CircHIPK3/miR-124 affects angiogenesis in early-onset preeclampsia via CPT1A-mediated fatty acid oxidation.

doi:10.21203/rs.3.rs-3332759/v1

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CircHIPK3/miR-124 affects angiogenesis in early-onset preeclampsia via CPT1A-mediated fatty acid oxidation.

https://doi.org/10.21203/rs.3.rs-3332759/v1

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published 21 Jun, 2024

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Multiple theories have been proposed to explain the pathogenesis of early-onset preeclampsia (EOPE), and angiogenic dysfunction is an important part of the pathogenesis. Carnitine palmitoyltransferase (CPT1A) is a key rate-limiting enzyme in the metabolic process of fatty acid oxidation (FAO). FAO regulates endothelial cell (EC) proliferation during vascular germination and is also essential for ab initio deoxyribonucleotide synthesis, but its role in EOPE needs to be further elucidated. In the present study, we investigated its functional role in EOPE by targeting the circHIPK3/miR-124-3p/CPT1A axis. In our study, reduced expression of circHIPK3 and CPT1A and increased expression of miR-124-3p in placental tissues from patients with EOPE were associated with EC dysfunction. Here, we confirmed that CPT1A regulates fatty acid oxidative activity, cell proliferation, and tube formation of ECs by regulating FAO. Functionally, knockdown of circHIPK3 suppressed EC angiogenesis by inhibiting CPT1A-mediated fatty acid oxidative activity, which was ameliorated by CPT1A overexpression. In addition, circHIPK3 regulates CPT1A expression by sponging miR-124-3p. Hence, circHIPK3 knockdown reduced the fatty acid oxidative process in ECs by sponging miR-124-3p in a CPT1A-dependent manner and inhibited EC proliferation and tube formation, which may have led to aberrant angiogenesis in EOPE. Thus, strategies targeting CPT1A-driven FAO may be a promising approach for the treatment of EOPE.

CPT1A

Endothelial cell༛Angiogenesis

Early-onset preeclampsia༛Fatty acid oxidation

Preeclampsia (PE) is a heterogeneous disease characterized by new-onset hypertension and proteinuria after 20 weeks of gestation. According to a recent Lancet report^[1], this disease affects 3%-5% of all pregnancies, and no treatments can significantly alters the progression of PE other than termination of pregnancy. Using the 34th week as a watershed, researchers have divided PE into early-onset PE (EOPE) and late-onset PE (LOPE). Numerous studies^[2] have confirmed that these two subtypes have different pathological mechanisms. Placental dysfunction and impaired endothelial cell (EC) angiogenesis play important roles in the pathogenesis of EOPE^[3], but the exact mechanism of their action remains to be elucidated^{[4, 5]}.

Until 2009, ECs were thought to be regulated only by provascular growth factor signaling. However, recent studies have shown that different metabolic pathways of ECs, such as glycolysis^[6], fatty acid oxidation (FAO), and glutamine/asparagine, control different functions of ECs and are involved in angiogenesis, and current studies have shown that EC metabolic pathways^{[7, 8]} are sufficient to alter vascular sprouting. In various diseases, targeting metabolism and metabolite delivery in abnormal ECs is a novel therapeutic approach to inhibit or stimulate vascular growth^[9]. FAO is a key bioenergy supply pathway for ECs, which provide a carbon source for ab initio nucleotide synthesis^[10]. Carnitine palmitoyltransferase 1A (CPT1A) belongs to the CPT1 family, which comprises key rate-limiting enzymes in the β-oxidation of fatty acids, catalyzing the conversion of long-chain fatty acids from lipid acyl CoA to acyl carnitine for translocation from the cytoplasm to the mitochondria. Among the three isoforms of CPT1, CPT1A has a higher affinity for carnitine and lower sensitivity to the endogenous CPT inhibitor malonyl coenzyme A, making it an effective FAO activator^[11]. CPT1A is widely expressed in human tissues and organs, such as the breast, ovary and liver, and plays a role in cell proliferation, apoptosis and tumor angiogenesis. Studies have shown that FAO metabolism is impaired in PE patients, but the exact mechanism is unknown^[12]. Therefore, further studies are needed to determine the mechanism by which CPT1A-mediated FAO exerts its pathological effects on PE.

