Mechanism of LEF1-AS1 regulating HUVEC cells by targeting miR-489-3p/S100A11 axis

Ethical statement

This study was approved by the Institutional Review Committee of the Third Affiliated Hospital of Zhengzhou University. Each participant provided written informed consent. The scheme was approved by the Scientific Research Ethics Committee of the Third Affiliated Hospital of Zhengzhou University ([2020] YLS No. 119).

Bioinformatics analysis

The target genes of miRs and miR-489-3p regulated downstream of lncRNA LEF1-AS1 were predicted using the StarBase database (http://starbase.sysu.edu.cn/index.php), an interactive analysis database of gene expression profiles.

Research objects

From January 2021 to March 2022, VM tissues and adjacent normal venous tissues were obtained from 38 patients diagnosed with VMs and treated surgically in the Third Affiliated Hospital of Zhengzhou University. Fresh tissues were temporarily placed in liquid nitrogen and stored permanently at −80 °C. Inclusion criteria for the experimental group were as follows: (1) VM was confirmed via clinical and pathological diagnosis; (2) surgical resection was performed without sclerosing agent injection, laser, interventional embolization, targeted drugs, or other treatments before the operation; (3) no basic diseases of important organs; (4) no special infections, such as hepatitis B, hepatitis C, syphilis, and AIDS. Inclusion criteria for the control group were as follows: (1) the incisal margin of the diseased tissue was confirmed as normal venous tissue via pathology; (2) the distance between the tissue taken and the cutting edge of the pathologically proven tissue was at least 3 cm; (3) other tissues were the same as those of the experimental group. The following were the exclusion criteria: (1) diagnosis of vascular malformations and vascular malformation syndrome other than VMs; (2) patients with autoimmune diseases; (3) patients with liver and kidney dysfunction; (4) medical records not well preserved and no follow-up record.

Cell culture and treatment

HUVECs (American Type Culture Collection, ATCC) cells were cultured in ECM (ScienCell, Carlsbad, CA, USA), which is a special endothelial cell culture medium containing 5% serum, and then placed in 37 °C, 5% CO₂, 95% humidity incubator (Thermo, Waltham, MA, USA). HUVECs in the logarithmic growth period were used to prepare the cell suspension and inoculated into a 6-well culture plate at the density of 1 × 10⁵ cells/well. After culturing for 24 h, the wells were randomly assigned to the NC, si-LEF1-AS1, miR inhibitor, and si lnc+miR inhibitor groups. Transfection was performed in accordance with the instructions of Lipofectamine3000 (Invitrogen, Waltham, MA, USA). After incubation for 6 h in an incubator at 37 °C under 5% CO₂ and 95% humidity, the transfection medium was replaced with a complete medium. The transfection was continued for 36 h, followed by the subsequent tests. The sequence of the primers used was as follows: NC justice chain: 5′-UUCUCCGAACGUGUCGUTT-3′, antisense chain: 5′-ACGUGACACGUUCGGAGAATT-3′; Si-LEF1-AS1 justice chain: 5′-ACAUUUCGUUUCUAGCCC-3′, antisense chain: 5′-GCUAGAAACGAAAUGUGA-3′. These were synthesized by Huzhou Hippo Biotechnology Co., Ltd., Huzhou, China.

Gene chip experiment

Agilent human lncRNA microarray 2019 (4 × 180 k, Design ID: 086188) chip was used to detect the samples. After sample quality control, data were standardized using the quantile algorithm, and the differential genes were subsequently screened.

(1) The total RNA of the sample was quantified using NanoDrop ND-2000, and RNA integrity was checked. After quantification of the RNA, sample labeling, hybridization, and chip elution were performed according to the standard process of the chip. First, the total RNA was reverse-transcribed into double-stranded complementary DNA (cDNA), and the cRNA was labeled with fluorescence using cyanine-3-CTP (Cy3). The labeled cRNA was hybridized with the gene chip, and the scanning results were obtained using Agilent Human lncRNA Microarray 2019 (4 * 180 k, Design ID:086188) after elution.

