miR-2467-3p/ABLIM1 Axis Mediates the Formation and Progression of Deep Vein Thrombosis by Regulating Inflammation and Oxidative Stress

Abstract

Deep vein thrombosis (DVT) is a common postoperative complication of orthopaedic surgery with a complex pathogenesis mechanism. The effect of the miR-2467-3p/acting-binding LIM protein 1 (ABLIM1) axis on thrombus formation and human vascular endothelial cells (HUVECs) progression was evaluated aiming to identify a novel potential biomarker of DVT. DVT rat models were established by inferior vena cava stenosis. The expression of the miR-2467-3p/ABLIM1 axis was analyzed by PCR. HUVECs were induced with oxidative low-density lipoprotein (ox-LDL). Cell growth and motility were assessed by cell counting kit 8 (CCK8) and Transwell assay. The inflammation and oxidative stress were estimated by proinflammatory cytokines and generation of MDA and reactive oxygen species (ROS). ABLIM1 was downregulated in DVT rats. Overexpressing ABLIM1 could suppress the formation of thrombosis and alleviate inflammation and oxidative stress. In HUVECs, ox-LDL induced significantly increased miR-2467-3p and decreased ABLIM1, and miR-2467-3p could negatively regulate ABLIM1. The knockdown of miR-2467-3p could alleviate the inhibited cell growth and motility by ox-LDL, and the inflammation and oxidative stress were also attenuated. While silencing could reverse the effect of miR-2467-3p on ox-LDL-induced HUVECs. The miR-2467-3p/ABLIM1 axis regulates the occurrence and development of DVT through modulating HUVECs inflammation and oxidative stress.

Deep vein thrombosis (DVT) refers to the abnormal clotting of blood in the deep vein system, which further results in the narrowing or occlusion of the venous cavity and obstructs venous return and venous hypertension. Recently, the incidence of DVT is gradually increasing, and data have reported that approximately 6% of DVT patients died within 30 days of onset, where pulmonary embolism was the main cause of death. ^{1 2} The pathogenic mechanism of DVT remains unclear, but there have been several risk factors explored, including obesity, trauma, fracture, tumor, hormone therapy, and some primary etiologies. ³ DVT has been considered a major complication of surgical treatments especially in orthopaedic surgery, such as hip fracture surgery, and knee replacement surgery. ^{4 5 6 7} Moreover, vascular endothelial cells, coagulation/anticoagulation systems, inflammation cytokines, and changes in blood flow have been demonstrated to be involved in the pathological process of DVT, where endothelial cell injury plays a vital role. ⁸

With the development of molecular biology, there have been several molecules revealed to regulate endothelial cell injury and mediate DVT progression. For example, silencing long non-coding RNA (lncRNA) LINC01123 could reduce thrombosis length and weight, alleviate inflammation, and further suppress the formation of DVT. ⁹ Transcriptional profiles have also been devoted to identifying candidate regulators for DVT. A recent study focused on the occurrence of venous thrombosis in osteomyelitis, and several hub genes were unearthed. ABLIM1 was observed to be downregulated in both vein thrombosis and osteomyelitis implying its potential involvement in the progression of these two diseases. ¹⁰ However, there was a lack of available data confirming this. Previously, the abnormal expression of ABLIM1 has been demonstrated to regulate the progression of human diseases, such as glioblastoma and hepatocellular carcinoma, ^{11 12} and mediate the function of non-coding RNA (ncRNAs). For example, ABLIM1 was revealed to mediate the enhancement of nasopharyngeal carcinoma progression by lncRNA ZNF667-AS1 through miR-1290. ¹³ Moreover, ABLIM1 was also reported to regulate cell progression, such as the differentiation of osteoclast and the migration of hepatocellular carcinoma cells. ^{12 14 15} ABLIM1 was speculated to regulate the progression of vascular endothelial cells and further mediate the development of DVT.

In mechanism, the function and regulatory effect of upstream ncRNAs, including long noncoding RNAs and microRNAs, also played critical roles in disease development and cellular processes. The interaction between miRNAs and target genes has also been considered a promising therapeutic target for human diseases. With the help of online prediction databases, miR-2467-3p was speculated as the direct upstream regulator of ABLIM1. Although the function of miR-2467-3p in DVT has not been reported, miR-2467-3p was demonstrated to be involved in human cancers, such as breast cancer, nonsmall cell lung cancer, and cervical cancer. ^{16 17 18} Therefore, the effects of miR-2467-3p on ABLIM1 and the regulation of vascular endothelial cells were also estimated in the present study aiming to complete the regulation chain of ABLIM1 in DVT progression.