Recently, many noncoding RNAs, including microRNAs (miRNAs), lncRNAs, and circRNAs, have attracted increasing attention due to their biological effects on the development of PE^{[13, 14]}. A previous study reported that some differentially expressed noncoding RNAs in the placenta affect placental growth and developmental processes and play an important role in PE^[15]. We used bioinformatics software to predict miRNAs that might bind to CPT1A. Among them, miR-124-3p was predicted to have binding sites through five software predictions (miRmap, microT, miRanda, PicTar, and TargetScan), indicating that it may directly bind to CPT1A to regulate its expression. Previous studies have shown that miR-124-3p plays a role in PE by inhibiting trophoblast migration and invasion^[16]. Other studies of miR-124-3p have also revealed its involvement in the regulation of cell proliferation, apoptosis, and angiogenesis^{[16, 17]}. We further predicted circRNAs that may bind to miR-124-3p using ENCORI and found that circHIPK3, which is associated with the occurrence and development of PE, has binding sites for miR-124-3p. CircHIPK3 is derived from exon 2 of the HIPK3 gene and is expressed in a variety of tissues, including lung, heart, and stomach, and is involved in the regulation of numerous pathophysiological processes, such as oxidative stress, the cell cycle, and tumor cell proliferation, metastasis, and invasion^[18]. Recent studies have shown that circHIPK3 also plays an important role in angiogenesis^[19]. CircHIPK3 has been reported to be involved in the development of PE by regulating trophoblast migration and invasion^[20]. However, no studies have confirmed whether circHIPK3 can influence EC function to play a role in the pathogenesis of EOPE. CircRNAs often function as competing endogenous RNAs (ceRNAs)^[21]. Therefore, we hypothesized that circHIPK3 may act as a molecular sponge for miR-124-3p and thus regulate the expression of downstream targets through the ceRNA mechanism. In the present study, we validated the important role of CPT1A-mediated FAO in EC angiogenesis in EOPE. Downregulation of circHIPK3 and CPT1A was found in EOPE patients, and we verified the interactions between circHIPK3 and miR-124-3p as well as miR-124-3p and CPT1A. The mechanism of the role of the circHIPK3/miR-124-3p/CPT1A axis in EOPE was elucidated, providing new insights for further study of the pathogenesis of EOPE as well as new ideas for the treatment of EOPE.

1. Differential expression of circHIPK3, miR-124-3p and CPT1A in the placenta of patients with EOPE

CircHIPK3 is the main circRNA isomer derived from exon 2 of the HIPK3 gene, as confirmed by Sanger sequencing (Fig. 1, A). Placental tissues were collected from pregnant women with EOPE and from healthy pregnant women who underwent cesarean section due to nonorganic diseases such as large fetus, small pelvis and scarred uterus. Real-time fluorescence quantitative PCR, immunoblotting (WB) and immunohistochemistry (IHC) were used to verify whether circHIPK3, CPT1A and miR-124-3p were differentially expressed in the EOPE and normal groups, and the results showed that the mRNA levels of circHIPK3 and CPT1A were significantly decreased in EOPE, while miR-124-3p was upregulated (Fig. 1, B-D). We examined CPT1A protein expression in these patients using WB and IHC analyses. CPT1A protein expression was decreased in the placental tissues from the patients with EOPE compared with the normal tissues (Fig. 1, E-F). The IHC results also showed that CPT1A was mainly localized in the cytoplasm (Fig. 1, F). In addition, we confirmed the subcellular localization of circHIPK3 and miR-124-3p. We isolated the nucleus and cytoplasm of ECs, and the results showed that circHIPK3 and miR-124-3p were mainly localized in the cytoplasm (Fig. 1, G).