(2) Feature extraction software was adopted to process the scanning result map. To eliminate the data differences between experiments, the quantile method was used for the standardized processing of the original data. Before performing the subsequent analysis, standardized data were screened and filtered. For comparison, in each group of samples, at least 75% of the samples labeled as “detected” were left for follow-up analysis. Differential gene screening was performed using the P-value and the multiple change value of the t-test. The screening criteria were upregulated or downregulated with multiple change values of ≥2.0 and a P-value of ≤0.05.

Detection of RNA expression using quantitative reverse transcription (qRT)-PCR

The total RNA of tissues and cells was extracted with TRLzol (Beijing Quanshijin Company, Beijing, China), and RNA was concentrated using isopropanol precipitation. The reverse transcription was performed according to the instructions of the ReversTra Ace qPCR RT Kit (Toyobo Company, Osaka, Japan). The RNA was reverse transcribed into cDNA, and qRT-PCR was performed according to the instructions of SYBR Green Realtime PCR Master Mix Kit (Toyobo Company, Osaka, Japan) for qPCR detection. The sequence of primers used for qRT-PCR are shown in Table 1 (Huzhou Hippo Biotechnology Co., Ltd., Huzhou, China). The reaction conditions were as follows: predenaturation at 95 °C for 60 s, denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 45 s for 40 cycles (the PCR instrument was from Roche, Switzerland). The primers were synthesized by Beijing Qingke Technology Co., Ltd. GAPDH and U6 were used as reference genes to compare the relative cycle threshold (Ct) values of LEF1-AS1, miR-489-3p, and S100A11. The relative expression of the target gene was calculated using the 2^−ΔΔCt method; three duplicate were set in each group, and each experiment was repeated thrice.

Western blotting of protein expression

The total protein of tissues and cells was extracted using the BCA protein (Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China) quantitative kit to detect the protein concentration and stored at −20 °C as a standby. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, American Abcam Company, ab9485, working concentration 1:1,000) and S100A11 antibody (American Abcam Company, ab169530, working concentration 1:1,000) were purchased. Subsequently, 15% SDS-PAGE gel (Wuhan Saiwei Biotechnology Co., Ltd., Wuhan, China) was prepared, and 40 µg of the protein was added to each well. Gel electrophoresis was performed to separate the protein, and it was transferred to a polyvinylidene difluoride (PVDF) membrane (membrane transfer condition: 300 mA, 150 min). The membrane was washed with Tris-buffered saline with 0.1% Tween 20 (TBST) three times, 10 min/time. The PVDF membrane was sealed with a rapid sealing solution for 1 h, washed with TBST three times, and strips corresponding to S100A11 and GAPDH were cut. The corresponding primary antibody was added and incubated at 4 °C overnight. The next day, the film was initially washed with TBST three times, 10 min/time, and then a fluorescent secondary antibody (Odyssey Company, Stamford, CT, USA, working concentration 1:10,000) was added and incubated in a dark shaking bed for 1 h. The film was washed with TBST buffer three times, 10 min/time, and finally detected using a protein imprint exposure imager. Image J software was used to measure the band gray value, the relative protein expression was calculated, and GraphPad Prism 9.0 was used to construct a histogram.

Double luciferase reporter gene assay

(1) The possible binding sites of miR-489-3p and LEF1-AS1 as well as those of miR-489-3p and S100A11 were predicted by searching the bioinformatics website StarBase database. The LEF1-AS1 3′-UTR sequences containing the binding sites of miR-489-3p were obtained. The 3′-UTR sequence of S100A11 and that of the mutant (Mut) (Huzhou Hippo Biotechnology Co., Ltd., Beijing, Chain.) synthesized using the chemical method with the corresponding binding sites were cloned into the vector.

