′-CCC AAT ACG ACC AAA TCC GTT-3′; miR376c, forward 5′-GCG CAA CAT AGA GGA AA-3′ and reverse 5′-GGT GCA GGG TCC GAG GT-3′; U6, forward 5′-CTC GCT TCG GCA GCA CA-3′ and reverse 5′-AAC GCT TCA CGA ATT TGC GT-3′. Relative gene expression levels were calculated using the 2−ΔΔCt method and normalized to GAPDH or U6. Western blot Total proteins were extracted using a radioimmunoprecipitation assay buffer (Sigma, St. Louis, MO, USA), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% fat-free milk, membranes were probed with specific primary antibodies against GADD45A and GAPDH (Abcam, Cambridge, UK) overnight at 4 °C. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Abcam) for 1 h at room temperature. Finally, the protein bands were visualized by using an enhanced chemiluminescence detection system (Thermo Fisher, San Jose, CA, USA) and quantified by Image J software. The relative protein expression was normalized to endogenous GAPDH. Cell viability assay Cell viability was assessed by using a Cell Counting kit-8 (CCK-8) assay (Sigma). Transfected cells were seeded into 96-well plates (3 × 103 cells/well) and incubated for 24 h. Then, 10 μL of CCK-8 solution was added into each well and incubated for another 2 h. The optical density of each well was then measured at a wavelength of 450 nm using a microplate reader.
Transwell migration and Matrigel invasion assays Cell invasion and migration were evaluated by using a 24-well transwell insert system (8 μm pore filters; Costar, Cambridge, MA, USA). Then, 1 × 105 cells were resuspended in 200 μL of serum-free medium and seeded into the upper transwell chambers pre-coated with (for invasion) or without (for migration) Matrigel. In the lower chamber, 800 μL of medium containing 10% fetal bovine serum was added to induce chemotaxis. After 24 h of incubation at 37 °C, chambers were disassembled, and the invaded and migratory cells were stained with 2% crystal violet and counted under the microscope in five random visual fields. Target prediction and dual-luciferase reporter assay Bioinformatics analysis was used to predict the potential targets of lncRNA DLX6-AS1and miR-376c, including DIANA-LncBase v2, microRNA.org, and miRDB. For the luciferase reporter assay, wild-type or mutant DLX6-AS1 and GADD45A fragments with or without the miR-376c binding element were respectively cloned into the luciferase reporter vector pGL3 (Promega, Madison, WI, USA). HEK293T cells were grown in 12-well plates to 70% confluency and then co-transfected with either pGL3-DLX6T-AS1-wt or pGL3-DLX6T-AS1-mut (pGL3-GADD45A-wt or pGL3-GADD45A-mut) and the miR-376c mimic or NC-mimic. Luciferase activity was determined 24 h after transfection by using the Dual-Luciferase Reporter Assay System (Promega). RNA pull-down assay Biotinylated wild-type miR-376c (miR-376c-Bio), biotinylated mutant miR-376c (miR-376c-MutBio), or biotinylated NC (NC-Bio) was transfected into the HTR-8/SVneo cells. After 48 h of transfection, the cells were lysed and incubated with M-280 streptavidin magnetic beads (Invitrogen). The bound RNAs were then purified with TRIzol reagent (Invitrogen) and
analyzed by RT–PCR. Statistical analysis Each experiment was performed in triplicate and repeated three times. All data for this study are expressed as the mean ± standard deviation (SD). One-way analysis of variance and Student's t tests were performed to evaluate statistical differences within or between the groups by using SPSS 22.0 software. A value of p < 0.05 was considered statistically significant.
Results lncRNA DLX6-AS1 was upregulated in placentas from women with PE To investigate the potential role of DLX6-AS1 in the progression of PE, we first measured DLX6-AS1 expression in placentas from patients with PE (n = 24) and from control pregnancies (n = 18). Table 1 presents the clinicopathologic data. Resultly, DLX6-AS1 levels were 2.16-fold higher in PE placentas, compared to normal ones (Fig. 1, p < 0.05), suggesting that upregulation of DLX6-AS1 in the placenta may be implicated in the pathogenesis of PE. DLX6-AS1 overexpression inhibited migration and invasion of trophoblastic cells To explore the biological functions of DLX6-AS1 in PE, the pcDNA3.1-DLX6-AS1 plasmid or empty vector was transfected into JEG3 and HTR-8/SVneo cells. Compared to the negative control, DLX6-AS1 was upregulated nearly 9-fold in both JEG3 and HTR-8/SVneo cells that were transfected with the pcDNA3.1-DLX6-AS1 vector (Fig. 2A, p < 0.05). Decreased cell viability was observed in both JEG3 and HTR-8/SVneo cells transfected with the pcDNA3.1-DLX6-AS1 vector (Fig. 2B, p < 0.05). Overexpression of DLX6-AS1 also significantly reduced migratory and invasive abilities of JEG3 and HTR-8/SVneo cells (Fig. 2C– 2D, p < 0.05). Taken together, upregulation of DLX6-AS1 impaired the proliferative, migratory,