2. CPT1A regulates fatty acid oxidative activity and EC functions

We used primary human umbilical vein ECs (HUVECs) to establish a model of vascular ECs, and the constructed CPT1A overexpression/knockdown plasmids were transfected into ECs for corresponding CPT1A functional studies. The efficiency of the CPT1A overexpression plasmid was first verified, as well as that of the CPT1A knockdown plasmid (Supplementary Fig. 1, A-C). Based on the results, sh-CPT1A-941 was selected as the plasmid for subsequent experiments (Supplementary Fig. 1, C). To verify the role of CPT1A as a key enzyme for FAO activity, we measured the relative fluorescence units during FAO in ECs (Fig. 2, A-B). The mitochondrial oxidative phosphorylation uncoupler carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 2.5 µM) was used to achieve maximum respiratory activity as a positive control, and cells treated with 40 µM etomoxir were used as a negative control. The overexpression group showed enhanced FAO of ECs, while the knockdown group showed the opposite effect. The proliferation, migration, and tube-forming abilities of ECs are crucial for angiogenesis. Therefore, CCK-8, Transwell, and tube formation assays were conducted to investigate the effect of CPT1A on EC function. The function of ECs was detected by CCK-8, Transwell, and tubule formation assays. The CCK-8 assay showed that overexpression of CPT1A increased cell proliferation, while CPT1A silencing produced the opposite effect (Fig. 2, C-D). Next, flow cytometry analysis was performed to examine whether CPT1A affects EC proliferation by altering cell cycle progression. Flow cytometry analysis showed that overexpression of CPT1A increased the proportion of cells in the S phase of the cycle, whereas cells in the knockdown group were more stagnant in the DNA replication phase (G0/G1) (Fig. 2, E-F), suggesting that CPT1A promotes EC proliferation by accelerating the cell cycle. We also performed Transwell assays on ECs with knockdown and overexpression of CPT1A and found that CPT1A did not affect the migration of ECs (Fig. 2, G-H), which is consistent with Schoors’s findings^[10]. Finally, we conducted tube formation assays and found that overexpression of CPT1A promoted the tube formation ability of ECs, which was strongly reduced after silencing CPT1A. In summary, overexpression of CPT1A increases FAO activity, proliferation, and tube formation of ECs. Overexpression of CPT1A promoted proliferation by accelerating the cell cycle, while silencing of CPT1A had the opposite effect (Fig. 2, I-J).

3. CircHIPK3 as a ceRNA for miR-124-3p regulates the expression of CPT1A in ECs

Our study (Fig. 1, A) showed that there was a difference in the expression of circHIPK3 and miR-124-3p in placental tissues of the EOPE patients. To verify the possible interactions between circHIPK3 and miR-124-3p, we first constructed a knockdown plasmid for circHIPK3 and selected sh-1 as the plasmid for subsequent studies based on the efficiency screening results shown (Supplementary Figure). First, the miR-124-3p levels was found to be elevated in ECs after knockdown of circHIPK3, while the expression of CPT1A was decreased, and the immunoblotting results also showed a decrease in its protein level (Fig. 3, A-B). Moreover, the miR-124-3p mimic reduced the CPT1A mRNA and protein levels (Fig. 3, C), consistent with the effect of sh- circHIPK3 on the expression of CPT1A. To further investigate whether circHIPK3 acts as a molecular sponge for miR-124-3p and thus regulates the expression of the downstream CPT1A protein, we used RNA hybridization prediction software and dual luciferase assays to predict the possible binding sites of miR-124-3p to circHIPK3 and to CPT1A. Transfection of miR-124-3p mimics decreased the luciferase intensity of wild type circHIPK3 as well as wild type CPT1A without affecting the mutant luciferase intensity of both molecules (Fig. 3, D-E). The results indicate that miR-124-3p has a direct binding site for both circHIPK3 and CPT1A. Further cotransfection of ECs transfected with circHIPK3 knockdown plasmid with miR-124-3p inhibitor revealed that the decreased expression of CPT1A due to decreased circHIPK3 expression rebounded (Fig. 3, F-G). Thus, circHIPK3 may regulate the expression of the downstream molecule CPT1A by binding miR-124-3p through its role as a molecular sponge.