(2) HUVEC cells were coated in 12-well plates and cotransfected with WT or Mut type and miR-489-3p mimic and Lipo3000 (Invitrogen, the United States) in HUVEC cells when the cell confluence reached 60–80%.

(3) Forty-eight hours after transfection, firefly luciferase (Fluc) and sea luciferase (Rluc) activities in each group were detected using the reporter luciferase assay kit (Dual-Luciferase Reporter Assays, Promega), and the relative expressions of each reporter gene were expressed using Fluc/Rluc values.

(4) Statistical analysis was performed to determine whether there were statistically significant differences between the groups.

Detection of cell proliferation using the CCK-8 assay

Each group of cells was inoculated into 96-well plates according to the standard of 100 µL of cell culture medium and 3000 cells in each well. The cells were incubated at 37 °C in a 5% CO₂ incubator for 24, 48, and 72 h, following which the supernatant was discarded. Subsequently, 100 µL of cell culture solution and 10 µL of CCK-8 solution were added (Toyo Textile Company, Tokyo, Japan). These solutions were mixed evenly and incubated for 2 h. Finally, the optical density of each well was measured at a wavelength of 450 nm with an automatic microplate reader.

Detection of cell migration using the transwell assay

In the upper chamber of the transwell plate (Corning Company, New York, NY, USA), HUVEC cells transfected for 36 h were added and resuspended in 200 µL (2 × 10⁴ cells/well) of serum-free medium. Later, 600 µL of RPMI1640 medium containing 20% fetal bovine serum was added to the 24-well plate (Beijing Solebar Technology Co., Ltd., Beijing, China), and the cells were added. Each group of cells was set with three reperforators and placed in an incubator at 37 °C with 5% CO₂ and 95% humidity for 36 h. The chamber was removed, the liquid in the upper chamber was sucked out, rinsed gently with phosphate-buffered saline (PBS), fixed with 4% methanol for 20 min, stained with crystal violet for 10 min, rinsed twice with PBS, the residual cells in the upper chamber were gently wiped off with a cotton swab, and the cells migrated to the lower layer of the microporous membrane were counted under an inverted microscope. The ImageJ software was used for statistical analysis.

Angiogenesis experiment

The matrix adhesive (BD Company, Franklin Lakes, NJ, USA) was added to the 24-well plate with a capacity of 300 µL/hole for solidification. The HUVEC cells were resuspended in ECM culture medium, inoculated on a Matrigel-coated 24-well plate at a density of 8 × 10⁴ cells/well, and incubated at 37 °C for 6 h. Each group of cells was set with three multiple wells. The growth was observed under an inverted microscope, and the tube formation of HUVEC cells was photographed. The ImageJ software was used to count the length of the assembly tube.

Statistical analysis

SPSS26.0 and GraphPad Prism 8.0 software were used for statistical analysis. Independent samples t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. The difference was considered statistically significant if P < 0.05.

LncRNA expression profile in VMs

To identify lncRNAs that are preferentially expressed in human VM tissues and possibly contribute to the progression of the disease, candidate lncRNA biomarkers were screened using chip and bioinformatics analysis. The expression profiles of differentially expressed lncRNAs in VM tissues and their paired normal tissues were evaluated. These lncRNAs were from five patients with VMs. The Agilent human lncRNA microarray was used, and the differentially expressed lncRNAs were identified by performing microarray analysis. The analysis detected 1651 lncRNAs that were differentially expressed in VMs, with at least twice the change. Of these, 1015 lncRNAs were upregulated and 636 were downregulated. The differentially expressed lncRNAs were selected with volcanic map filtering (fold change ≥ 2 and P-value ≤ 0.05). As depicted in the volcanic map (Fig. 1A), the two vertical lines represented the double change boundary and the horizontal line denoted the statistically significant boundary (P < 0.05). The upregulated and downregulated genes with multiple changes ≥2 and statistical significance were marked with red dots and blue dots, respectively. Candidate lncRNA biomarkers were selected based on the results of microarray and bioinformatics analyses. The microarray results showed that the NC sample was the normal tissue of the patient. Compared with normal vascular tissues, multiple lncRNAs were abnormally expressed in VMs. The cluster diagram of the lncRNA profile comparison between VMs and paired normal venous vascular samples is shown in Fig. 1B. In the figure, each row represents an lncRNA and each column represents a tissue sample. According to the color code description of relative lncRNA expression, red represents upregulation, blue represents downregulation, and the numbers 0.8, 0, and −0.8 signify multiple changes in the corresponding spectrum. The hierarchical clustering of the expressions of 1651 lncRNAs clearly distinguished VMs from normal tissues.