and invasive capacities of trophoblastic cells. DLX6-AS1 decreased expression of miR-376c in trophoblastic cells Bioinformatics analyses were performed to investigate the molecular mechanism by which DLX6-AS1 regulates trophoblast behavior. MiR-376c, a microRNA associated with PE[11], was found to have complementary binding sites with DLX6-AS1 (Fig. 3A). A luciferase reporter assay showed that miR-376c significantly suppressed luciferase activity fused to the wild-type DLX6-AS1 fragments, but it failed to repress luciferase activity fused to the mutated one (Fig. 3B, p < 0.05). A biotin-based pull-down assay showed that DLX6-AS1, but not the deletion mutants, was efficiently pulled down by miR-376c (Fig. 3C, p < 0.05). The expression of miR-376c in placentas from patients with PE was significantly downregulated (Fig. 3D, p < 0.05) and negatively correlated with DLX6-AS1 expression (Fig. 3E, r = – 0.65). Moreover, a marked decrease in expression of miR-376c was noted in HTR-8/SVneo cells transfected with pcDNA3.1-DLX6-AS1 (Fig. 3F, p < 0.05). These results suggest that DLX6-AS1 may interact with miR-376c and decreased its expression in human trophoblasts. MiR-376c targeted GADD45A to modulate proliferation, migration and invasion of trophoblastic cells Bioinformatic analysis indicated that GADD45A, a molecule linked with PE progression[12], contains a miR-376c binding site in its 3′ untranslated region (UTR), as shown in Fig. 4A. MiR-376c significantly reduced the luciferase activity of HEK293T cells transfected with the wild-type GADD45A plasmid, but it failed to suppress luciferase activity in cells transfected with the mutant plasmid (Fig. 4B, p < 0.05). Both mRNA and protein expression of GADD45A was markedly reduced in HTR-8/SVneo cells transfected with the miR-376c mimic (Fig. 4C–4D, p < 0.05). Overexpression of miR-376c also dramatically enhanced proliferation,
migration, and invasion capacities of HTR-8/SVneo cells (Fig. 4E–4G, p < 0.05). However, overexpression of GADD45A significantly inhibited the miR-376c-induced increase in proliferation, migration, and invasion of HTR-8/SVneo cells (Fig. 4E–4G, p < 0.05). Collectively, miR-376c promoted cell proliferation, migration, and invasion by downregulating GADD45A in human trophoblasts. DLX6-AS1
inhibited
trophoblastic
migration
and
invasion
through
the
miR-376c/GADD45A axis Because GADD45A is a target of miR-376c and because DLX6-AS1 can interact with miR-376c in human trophoblasts, we speculated that DLX6-AS1 may affect PE progression by regulating the miR-376c/GADD45A axis. Overexpression of DLX6-AS1 in HTR-8/SVneo cells resulted in an obvious increase in both mRNA and protein expression of GADD45A (Fig. 5A–5B, p < 0.05). The inhibition effects of DLX6-AS1 on trophoblast proliferation, migration, and invasion were effectively neutralized by GADD45A knockdown (Fig. 5C–5E, p < 0.05). Similarly, miR-376c overexpression also markedly attenuated DLX6-AS1-induced inhibition on trophoblast proliferation, migration and invasion (Fig. 5C–5E, p < 0.05). Collectively, DLX6-AS1 may contribute to PE by impairing the proliferative, migratory, and invasive abilities of trophoblasts via the miR-376c/GADD45A axis.
Discussion Numerous lncRNAs exhibit abnormal expression in human placentas in pregnancies complicated by PE, and play functional roles in the pathogenesis of PE[1, 4, 7]. For example, lncRNA RPAIN is upregulated in PE placentas and inhibits trophoblast proliferation and invasion[8]. However, lncRNA MALAT1 is downregulated in PE placentas, and overexpression of MALAT1 can increase the migration rate and invasiveness of trophoblasts[6]. In this study,
we show that lncRNA DLX6-AS1 was upregulated in placentas from patients with PE and contributes to PE by regulating the miR-376c/GADD45A axis. LncRNA DLX6-AS1, which is located on chr7: 96955141-97014065, is upregulated in several tumor tissues, including renal cell carcinoma, hepatocellular carcinoma, and lung adenocarcinoma[13-15]. In the present study, the expression of DLX6-AS1 was markedly increased in the placentas of 24 patients with PE, compared with those pregnant women who underwent caesarean section because of abnormal fetal position with delivery at < 37 weeks gestation. This result is consistent with a previous study on expression profiles of lncRNAs in PE and normal placentas[7], suggesting that upregulation of DLX6-AS1 in the placenta may be implicated in the pathogenesis of PE.Trophoblast proliferation and invasion are essential in normal pregnancies and regulated by various factors[16, 17]. Reduced trophoblast proliferation, migration, and invasion are critical events that contribute to PE[11]. LncRNA DLX6-AS1 is reported to be a carcinogenic factor that promotes the proliferation, migration, and invasion of tumor cells[13, 14]. Our data revealed that overexpression of DLX6-AS1 also dramatically decreased the proliferation, migration, and invasion abilities of trophoblasts. These findings indicate that upregulated DLX6-AS1 might contribute to insufficient or incomplete trophoblastic invasion, thereby leading to placental defects and subsequent preeclampsia. Evidence suggests that many lncRNAs act as endogenous microRNA (miRNA) sponges by binding to miRNAs and causing malfunction through various pathological and physiological processes[18]. For example, lncRNA DLX6-AS1 inhibits the proliferation, migration, and invasion of hepatocellular carcinoma cells by modulating the miR-203a/MMP-2 pathway[14]. Similarly, lncRNA DLX6-AS1 promotes renal cell carcinoma by regulating the miR-26a/PTEN axis[13]. In this study, we found that miR-376c has complementary binding sites with