4. CircHIPK3 regulates FAO activity and EC angiogenesis through CPT1A

To verify the effect of circHIPK3 on CPT1A and explore its effect on EC functions, we divided the ECs into four groups: sh-NC, sh-circHIPK3, sh-NC + oe-CPT1A, and sh-circHIPK3 + oe-CPT1A. The sh-circHIPK3 group showed decreased FAO activity and proliferation of ECs, a delayed cell cycle, and inhibited tube formation of ECs (Fig. 4, C-F). In contrast, CPT1A overexpression promoted FAO activity, EC proliferation, and tube formation (Fig. 4, C-F). However, in the cotransfected group, CPT1A overexpression was found to reverse the effects of circHIPK3 downregulation (Fig. 4, C-F). Thus, circHIPK3 knockdown reduces FAO activity and contributes to inhibiting the angiogenesis of ECs through the FAO rate-limiting enzyme CPT1A, whereas overexpression of CPT1A reverses these changes.

The pathogenesis of EOPE is thought to primarily involve placental defects^[22], where the diseased placenta gradually secretes high levels of antiangiogenic factors, leading to vascular inflammation, endothelial dysfunction^[23], and ultimately insufficient angiogenesis of the placenta. ECs play an important role in this mechanism, and normal EC function cannot be established without stable metabolism; glucose metabolism has been shown to play a key role in EOPE^[6], lipid metabolism plays a key role in ab initio nucleotide synthesis^[10], and the role of FAO pathways in the pathogenesis of EOPE has rarely been studied. Pregnant women with PE have impaired lipid metabolism, and CPT1A, a key rate-limiting enzyme in the fatty acid oxidative metabolic pathway, can be used as a metabolic target for antitumor therapy^[24]. This molecule has a key role in angiogenesis, and blocking CPT1A can be a new strategy to block angiogenesis^[10], but whether CPT1A is involved in the pathogenesis of EOPE has not been confirmed.

In this study, we confirmed that placental tissues from EOPE patients have differential expression of CPT1A and confirmed the key role of CPT1A in the oxidative metabolism of fatty acids. CPT1A overexpression promotes FAO activity in ECs, which promotes cell proliferation by facilitating the transition from G0/G1 to S phase, mainly because CPT1A increases the nucleoside content of metabolic intermediates^[25] and promotes tube formation. Here, we also found a decrease in circHIPK3 expression in EOPE. CircHIPK3 knockdown inhibited CPT1A expression and caused cell cycle blockade in the G0/G1 phase, thereby inhibiting cell proliferation and in turn playing a role in the impaired angiogenesis of EOPE, whereas CPT1A overexpression reversed the effects of circHIPK3 knockdown. The ceRNA hypothesis describes how lncRNAs act as molecular sponges for miRNAs to regulate mRNA expression levels and may have important functions in pathophysiological processes. Many studies have shown that ceRNA is an important mechanism for the regulatory function of circHIPK3^[26]. Although related miRNAs have been shown to regulate or be regulated by circHIPK3^[27], they are rarely reported in PE. In vitro experimental studies have shown that reduced expression of circHIPK3 downregulates CPT1A expression by acting as a ceRNA of miR-124-3p in HUVECs, thereby affecting FAO activity. The binding sites of circHIPK3 in miR-124-3p and miR-124-3p in CPT1A were confirmed by dual luciferase assays. Overexpression of CPT1A ameliorated the impaired angiogenesis caused by reduced circHIPK3, based on which the angiogenic regulatory role of the circHIPK3/miR-124-3p/CPT1A axis in EOPE was confirmed.