Materials and Methods

Deep Vein Thrombosis Animal Modeling

This study had been approved by the Ethics Committee of Zibo Central Hospital. Clean Sprague–Dawley rats were obtained from SPF Biotech Co. (Beijing, China) and maintained at 25°C and 50% humidity with free access to food and drink. Animals were allowed to adjust to the environment for 7 hours and were fasted overnight before the experiment.

DVT rat models were established by the ligation of the inferior vena cava according to previous studies. ^{19 20} Rats were first anesthetized with 1% pentobarbital sodium via intraperitoneal injection. A 2-cm incision was taken along the belly line under 5 cm of the episternum. The skin, subcutaneous muscle layer, and peritoneum of rats were incised layer-by-layer to expose the inferior vena cava. The inferior vena cava was slowly separated. Then, the 5–0 suture line was placed parallel to the inferior vena cava and was close to the vascular wall. The inferior vena cava was then ring-ligated with the 4–0 suture adjacent to the renal vein, and the 5–0 suture was slowly extracted. The contents of the abdominal cavity were carefully returned, and the abdomen was closed layer-by-layer. In the sham group, only the inferior vena cava was exposed with similar methods as above. All rats were injected with antibiotics preventing injection.

ABLIM1-overexpression plasmid was transfected before DVT animal modeling using Entranster in vivo DNA transfection reagent (Engreen Biosystem, United States).

Sample Collection

Rats were euthanized after 24 hours of modeling, and the abdominal cavity was opened along the original incision. The inferior vena cava was separated and collected. The condition of thrombosis formation and inferior vena cava changes were evaluated. The vascular tissues were stored at −80°C for the following analyses.

Cell Culture and Injured Cell Modeling

Human vascular endothelial cells (HUVECs) were obtained from American Type Culture Collection (ATCC) and maintained with HUVECs culture medium (Procell Life Science and Technology, China) at 37°C. Cells were available for modeling after the confluence reached 80 to 90%. Injured cells were induced by 20 μg/mL oxidative low-density lipoprotein (ox-LDL, Shanghai Yuanye Bio-Technology Co., China) for 24 hours. Then, cells were collected for the following experiments.

Cell Transfection

miR-2467-3p mimic, inhibitor, corresponding negative controls, pcDNA 3.1-NC, and pcDNA 3.1-ABLIM1 were transfected into HUVECs using Lipofectamine 2000 (Invitrogen, United States) according to manufacturer's instructions. Transfected cells were harvested after 48 hours of transfection for the following analyses.

Real-time Quantitative PCR

Total RNA was extracted with Buffer RZ (TIANGEN Biotech, China) and trichloromethane and evaluated by the value of obsorbance (OD) 260/280 using a microplate reader. Isolated RNA was reverse-transcribed to cDNA and quantitative PCR was performed with SYBR Green reagent on the 7500 PCR system for the evaluation of miR-2467-3p and ABLIM1 expression with cel-miR-39 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference, respectively. The 2 ^−ΔΔCT method was used to calculate the relative expression levels.

Dual-luciferase Reporter Assay

Wild-type and mutant-type ABLIM1 vectors were constructed by cloning wild- or mutant-binding sites into the pGL3 vectors (Promega, United States). Cells were transfected with wild-ABLIM1 or mutant-ABLIM1 and miR-2476-3p mimic or inhibitor using Lipofectamine 2000 (Invitrogen, United States). The relative luciferase activity of ABLIM1 was analyzed on the luciferase system (Promega, United States) with Renilla as the internal reference.

Cell Proliferation

Logarithmic transfected and untransfected cells were seeded into 96-well plates supplied with a completed culture medium and incubated overnight at 37°C. Cell density was 5 × 10 ³ cells/well with five repeat wells of each treatment. Then cells were treated with 20 μg/mL ox-LDL. Plates were further incubated at 37°C for 24, 48, 72, and 96 hours followed by adding CCK8 reagent to each well. After 4 hours of CCK8 addition, plates were measured with a microplate reader for absorbance at 450 nm. The time–OD curve was plotted for the assessment of cell proliferation.

Cell Motility

Cells were seeded into the upper chamber of 24-well Transwell plates and incubated at 37°C. Ten percent fetal bovine serum (FBS)-containing culture medium was used as a chemoattractant placed in the bottom chamber. After 24 hours of incubation, cells in the upper chamber were removed, and the subsurface cells were fixed and stained. Cells were viewed with an optic microscope with five random fields of each treatment.