DLX6-AS1. MiR-376c also reportedly regulates many biological processes, including cell proliferation, invasion and migration[19-21]. The expression of miR-376c is downregulated in both placental and plasma samples collected from preeclamptic patients[11]. Consistent with this research, our data revealed that miR-376c was markedly downregulated and negatively correlated with DLX6-AS1 expression in placentas from patients with PE. Moreover, overexpression of DLX6-AS1 markedly decreased miR-376c expression in HTR-8/SVneo cells, suggesting that DLX6-AS1 may work as an endogenous sponge RNA to interact with miR-376c in human trophoblasts. GADD45A, a stress sensor in PE[22], is the predicted target of miR-376c in trophoblasts. Our data also revealed that overexpression of DLX6-AS1 increased the expression of GADD45A in HTR-8/SVneo cells. Migration and invasion abilities are much higher in GADD45A-deficient cells[23], and overexpression of GADD45A can inhibit migration and invasion of human trophoblasts [12]. Intriguingly, our data showed that overexpression of GADD45A effectively reduced the miR-376c-induced increase in proliferation, migration and invasion of HTR-8/SVneo cells. As the first well-defined p53 downstream gene, GADD45A can be induced by numerous stresses and growth arrest signals[23], such as hypoxia and oxidative stress, which are implicated in preeclamptic pathology[22]. Moreover, GADD45A expression is increased in the placentas of patients with PE and in hypoxia/reoxygenation-treated trophoblasts [12, 24]. Because miR-376c is downregulated by DLX6-AS1 and GADD45A is downregulated by miR-376c in human trophoblasts, we concluded that upregulation of DLX6-AS1 in placentas of patients with PE might result in decreased expression of miR-376c and increased expression of GADD45A. As
expected, overexpression of DLX6-AS1 in HTR-8/SVneo cells resulted in an increase in both mRNA and protein expression of GADD45A. Moreover, DLX6-AS1-induced inhibition of trophoblast proliferation, migration, and invasion was effectively neutralized by GADD45A knockdown and miR-376c overexpression. These findings suggest that DLX6-AS1 may contribute to PE by impairing the proliferative, migratory, and invasive abilities of trophoblasts via the miR-376c/GADD45A axis. In conclusion, our study demonstrated that lncRNA DLX6-AS1 was significantly upregulated in human placentas in pregnancies complicated by PE. Upregulation of DLX6-AS1 dramatically reduced the proliferation, migration and invasion of trophoblasts by regulating the miR-376c/GADD45A axis. These findings suggest that the DLX6-AS1/miR-376c/GADD45A pathway may be involved in the pathogenesis of PE and thus may be a diagnostic and therapeutic target for PE.
Conflict of Interests The authors declare that they have no competing interests.
Acknowledgments This study was funded by Scientific Research Project of Shenzhen Health and Family Planning System，Guangdong Province, China (No. 201606027).
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Tab.1. Clinical characteristics of preeclampsia and normal pregnant women enrolled in this study. control
Preeclampsia
(n=18)
(n=24)
Maternal age (years)
27.78±1.80
28.29±1.83
0.37
Gestational age (weeks)
35.32±1.14
34.62±1.95
0.18
Systolic blood pressure (mmHg)
108.16±7.16
163.08±9.34
< 0.01*
Diastolic blood pressure (mmHg)
73.22±5.75
106.04±7.08
< 0.01*
Proteinuria (g/24h)
0.02±0.03
3.71±1.38
< 0.01*
Parameters
P value
Values represent as mean±SD. *p < 0.05, significant difference between preeclampsia and control patients.
Fig. 1. lncRNA DLX6-AS1 expression is increased in placentas from patients with preeclampsia. The relative expression of lncRNA DLX6-AS1 in placental tissues from control pregnant women (n=18) and from women with preeclampsia (n=24) was assessed by qRT-PCR and normalized to GAPDH. Values represent as mean ± SD. *p < 0.05 versus the control group. Fig. 2. Effects of DLX6-AS1 on proliferation, migration, and invasion of trophoblasts. JEG3 and HTR-8/SVneo cells were transfected with pcDNA3.1-DLX6-AS1 plasmid or an empty vector. (A) DLX6-AS1 expression was measured by real-time PCR 48 hours after transfection. (B) Cell viability was measured by CCK-8 assay 24 h after transfection. (C) A transwell migration assay was performed to assess the migration ability of JEG3 and HTR-8/SVneo cells. (D) Invasion ability of JEG3 and HTR-8/SVneo cells was measured by Matrigel-based transwell invasion assay. Values represent as mean ± SD. *p < 0.05 versus the control group. Fig. 3. DLX6-AS1 interacts with miR-376c and regulates its expression. (A) Predicted binding sites between DLX6-AS1 and miR-376c. (B) The relative luciferase activities in HEK293T cells 24 h after co-transfection with pGL3-DLX6-AS1-wt or pGL3-DLX6-AS1-mut and miR-376c mimic or miR-NC mimic. (C) The bound levels of DLX6-AS1 were analyzed by RT-PCR after performing a biotin-based pulldown assay. D) The relative expression of miR-376c in placental tissues from patients with preeclampsia or control pregnant woman was assessed by qRT-PCR and normalized to GAPDH. (E) The correlation between DLX6-AS1 and miR-376c level in placental tissues from patients with preeclampsia. (F) The relative expression of miR-376c in HTR-8/SVneo cells 24 h after transfection with pcDNA3.1-DLX6-AS1 plasmid or an empty vector. Values represent as mean ± SD. *p < 0.05 versus the miR-NC, normal, NC-bio, or control group.