Gestation is a period of vigorous lipid metabolism. The human placenta uses fatty acids as an important metabolic fuel and obtains energy from FAO, which is an important metabolic energy source for the placenta during gestation. FAO affects a variety of physiological metabolic activities, and CPT1A has been shown to bind to and regulate succinylation of lactate dehydrogenase to promote cell proliferation in the presence of glutamine depletion^[28]. The role of FAO is increasingly being recognized in the metabolic aspects of disease, such as the "reprogramming" of the FAO pathway in cancer. Many types of cancer appear to rely on FAO as an important source of ATP for rapid growth^[29]. In breast cancer cells, downregulation of CPT1 regulates histone acetylation levels and induces histone deacetylase (HDAC) inhibition, promoting apoptosis, inhibiting angiogenesis, and downregulating genes associated with poor prognosis, invasion and metastasis^[30]. Impaired proliferation and migration of lymphatic ECs in the absence of CPT1A in vivo can lead to lymphoid defects^[31], demonstrating its epigenetic regulatory function. In mitochondrial function, CPT1A plays an important role by regulating energy metabolism and reactive oxygen species (ROS) levels^[32]. In CPT1A-inactivated mice, FAO-controlled CPT1A endothelial deficiency promoted EC dysfunction (leukocyte infiltration, barrier disruption) by increasing endothelial oxidative stress. Therefore, based on the important role of FAO in angiogenesis, many tumor therapy studies have blocked tumor angiogenesis by targeting inhibition of this process^[10], for example, using the CPT1A inhibitor etomoxir^[33], which activates the Ca2+/CamKII/AMPK pathway and has shown that CPT1A activity is required to maintain Ca2 + homeostasis^[34]. However, clinical trials of this treatment have shown hepatotoxicity, and high doses may lead to adverse side effects^[35]. In addition, some studies have shown that danchi pill (DQP) can upregulate CPT1A and CD36 expression in the infarct marginal zone of cardiac tissue and in ROS-stimulated ECs, suggesting that DQP can promote the development of FAO by activating key enzymes under ischemic conditions. Emerging evidence emphasizes the impact of genetic and metabolic reprogramming on placental function^[36], regulating angiogenesis. Pharmacological activation of CPT1A may provide an alternative option for therapeutic angiogenesis in ischemic diseases^[37].

Therefore, we suggest that the regulation of angiogenesis by targeting CPT1A-mediated FAO in ECs should be further investigated as a promising therapeutic strategy.

Tissue collection and primary cell extraction

Placental chorionic tissues were collected from patients with EOPE after cesarean section (16 cases) and from healthy pregnant women who underwent cesarean section due to nonorganic diseases such as large fetus, small pelvis and scarred uterus (16 cases), and the collection site was approximately 3 cm from the placental cord, part of which was fixed with paraformaldehyde for IHC. Inclusion criteria were defined according to the American College of Obstetricians and Gynecologists (ACOG): PE was defined as hypertension with proteinuria occurring after 20 weeks of gestation. EOPE was defined as PE that developed prior to 34 weeks of gestation. The age of these patients was 26–37 years. Patients with smoking or multiple pregnancies, fetal structural or genetic abnormalities, maternal infections and any other confounding pathology (diabetes mellitus, renal disease, chronic hypertension, hyperthyroidism and hypothyroidism) were excluded. The remaining samples were snap-frozen in liquid nitrogen using lyophilization tubes and stored in a liquid nitrogen tank for RNA and protein extraction. A 10-cm umbilical cord from the placental remnant of a healthy woman delivered by full-term cesarean section was selected for extraction of primary HUVECs for subsequent experiments using a previously reported method^[6]. Informed consent was obtained with approval from the Medical Ethics Committee of the Xiangya Hospital Central South University (Ethics number: 202307152). All patients signed an informed consent form before enrollment in the study.

Cell culture

This study used primary HUVECs, which were isolated from freshly obtained human umbilical cords of healthy mothers, as a model of vascular ECs in vitro. The umbilical cords were washed inside and outside with saline in a sterile station, and type I collagenase (0.1%; Solarbio, China) was digested at 37°C for 10 min before addition of a terminating digestion solution (ECM complete medium: 10% fetal bovine serum, 1% epidermal growth factor) and 5 ml of saline rinse. The rinse solution was then centrifuged at 1200 rpm for 5 min to separate the HUVECs from the collagenase, and the HUVECs were resuspended in ECM after measuring the concentration. Cells were seeded at the appropriate concentration in culture flasks and incubated at 37°C in a 5% CO₂ incubator. Primary ECs were cultured to the third generation (stable cell state) and then used for experiments.

Real-time quantitative PCR

Total RNA was extracted from cells and tissue using the Steadypure RNA Extraction Kit (Accurate Biotechnology, Hunan). The cDNA templates of miRNAs were synthesized with an Evo M-MLV RT Mix Kit (Accurate Biotechnology, Hunan), while miRNA was synthesized with TransScript miRNA First-Strand cDNA Synthesis SuperMix (+ Dye I/+Dye II). RT‒qPCR was performed using a SYBR Green Premix Pro Taq Hs qPCR Kit (Accurate Biotechnology, Hunan) for miRNA and PerfectStart Green qPCR SuperMix (TransGen Biotech, Beijing) for miRNA. Quantitative real-time PCR was performed using a QuantStudioTM5 system (Applied Biosystems, CA, USA) with a preset PCR program. The primer sequences purchased from Tsingke Biotechnology (Changsha, China) for real-time fluorescent quantitative PCR are shown in Table S1.