The lncRNA LEF1-AS1-miR-489-3p-S100A11 interaction network

This study aimed to determine the role of the lncRNA LEF1-AS1 in VMs. The downstream regulatory miRs of LEF1-AS1 were predicted using the StarBase online prediction website, and the binding sites of LEF1-AS1 and miR-489-3p (Fig. 2A) were obtained. The upper sequence was LEF1-AS1, and the lower sequence was miR-489-3p. The downstream regulatory genes of miR-489-3p were explored in the StarBase database, and the binding sites of miR-489-3p and S100A11 were obtained (Fig. 2B). The upper sequence was S100A11, and the lower sequence was miR-489-3p. The findings revealed that the lncRNA LEF1-AS1-miR-489-3p-S100A11 axis may be involved in the occurrence and development of VMs.

The high expression of lncRNAs LEF1-AS1 and S100A11 and the low expression of miR-489-3p in VMs

RT-qPCR was used to detect the VM tissues of 38 patients. The results showed that compared with the expressions of LEF1-AS1, miR-489-3p, and S100A11 in normal veins, the expression of LEF1-AS1 in VM tissues was significantly higher (Fig. 3A, P < 0.0001), and the difference was statistically significant. The expression of S100A11 was also significantly increased (Fig. 3C, P < 0.0001). On the contrary, the expression of miR-489-3p decreased significantly (Fig. 3B, P < 0.0001), and the difference was statistically significant.

Regulation of the expression of S100A11 by LEF1-AS1 via adsorption onto miR-489-3p

The results of the above tissue experiments verified the ceRNA regulatory mechanism of the lncRNA LEF1-AS1, and the findings indicated that LEF1-AS1 may be involved in regulating the occurrence and development of VM. However, the specific mechanism of action needs to be further explored by performing cell culture experiments. After transfection of HUVEC cells, whether LEF1-AS1 and miR-489-3p were silenced successfully was determined using qRT-PCR assay. The results showed that the relative expression of LEF1-AS1 in the si-LEF1-AS1 group was significantly lower than that in the si-NC group (Fig. 4A, P < 0.0001), and the difference was statistically significant, which indicated the successful silencing of LEF1-AS1 in HUVEC cells. HUVEC can be used for subsequent experiments. The relative expression of miR-489-3p in the miR-inhibitor group was significantly lower than that in the miR-NC group (Fig. 4B, P < 0.0001), and the difference was statistically significant. This result indicated that the expression of miR-489-3p was successfully inhibited and that these cells could be used for subsequent experiments. Bioinformatics prediction results showed that LEF1-AS1 and miR-489-3p possessed complementary binding sites, and qRT-PCR results showed that LEF1-AS1 silencing increased the expression of miR-489-3p in HUVEC cells (Fig. 4C, P < 0.0001). This observation indicated that miR-489-3p interacted with LEF1-AS1, which in turn negatively regulated the expression of miR-489-3p. Furthermore, bioinformatics prediction results demonstrated the presence of a binding site between the miR-489-3p sequence and the 3′UTR region of S100A11. In addition, qRT-PCR detection results showed that inhibiting the expression of miR-489-3p significantly increased the expression of S100A11 (Fig. 4D, P < s0.0001). These results signified that the binding sites of miR-489-3p and S100A11 were bound to each other and negatively correlated. The ceRNA mechanism of LEF1-AS1 in HUVEC cells was further confirmed by performing a rescue experiment. The findings showed that a single transfection with si-LEF1-AS1 reduced the expression of S100A11, whereas a single transfection with miR-489-3p inhibitor increased the expression of S100A11. Cotransfection with si-LEF1-AS1 and miR-489-3p inhibitor partially reversed the inhibition of S100A11 expression caused by LEF1-AS1 silencing. The outcomes of qRT-PCR and Western blotting implied that the inhibition of LEF1-AS1 expression reduced the expression of S100A11 at both mRNA and protein levels and that the inhibition was negatively regulated by miR-489-3p (Figs. 4E–4G). In conclusion, LEF1-AS1 can regulate the expression of S100A11 by competitively binding to miR-489-3p.