Fig. 4. MiR-376c targets GADD45A and promotes proliferation, migration, and invasion of trophoblasts. (A) The putative miR-376c binding sites in the 3' untranslated region of GADD45A. (B) The relative luciferase activities in HEK293T cells 24 h after co-transfection with pGL3-GADD45A-wt or pGL3-GADD45A-mut and miR-376c mimic or miR-NC mimic. (C) The mRNA expression of GADD45A in HTR-8/SVneo cells transfected with miR-376c mimic or miR-NC mimic was detected by qRT-PCR. (D) The protein expression of GADD45A in HTR-8/SVneo cells transfected with miR-376c mimic or NC-mimic was detected by western blot. (E) Cell viability was measured by CCK-8 assay 24 h after transfection. (F) The migration ability of HTR-8/SVneo cells was assayed by transwell migration assay. (G) The invasion ability of HTR-8/SVneo cells was measured by Matrigel-based transwell invasion assay . Values represent as mean ± SD. *p < 0.05 versus the control group. #p < 0.05 versus the miR-376c group. Fig. 5. DLX6-AS1 modulates trophoblast proliferation, migration and invasion via the miR-376c/GADD45A axis. (A) The mRNA expression of GADD45A in HTR-8/SVneo cells transfected with pcDNA3.1-DLX6-AS1 plasmid or an empty vector was detected by qRT-PCR. (B) The protein expression of GADD45A in HTR-8/SVneo cells was detected by western blot. (C) Cell viability was measured by CCK-8 assay. (D) The migration ability of HTR-8/SVneo cells was assayed by transwell migration assay. (E) The invasion ability of HTR-8/SVneo cells was measured by Matrigel-based transwell invasion assay. Values represent as mean ± SD. *p < 0.05 versus the control group. #p < 0.05 versus the DLX6-AS1 group. Highlights
lncRNA DLX6-AS1 is upregulated in placentas from women with preeclampsia DLX6-AS1 inhibits trophoblastic migration and invasion DLX6-AS1 affects trophoblastic migration and invasion via miR-376c/GADD45A pathway
PII: DOI: Reference:
S0014-4827(18)30579-2 https://doi.org/10.1016/j.yexcr.2018.07.039 YEXCR11143
To appear in: Experimental Cell Research Received date: 14 March 2018 Revised date: 23 July 2018 Accepted date: 24 July 2018 Cite this article as: Yan Tan, Di Xiao, Yanbin Xu and Chenhong Wang, Long non-coding RNA DLX6-AS1 is upregulated in preeclampsia and modulates migration and invasion of trophoblasts through the miR-376c/GADD45A axis, Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2018.07.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Long non-coding RNA DLX6-AS1 is upregulated in preeclampsia and modulates migration and invasion of trophoblasts through the miR-376c/GADD45A axis Yan Tan1, Di Xiao2, Yanbin Xu1, Chenhong Wang1,3* 1
Department of Gynecology and Obstetrics, Affiliated Shenzhen Maternity & Child Healthcare
Hospital, Southern Medical University (Shenzhen Maternity & Child Healthcare Hospital), Shenzhen 518028, Guangdong, China 2
Department of Gynecology and Obstetrics, The Third Affiliated Hospital, Southern Medical
University, Guangzhou 510150, Guangdong, China 3
Department of Gynecology and Obstetrics, Shenzhen Hospital, Southern Medical University,
Shenzhen 518000, Guangdong, China *Correspondence to: Chenhong Wang, Present address: Department of Gynecology and Obstetrics, Shenzhen Hospital of Southern Medical University, No.1333, Xinhu Road, Baoan district, Shenzhen 518000, Guangdong Province, China. Tel./ Fax: +86-0755-23360520. [email protected] Abstract Long non-coding RNA (lncRNA) are key regulatory molecules that are implicated in diverse biological processes and human diseases, including preeclampsia. However, their expression and functions in the progression of preeclampsia remains largely unclear. In this study, lncRNA DLX6-AS1 was confirmed to be significantly upregulated in the placentas of patients with preeclampsia, compared with normal controls. Overexpression of DLX6-AS1 dramatically decreased proliferation, migration and invasion in trophoblast JEG3 and HTR-8/SVneo cells. Luciferase activity and RNA pull-down assays showed that DLX6-AS1 interacted with miR-376c, and that overexpression of DLX6-AS1 significantly reduced expression of miR-376c
in HTR-8/SVneo cells. Also, miR-376c targeted and downregulated GADD45A in HTR-8/SVneo cells. Overexpression of GADD45A effectively attenuated a miR-376c-induced increase in the proliferation, migration and invasion of HTR-8/SVneo cells. Further research showed that DLX6-AS1-induced inhibition of trophoblast proliferation, migration and invasion was effectively neutralized by GADD45A knockdown and miR-376c overexpression. Taken together, these findings suggest that DLX6-AS1 may contribute to preeclampsia by impairing proliferative, migratory and invasive abilities of trophoblasts via the miR-376c/GADD45A axis. Keywords Preeclampsia, DLX6-AS1, trophoblast, GADD45A, miR-376c
Introduction Preeclampsia (PE), a pregnancy-specific syndrome, remains a leading cause of maternal and neonatal morbidity and mortality[1]. This multi-systemic disorder clinically manifests as new-onset hypertension (≥ 140/90 mmHg) and overt proteinuria (≥ 300 mg/24 h) after 20 weeks of gestation[2]. Trophoblastic dysfunction, such as inadequate migration and invasion, are well-known to be correlated with PE[2, 3]. However, the molecular mechanism implicated in the pathogenesis of PE and the regulation of trophoblast behavior are still largely unknown. Long non-coding RNAs (lncRNAs) are a class of RNA transcripts longer than 200 nucleotides in length which play multiple and crucial roles in many fundamental biochemical and disease processes[4, 5]. LncRNAs help regulate gene expression through chromosome dosage compensation, genomic imprinting, and transcriptional and epigenetic regulation[1, 5, 6]. Numerous lncRNAs are differentially expressed in the placentas from healthy subjects and in patients with PE, suggesting that aberrant expression of lncRNA may contribute to PE[4, 7].