Protein extraction and immunoblotting

Protein extraction from tissues and cells was performed using RIPA lysis buffer (Bioss, China), and the concentration was determined using a BCA protein assay kit (Bioss, China). The protein samples were incubated for 5 min at 95°C; then, proteins (20 mg) were subjected to 8% SDS‒PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (0.45 mm pore size; Millipore, MA, USA). The primary antibodies used were rabbit anti-CPT1A (15184-1-AP) and rabbit anti-β-actin from Proteintech. All primary antibodies were used at a 1:1,000 dilution, with secondary HRP antibodies (Abiowell, China) used at 1:8000. Protein strips were developed using an ImageQuant 800 instrument and analyzed by ImageJ.

IHC

Placental tissues were embedded in paraffin. Sections were dewaxed in water, and antigen repair was performed in a microwave oven in a repair cassette filled with citrate antigen repair buffer (pH 6.0) in a microwave oven, washed and blocked for endogenous peroxidase. After serum blocking, a primary antibody against CPT1A (1:300, 15184-1-AP, Proteintech) was added and incubated overnight at 4°C. After incubation with secondary antibodies, the staining was repeated with DAB and hematoxylin.

Subcellular fractionation assay

RNA isolated from the cytoplasmic and nuclear extracts obtained from HUVECs by an Invitrogen PARIS nuclear and cytoplasmic extraction kit (AM1921, Invitrogen, Thermo Fisher Scientific) was subjected to RT‒qPCR analysis; see the previous section for specific steps. The levels of U6 (nuclear control), GAPDH (cytoplasmic control), circHIPK and miR-124-3p were determined.

Cell transfection

The pcDNA3.1+/BamHI EcoRI vector (GenePharma, Shanghai, China) was used for CPT1A overexpression, the empty plasmid vector was used as a control, the pGPU7/GFP/Neo vector (GenePharma, Shanghai, China) was used for CPT1A silencing, and sh-NC was used as the NC. GenePharma designed and synthesized four sh-RNAs targeting the CPT1A gene and two sh-RNAs targeting the circHIPK3 gene. sh-CPT1A (sh-941) and sh-circHIPK3 (sh-1), which were identified as the most effective by real-time fluorescence quantitative PCR, were used for further experiments. MiR-124-3p overexpression and repression were achieved using miR-124-3p mimics and miR-124-3p inhibitors, respectively, with NC mimics and NC inhibitors serving as controls (GenePharma). These plasmids were transfected into cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) as needed. Cells were transfected for 48 h and then collected for subsequent analysis.

FAO assay

The FAO assay followed the manufacturer's instructions for the Fatty Acid Oxidation Assay Kit (ab217602, Abcam). A total of 3×10⁴ cells per well were seeded in a 96-well plate and cultured overnight. Then, the cells were rinsed twice with 100 µl of prewarmed FA-free medium and mixed with 90 µl of prewarmed FA measurement medium. The cell-free wells were used as signal controls. Wells were supplemented with 85 µl of FA-free measurement medium and 5 µl of BSA control as the FA-free control. Each well was spiked with 10 µl of extracellular O₂ consumption reagent from the Extracellular Oxygen Consumption Assay kit (ab197243, Abcam) except for the blank control. The FAO activator FCCP (0.5 µM) and the inhibitor etomoxir (40 µM) were used as positive and negative controls, respectively, and the concentrations were used as recommended in the instructions and according to a previously described protocol^[38]. The wells were sealed with two drops of prewarmed mineral oil, and the fluorescence signal was immediately detected using an instrument (Biotek, Cytation5).

Cell proliferation assay

Cell proliferation assays were performed using CCK-8 assays (K101, APE). Transfected cells were seeded in 96-well plates at a density of 3\(\times\)10³ cells/100 ml per well. Proliferation levels were measured at 0, 24, 48 and 72 h post-transfection. CCK-8 solution (10 ml) was added to each well and then incubated for 1 h at 37°C in the dark. The absorbance at 450 nm was measured using a multimode reader (Infinite M200 Pro; Tecan).