The targeting of miR-489-3p by LEF1-AS1, and S100A11 acting as the target gene of miR-489-3p

The above bioinformatics prediction results revealed the possible presence of complementary binding sites between LEF1-AS1 and miR-489-3p. Furthermore, there were possible complementary binding sites between miR-489-3p and S100A11. Meanwhile, the above PCR results denoted that LEF1-AS1 silencing increased the expression of miR-489-3p in HUVEC cells (Fig. 4C, P < 0.0001) and that LEF1-AS1 negatively regulated the expression of miR-489-3p. Moreover, the PCR results signified that inhibiting the expression of miR-489-3p enhanced the expression of S100A11 (Fig. 4D, P < 0.0001) and that miR-489-3p negatively regulated the expression of S100A11. The results of the double luciferase assay showed that after transfection of the wild-type LEF1-AS1 sequence, the activity of luciferase was significantly inhibited in the miR-489-3p mimic group, but the fluorescence intensity of the mutant plasmid did not change significantly (Fig. 5A, P < 0.001). After transfection of the wild-type S100A11 sequence, luciferase activity was significantly inhibited in the Mir-489-3p mimic group. Although the activity of luciferase was significantly inhibited in the miR-489-3p mimic group, the fluorescence intensity of mutant plasmids did not change significantly (Fig. 5B, P < 0.001). Based on the above bioinformatics prediction results and those of qRT-PCR, the results of dual luciferase assay better demonstrate that LEF1-AS1 in HUVEC cells can target miR-489-3p and that S100A11 is the target gene of miR-489-3p.

Effects of LEF1-AS1 on proliferation, migration, and angiogenesis of HUVEC cells via the miR-489-3p/S100A11 axis

The above results confirmed the interaction among the three factors LEF1-AS1, miR-489-3p, and S100A11. In HUVEC cells, LEF1-AS1 regulated the expression of S100A11 by regulating miR-489-3p. Subsequently, the effect of the ceRNA mechanism exerted by LEF1-AS1 on the proliferation, migration, and angiogenesis of HUVECs was examined. After transfection with si-LEF1-AS1 alone, the proliferation and migration as well as in vitro angiogenesis of HUVEC cells were decreased. On the contrary, after transfection with miR-489-3p inhibitor alone, the proliferation, migration, and in vitro angiogenesis was enhanced. The inhibitory effect of LEF1-AS1 silencing on HUVEC proliferation, migration, and angiogenesis was negatively regulated by miR-489-3p. Cotransfection with si-LEF1-AS1 and miR-489-3p inhibitor partially reversed the proliferation (Fig. 6A), migration (Figs. 6B and 6C), and angiogenesis (Figs. 6D and 6E) of HUVEC cells caused by LEF1-AS1 silencing, and the differences were statistically significant. Combined with the present experimental results, it was concluded that the high expression of LEF1-AS1 in VMs results in the adsorption of miR-489-3p to venous endothelial cells, thereby increasing the expression of S100A11. Consequently, the proliferation, migration, and angiogenesis of HUVEC cells are enhanced. This ceRNA mechanism of action is employed by LEF1-AS1 to promote the development of VMs.