Some lncRNAs, such as MALAT-1, RPAIN and Uc.187 has been proved to be involved in regulating trophoblast behavior[6, 8, 9]. For instance, high expression of lncRNA SPRY4-IT1 in placentas from women with PE correlates with poor prognosis and contributes to PE by impairing trophoblast migration, invasion, and epithelial-mesenchymal transition[3, 10]. Therefore, increased understanding of the lncRNAs aberrantly expressed in PE patients are beneficial for understanding the pathogenesis and progression of PE, thereby proposing more therapeutic interventions. In the present study, lncRNA DLX6-AS1 was found to be upregulated in placentas from patients with PE, compared to those from women with normal pregnancies. Moreover, overexpression of DLX6-AS1 markedly repressed proliferation, migration and invasion of trophoblasts. Further investigation suggested that the inhibition effect of DLX6-AS1 on trophoblasts was related to the miR-376c/GADD45A axis. These findings indicate that DLX6-AS1 contributes to PE by regulating trophoblastic proliferation, migration and invasion via the miR-376c/GADD45A axis.
Materials and Methods Ethics statement and tissue samples Collection and experimentation of placental tissues in this study was approved by the Ethics Committee of the Affiliated Shenzhen Maternity & Child Healthcare Hospital of Southern Medical University (Guangdong, China). Subjects enrolled 24 patients with preeclampsia and 20 control patients who received a Caesarean section. Informed consent from all subjects was obtained before enrollment. Preeclampsia was diagnosed as systolic pressure ≥140 mm Hg and/or diastolic pressure ≥90 mm Hg, accompanied by proteinuria (urine protein ≥ 300 mg/24 h).
Control patients were those pregnant women who underwent caesarean section because of abnormal fetal position with delivery at < 37 weeks gestation. No subject was a smoker or had a history of any chronic diseases. Placental tissues were collected immediately after parturition and stored frozen in liquid nitrogen until use. Cell culture and transfection The human trophoblast cell lines JEG3 and HTR-8/SVneo were provided by American Type Culture Collection (Manassas, VA, USA) and cultivated in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and 100 U/mL of penicillin/streptomycin (Gibco) in a humidified atmosphere of 5% CO2 at 37 °C. Cells were plated in 24-well plates (2 × 104 cells/well), grown to 70% confluence, and then transfected with LipofectamineTM3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Overexpression
of
DLX6-AS1
was
achieved
using
pcDNA3.1-DLX6-AS1
plasmid
(GenePharma Co. Ltd., Shanghai, China) transfection, with an empty vector as a control. The pcDNA3.1-GADD45A and empty vectors, the miR-376c mimic and miR-NC mimic, and the GADD45A siRNA and scramble siRNA also were synthesized and provided by GenePharma. Cells were harvested 48 h after transfection for further studies. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted with Trizol reagent (Invitrogen) from placental tissue or trophoblasts, and cDNA was generated by using a Reverse Transcription Kit (Invitrogen) following the manufacturer’s instructions. RT-PCR was performed by using Syber Green PCR Mastermix (Applied Biosystems, Foster City, CA, USA). The specific primers were as follows: DLX6-AS1, forward 5′-CCA AAT GCT ACC ATC CAG CC-3′ and reverse 5′-TCT GGC TTC CCT TAA CCA AA-3′; GADD45A, forward 5′- GAG CAG AAG ACC GAA AGC GAC-3′ and
reverse 5′-GAA TGT GGA TTC GTC ACC AGC-3′; GAPDH, forward 5′-TTC GAC AGT CAG CCG CAT CTT-3′ and reverse 5′-CCC AAT ACG ACC AAA TCC GTT-3′; miR376c, forward 5′-GCG CAA CAT AGA GGA AA-3′ and reverse 5′-GGT GCA GGG TCC GAG GT-3′; U6, forward 5′-CTC GCT TCG GCA GCA CA-3′ and reverse 5′-AAC GCT TCA CGA ATT TGC GT-3′. Relative gene expression levels were calculated using the 2−ΔΔCt method and normalized to GAPDH or U6. Western blot Total proteins were extracted using a radioimmunoprecipitation assay buffer (Sigma, St. Louis, MO, USA), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% fat-free milk, membranes were probed with specific primary antibodies against GADD45A and GAPDH (Abcam, Cambridge, UK) overnight at 4 °C. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Abcam) for 1 h at room temperature. Finally, the protein bands were visualized by using an enhanced chemiluminescence detection system (Thermo Fisher, San Jose, CA, USA) and quantified by Image J software. The relative protein expression was normalized to endogenous GAPDH. Cell viability assay Cell viability was assessed by using a Cell Counting kit-8 (CCK-8) assay (Sigma). Transfected cells were seeded into 96-well plates (3 × 103 cells/well) and incubated for 24 h. Then, 10 μL of CCK-8 solution was added into each well and incubated for another 2 h. The optical density of each well was then measured at a wavelength of 450 nm using a microplate reader.