Flow cytometry analysis of the cell cycle

The transfected cells were collected by digestion, fixed and stained according to the protocol of the cell cycle assay kit (E-CK-A351, Elabscience) and then detected by flow cytometry (DxpAthena Cytek, USA). The percentages of cells in the G0/G1, S, and G2/M phases were calculated using ModFit software.

Cell migration assay

A Transwell 24-well filter insert (Corning, 8.0 µm pore size) was used to investigate cell migration. In each well, transfected HUVECs (5x104 cells/well) were resuspended in 200 ml of serum-free ECM and placed in the upper chamber, and 500 ml of complete medium was added to the lower chamber. Transwell units were incubated for 24 h at 37°C; the chambers were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 0.2% crystal violet for 20 min at 37°C. Five areas were randomly selected for counting under an inverted microscope (Leica, DM4B).

Tube-formation assay

The Matrigel gel was melted overnight at 4°C in advance, and the 96-well plates and tips were precooled 2 h in advance. Sixty microliters of Matrigel was added to each well of 96-well plates and incubated in a 37°C incubator for 0.5 h. HUVECs (1.5 X 10⁴ cells/well) were resuspended in 100 ml of conditioned medium and then seeded in wells. After incubation for 3–6 h at 37°C, tube-like structures formed and were imaged using a microscope (100x magnification; Olympus). Photomicrographs from each well were captured, and the total length of tubes was analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Luciferase reporter assay

CPT1A or circHIPK3 containing the miR-124-3p putative binding site was cloned into the pmirGlo vector (GenePharma). Cells were plated in 24-well plates at a density of 5*105 cells per well 1 day prior to transfection. Cells were cotransfected with WT or MUT luciferase vector and miR-124-3p or control using Lipofectamine 3000 reagent (Thermo Fisher Scientific). After 48 h of incubation, a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and Multifunctional Enzyme Labeler (Synergy HTX, USA) were used.

Statistics

All data are presented as the mean ± SD. Statistical analyses were performed using GraphPad Prism 9.4.1 software. Unpaired Student’s t test was used to determine the significance of differences between two groups, and one-way ANOVA was used for multiple groups. Statistical significance was defined as P < 0.05. All data were obtained from > 3 independent experiments.

Funding

This work was supported by the Postdoctoral Research Foundation of China (2022M723555), the Changsha Municipal Natural Science Foundation (kq2208375), and the Hunan Provincial Natural Science Foundation (2023JJ40958).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yanying Wu, Jingrui Huang, Lijuan Liu and Xiaowen Zhang. The first draft of the manuscript was written by Yanying Wu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during the current study are not publicly available but are available from the corresponding author on reasonable request.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the the Medical Ethics Committee of the Xiangya Hospital Central South University (Ethics number: 202307152).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

Our manuscript doesn’t contain any individual person’s data in any form.

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published 21 Jun, 2024

Read the published version in Journal of Molecular Medicine →

Editorial decision: Major Revisions Needed
21 Dec, 2023
Reviewers invited by journal
21 Sep, 2023
Editor assigned by journal
08 Sep, 2023
First submitted to journal
07 Sep, 2023

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CircHIPK3/miR-124 affects angiogenesis in early-onset preeclampsia via CPT1A-mediated fatty acid oxidation.

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Journal Publication

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Abstract

Figures

Introduction

Results

1. Differential expression of circHIPK3, miR-124-3p and CPT1A in the placenta of patients with EOPE

2. CPT1A regulates fatty acid oxidative activity and EC functions

3. CircHIPK3 as a ceRNA for miR-124-3p regulates the expression of CPT1A in ECs

4. CircHIPK3 regulates FAO activity and EC angiogenesis through CPT1A

Discussion

Materials and Methods

Tissue collection and primary cell extraction

Cell culture

Real-time quantitative PCR

Protein extraction and immunoblotting

IHC

Subcellular fractionation assay

Cell transfection

FAO assay

Cell proliferation assay

Flow cytometry analysis of the cell cycle

Cell migration assay

Tube-formation assay

Luciferase reporter assay

Statistics

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1