Transwell migration and Matrigel invasion assays Cell invasion and migration were evaluated by using a 24-well transwell insert system (8 μm pore filters; Costar, Cambridge, MA, USA). Then, 1 × 105 cells were resuspended in 200 μL of serum-free medium and seeded into the upper transwell chambers pre-coated with (for invasion) or without (for migration) Matrigel. In the lower chamber, 800 μL of medium containing 10% fetal bovine serum was added to induce chemotaxis. After 24 h of incubation at 37 °C, chambers were disassembled, and the invaded and migratory cells were stained with 2% crystal violet and counted under the microscope in five random visual fields. Target prediction and dual-luciferase reporter assay Bioinformatics analysis was used to predict the potential targets of lncRNA DLX6-AS1and miR-376c, including DIANA-LncBase v2, microRNA.org, and miRDB. For the luciferase reporter assay, wild-type or mutant DLX6-AS1 and GADD45A fragments with or without the miR-376c binding element were respectively cloned into the luciferase reporter vector pGL3 (Promega, Madison, WI, USA). HEK293T cells were grown in 12-well plates to 70% confluency and then co-transfected with either pGL3-DLX6T-AS1-wt or pGL3-DLX6T-AS1-mut (pGL3-GADD45A-wt or pGL3-GADD45A-mut) and the miR-376c mimic or NC-mimic. Luciferase activity was determined 24 h after transfection by using the Dual-Luciferase Reporter Assay System (Promega). RNA pull-down assay Biotinylated wild-type miR-376c (miR-376c-Bio), biotinylated mutant miR-376c (miR-376c-MutBio), or biotinylated NC (NC-Bio) was transfected into the HTR-8/SVneo cells. After 48 h of transfection, the cells were lysed and incubated with M-280 streptavidin magnetic beads (Invitrogen). The bound RNAs were then purified with TRIzol reagent (Invitrogen) and
analyzed by RT–PCR. Statistical analysis Each experiment was performed in triplicate and repeated three times. All data for this study are expressed as the mean ± standard deviation (SD). One-way analysis of variance and Student's t tests were performed to evaluate statistical differences within or between the groups by using SPSS 22.0 software. A value of p < 0.05 was considered statistically significant.
Results lncRNA DLX6-AS1 was upregulated in placentas from women with PE To investigate the potential role of DLX6-AS1 in the progression of PE, we first measured DLX6-AS1 expression in placentas from patients with PE (n = 24) and from control pregnancies (n = 18). Table 1 presents the clinicopathologic data. Resultly, DLX6-AS1 levels were 2.16-fold higher in PE placentas, compared to normal ones (Fig. 1, p < 0.05), suggesting that upregulation of DLX6-AS1 in the placenta may be implicated in the pathogenesis of PE. DLX6-AS1 overexpression inhibited migration and invasion of trophoblastic cells To explore the biological functions of DLX6-AS1 in PE, the pcDNA3.1-DLX6-AS1 plasmid or empty vector was transfected into JEG3 and HTR-8/SVneo cells. Compared to the negative control, DLX6-AS1 was upregulated nearly 9-fold in both JEG3 and HTR-8/SVneo cells that were transfected with the pcDNA3.1-DLX6-AS1 vector (Fig. 2A, p < 0.05). Decreased cell viability was observed in both JEG3 and HTR-8/SVneo cells transfected with the pcDNA3.1-DLX6-AS1 vector (Fig. 2B, p < 0.05). Overexpression of DLX6-AS1 also significantly reduced migratory and invasive abilities of JEG3 and HTR-8/SVneo cells (Fig. 2C– 2D, p < 0.05). Taken together, upregulation of DLX6-AS1 impaired the proliferative, migratory,
and invasive capacities of trophoblastic cells. DLX6-AS1 decreased expression of miR-376c in trophoblastic cells Bioinformatics analyses were performed to investigate the molecular mechanism by which DLX6-AS1 regulates trophoblast behavior. MiR-376c, a microRNA associated with PE[11], was found to have complementary binding sites with DLX6-AS1 (Fig. 3A). A luciferase reporter assay showed that miR-376c significantly suppressed luciferase activity fused to the wild-type DLX6-AS1 fragments, but it failed to repress luciferase activity fused to the mutated one (Fig. 3B, p < 0.05). A biotin-based pull-down assay showed that DLX6-AS1, but not the deletion mutants, was efficiently pulled down by miR-376c (Fig. 3C, p < 0.05). The expression of miR-376c in placentas from patients with PE was significantly downregulated (Fig. 3D, p < 0.05) and negatively correlated with DLX6-AS1 expression (Fig. 3E, r = – 0.65). Moreover, a marked decrease in expression of miR-376c was noted in HTR-8/SVneo cells transfected with pcDNA3.1-DLX6-AS1 (Fig. 3F, p < 0.05). These results suggest that DLX6-AS1 may interact with miR-376c and decreased its expression in human trophoblasts. MiR-376c targeted GADD45A to modulate proliferation, migration and invasion of trophoblastic cells Bioinformatic analysis indicated that GADD45A, a molecule linked with PE progression[12], contains a miR-376c binding site in its 3′ untranslated region (UTR), as shown in Fig. 4A. MiR-376c significantly reduced the luciferase activity of HEK293T cells transfected with the wild-type GADD45A plasmid, but it failed to suppress luciferase activity in cells transfected with the mutant plasmid (Fig. 4B, p < 0.05). Both mRNA and protein expression of GADD45A was markedly reduced in HTR-8/SVneo cells transfected with the miR-376c mimic (Fig. 4C–4D, p < 0.05). Overexpression of miR-376c also dramatically enhanced proliferation,
migration, and invasion capacities of HTR-8/SVneo cells (Fig. 4E–4G, p < 0.05). However, overexpression of GADD45A significantly inhibited the miR-376c-induced increase in proliferation, migration, and invasion of HTR-8/SVneo cells (Fig. 4E–4G, p < 0.05). Collectively, miR-376c promoted cell proliferation, migration, and invasion by downregulating GADD45A in human trophoblasts. DLX6-AS1
inhibited
trophoblastic
migration
and
invasion
through
the
miR-376c/GADD45A axis Because GADD45A is a target of miR-376c and because DLX6-AS1 can interact with miR-376c in human trophoblasts, we speculated that DLX6-AS1 may affect PE progression by regulating the miR-376c/GADD45A axis. Overexpression of DLX6-AS1 in HTR-8/SVneo cells resulted in an obvious increase in both mRNA and protein expression of GADD45A (Fig. 5A–5B, p < 0.05). The inhibition effects of DLX6-AS1 on trophoblast proliferation, migration, and invasion were effectively neutralized by GADD45A knockdown (Fig. 5C–5E, p < 0.05). Similarly, miR-376c overexpression also markedly attenuated DLX6-AS1-induced inhibition on trophoblast proliferation, migration and invasion (Fig. 5C–5E, p < 0.05). Collectively, DLX6-AS1 may contribute to PE by impairing the proliferative, migratory, and invasive abilities of trophoblasts via the miR-376c/GADD45A axis.
Discussion Numerous lncRNAs exhibit abnormal expression in human placentas in pregnancies complicated by PE, and play functional roles in the pathogenesis of PE[1, 4, 7]. For example, lncRNA RPAIN is upregulated in PE placentas and inhibits trophoblast proliferation and invasion[8]. However, lncRNA MALAT1 is downregulated in PE placentas, and overexpression of MALAT1 can increase the migration rate and invasiveness of trophoblasts[6]. In this study,
we show that lncRNA DLX6-AS1 was upregulated in placentas from patients with PE and contributes to PE by regulating the miR-376c/GADD45A axis. LncRNA DLX6-AS1, which is located on chr7: 96955141-97014065, is upregulated in several tumor tissues, including renal cell carcinoma, hepatocellular carcinoma, and lung adenocarcinoma[13-15]. In the present study, the expression of DLX6-AS1 was markedly increased in the placentas of 24 patients with PE, compared with those pregnant women who underwent caesarean section because of abnormal fetal position with delivery at < 37 weeks gestation. This result is consistent with a previous study on expression profiles of lncRNAs in PE and normal placentas[7], suggesting that upregulation of DLX6-AS1 in the placenta may be implicated in the pathogenesis of PE.Trophoblast proliferation and invasion are essential in normal pregnancies and regulated by various factors[16, 17]. Reduced trophoblast proliferation, migration, and invasion are critical events that contribute to PE[11]. LncRNA DLX6-AS1 is reported to be a carcinogenic factor that promotes the proliferation, migration, and invasion of tumor cells[13, 14]. Our data revealed that overexpression of DLX6-AS1 also dramatically decreased the proliferation, migration, and invasion abilities of trophoblasts. These findings indicate that upregulated DLX6-AS1 might contribute to insufficient or incomplete trophoblastic invasion, thereby leading to placental defects and subsequent preeclampsia. Evidence suggests that many lncRNAs act as endogenous microRNA (miRNA) sponges by binding to miRNAs and causing malfunction through various pathological and physiological processes[18]. For example, lncRNA DLX6-AS1 inhibits the proliferation, migration, and invasion of hepatocellular carcinoma cells by modulating the miR-203a/MMP-2 pathway[14]. Similarly, lncRNA DLX6-AS1 promotes renal cell carcinoma by regulating the miR-26a/PTEN axis[13]. In this study, we found that miR-376c has complementary binding sites with
DLX6-AS1. MiR-376c also reportedly regulates many biological processes, including cell proliferation, invasion and migration[19-21]. The expression of miR-376c is downregulated in both placental and plasma samples collected from preeclamptic patients[11]. Consistent with this research, our data revealed that miR-376c was markedly downregulated and negatively correlated with DLX6-AS1 expression in placentas from patients with PE. Moreover, overexpression of DLX6-AS1 markedly decreased miR-376c expression in HTR-8/SVneo cells, suggesting that DLX6-AS1 may work as an endogenous sponge RNA to interact with miR-376c in human trophoblasts. GADD45A, a stress sensor in PE[22], is the predicted target of miR-376c in trophoblasts. Our data also revealed that overexpression of DLX6-AS1 increased the expression of GADD45A in HTR-8/SVneo cells. Migration and invasion abilities are much higher in GADD45A-deficient cells[23], and overexpression of GADD45A can inhibit migration and invasion of human trophoblasts [12]. Intriguingly, our data showed that overexpression of GADD45A effectively reduced the miR-376c-induced increase in proliferation, migration and invasion of HTR-8/SVneo cells. As the first well-defined p53 downstream gene, GADD45A can be induced by numerous stresses and growth arrest signals[23], such as hypoxia and oxidative stress, which are implicated in preeclamptic pathology[22]. Moreover, GADD45A expression is increased in the placentas of patients with PE and in hypoxia/reoxygenation-treated trophoblasts [12, 24]. Because miR-376c is downregulated by DLX6-AS1 and GADD45A is downregulated by miR-376c in human trophoblasts, we concluded that upregulation of DLX6-AS1 in placentas of patients with PE might result in decreased expression of miR-376c and increased expression of GADD45A. As
expected, overexpression of DLX6-AS1 in HTR-8/SVneo cells resulted in an increase in both mRNA and protein expression of GADD45A. Moreover, DLX6-AS1-induced inhibition of trophoblast proliferation, migration, and invasion was effectively neutralized by GADD45A knockdown and miR-376c overexpression. These findings suggest that DLX6-AS1 may contribute to PE by impairing the proliferative, migratory, and invasive abilities of trophoblasts via the miR-376c/GADD45A axis. In conclusion, our study demonstrated that lncRNA DLX6-AS1 was significantly upregulated in human placentas in pregnancies complicated by PE. Upregulation of DLX6-AS1 dramatically reduced the proliferation, migration and invasion of trophoblasts by regulating the miR-376c/GADD45A axis. These findings suggest that the DLX6-AS1/miR-376c/GADD45A pathway may be involved in the pathogenesis of PE and thus may be a diagnostic and therapeutic target for PE.
Conflict of Interests The authors declare that they have no competing interests.
Acknowledgments This study was funded by Scientific Research Project of Shenzhen Health and Family Planning System，Guangdong Province, China (No. 201606027).
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Tab.1. Clinical characteristics of preeclampsia and normal pregnant women enrolled in this study. control
Preeclampsia
(n=18)
(n=24)
Maternal age (years)
27.78±1.80
28.29±1.83
0.37
Gestational age (weeks)
35.32±1.14
34.62±1.95
0.18
Systolic blood pressure (mmHg)
108.16±7.16
163.08±9.34
< 0.01*
Diastolic blood pressure (mmHg)
73.22±5.75
106.04±7.08
< 0.01*
Proteinuria (g/24h)
0.02±0.03
3.71±1.38
< 0.01*
Parameters
P value
Values represent as mean±SD. *p < 0.05, significant difference between preeclampsia and control patients.
Fig. 1. lncRNA DLX6-AS1 expression is increased in placentas from patients with preeclampsia. The relative expression of lncRNA DLX6-AS1 in placental tissues from control pregnant women (n=18) and from women with preeclampsia (n=24) was assessed by qRT-PCR and normalized to GAPDH. Values represent as mean ± SD. *p < 0.05 versus the control group. Fig. 2. Effects of DLX6-AS1 on proliferation, migration, and invasion of trophoblasts. JEG3 and HTR-8/SVneo cells were transfected with pcDNA3.1-DLX6-AS1 plasmid or an empty vector. (A) DLX6-AS1 expression was measured by real-time PCR 48 hours after transfection. (B) Cell viability was measured by CCK-8 assay 24 h after transfection. (C) A transwell migration assay was performed to assess the migration ability of JEG3 and HTR-8/SVneo cells. (D) Invasion ability of JEG3 and HTR-8/SVneo cells was measured by Matrigel-based transwell invasion assay. Values represent as mean ± SD. *p < 0.05 versus the control group. Fig. 3. DLX6-AS1 interacts with miR-376c and regulates its expression. (A) Predicted binding sites between DLX6-AS1 and miR-376c. (B) The relative luciferase activities in HEK293T cells 24 h after co-transfection with pGL3-DLX6-AS1-wt or pGL3-DLX6-AS1-mut and miR-376c mimic or miR-NC mimic. (C) The bound levels of DLX6-AS1 were analyzed by RT-PCR after performing a biotin-based pulldown assay. D) The relative expression of miR-376c in placental tissues from patients with preeclampsia or control pregnant woman was assessed by qRT-PCR and normalized to GAPDH. (E) The correlation between DLX6-AS1 and miR-376c level in placental tissues from patients with preeclampsia. (F) The relative expression of miR-376c in HTR-8/SVneo cells 24 h after transfection with pcDNA3.1-DLX6-AS1 plasmid or an empty vector. Values represent as mean ± SD. *p < 0.05 versus the miR-NC, normal, NC-bio, or control group.
Fig. 4. MiR-376c targets GADD45A and promotes proliferation, migration, and invasion of trophoblasts. (A) The putative miR-376c binding sites in the 3' untranslated region of GADD45A. (B) The relative luciferase activities in HEK293T cells 24 h after co-transfection with pGL3-GADD45A-wt or pGL3-GADD45A-mut and miR-376c mimic or miR-NC mimic. (C) The mRNA expression of GADD45A in HTR-8/SVneo cells transfected with miR-376c mimic or miR-NC mimic was detected by qRT-PCR. (D) The protein expression of GADD45A in HTR-8/SVneo cells transfected with miR-376c mimic or NC-mimic was detected by western blot. (E) Cell viability was measured by CCK-8 assay 24 h after transfection. (F) The migration ability of HTR-8/SVneo cells was assayed by transwell migration assay. (G) The invasion ability of HTR-8/SVneo cells was measured by Matrigel-based transwell invasion assay . Values represent as mean ± SD. *p < 0.05 versus the control group. #p < 0.05 versus the miR-376c group. Fig. 5. DLX6-AS1 modulates trophoblast proliferation, migration and invasion via the miR-376c/GADD45A axis. (A) The mRNA expression of GADD45A in HTR-8/SVneo cells transfected with pcDNA3.1-DLX6-AS1 plasmid or an empty vector was detected by qRT-PCR. (B) The protein expression of GADD45A in HTR-8/SVneo cells was detected by western blot. (C) Cell viability was measured by CCK-8 assay. (D) The migration ability of HTR-8/SVneo cells was assayed by transwell migration assay. (E) The invasion ability of HTR-8/SVneo cells was measured by Matrigel-based transwell invasion assay. Values represent as mean ± SD. *p < 0.05 versus the control group. #p < 0.05 versus the DLX6-AS1 group. Highlights
lncRNA DLX6-AS1 is upregulated in placentas from women with preeclampsia DLX6-AS1 inhibits trophoblastic migration and invasion DLX6-AS1 affects trophoblastic migration and invasion via miR-376c/GADD45A pathway