Inflammation and Oxidative Stress Evaluation

Inflammation factors, including IL-1β, IL-6, and IL-8 were analyzed with corresponding ELISA kits according to the manufacturer's protocol. Oxidative stress was assessed by the levels of MDA and ROS with corresponding commercial kits according to the previously described method.

Statistical Analyses

Statistical analyses were conducted with the SPSS 26.0 and GraphPad Prism 9.0 software, and data were represented as mean ± standard deviation ( n  = 3). Comparison among multiple groups was performed using one-way analysis of variance, and Student's t -test was employed for comparison between the two groups ( p  < 0.05).

Results

Regulatory Effect of ABLIM1 on Thrombosis Formation, Inflammation, and Oxidative Stress of Deep Vein Thrombosis Rats

Established DVT rats showed significant formation of thrombosis as the length ( Fig. 1A ) and weight ( Fig. 1B ) of thrombosis were significantly higher than the sham rats. Moreover, DVT modeling also induced significantly increasing levels of proinflammatory cytokines, including IL-1β, IL-6, and IL-8 ( Fig. 1C ), and increasing levels of oxidative products, including MDA ( Fig. 1D ) and ROS ( Fig. 1E ).

Fig. 1.

The ligation of the inferior vena cava promoted the formation of DVT with increasing thrombosis length ( A ), weight ( B ), proinflammatory cytokines ( C ), MDA ( D ), and ROS ( E ). **** p  < 0.0001. DVT, deep vein thrombosis.

ABLIM1 was found to be significantly downregulated in DVT rats, which was elevated by its overexpression plasmid ( Fig. 2A ). The overexpression of ABLIM1 significantly inhibited the formation of thrombosis, where the length ( Fig. 2B ) and weight ( Fig. 2C ) of thrombosis in DVT rats were decreased. Additionally, the levels of IL-1β, IL-6, IL-8, MDA, and ROS were also dramatically reduced by the overexpression of ABLIM1 ( Fig. 2D–F ), indicating the suppressed inflammation and oxidative stress in DVT rats.

Fig. 2.

ABLIM1 was downregulated in DVT rats ( A ), and its overexpression significantly suppressed the length ( B ) and weight ( C ) of thrombosis, inflammation factor levels ( D ), the levels of MDA ( E ), and ROS ( F ). ^ns p  > 0.05, **** p  < 0.0001. DVT, deep vein thrombosis.

miR-2467-3p Negatively Modulated ABLIM1 and Mediated the Function of ABLIM1 in HUVECs

The upstream miRNAs of ABLIM1 were predicted from miRDB (a total of 38 miRNAs were enriched), miRWalk (a total of 2,257 miRNAs were enriched), and TargetScan (a total of 25 miRNAs were filtered) databases, and miR-2467-3p was enriched in the intersection of these databases ( Fig. 3A ). miR-2467-3p could negatively regulate the luciferase activity of ABLIM1 with several predicted binding sites ( Fig. 3B ).

Fig. 3.

The upstream miRNAs of ABLIM1 were predicted from miRDB (Target score ≥80), miRWalk (binding score ≥0.8), and TargetScan (|total context + + score| ≥0.2) databases, and only miR-2467-3p was enriched in the intersection ( A ). miR-2467-3p could negatively regulate the luciferase activity of ABLIM1 in HUVECs ( B ). miR-2467-3p was upregulated in ox-LDL-induced HUVECs, which was suppressed by the transfection of miR-2467-3p inhibitor ( C ). ABLIM1 was downregulated in ox-LDL-induced HUVECs, which was elevated by miR-2467-3p knockdown ( D ). The transfection of ABLIM1 siRNA could reverse the effect of miR-2467-3p silencing. ^ns p  > 0.05, *** p  < 0.001, **** p  < 0.0001. HUVEC, human vascular endothelial cell; ox-LDL, oxidative low-density lipoprotein.

HUVECs were treated with ox-LDL to mimic the vascular environment of DVT. It was found that miR-2467-3p was significantly upregulated in ox-LDL-treated HUVECs. miR-2467-3p was significantly suppressed by the transfection of the inhibitor, but the siRNA of ABLIM1 showed no significant effect on the expression of miR-2467-3p ( Fig. 3C ). In contrast, significantly reduced expression of ABLIM1 was observed in ox-LDL-treated HUVECs. The knockdown of miR-2467-3p significantly elevated the expression of ABLIM1 in ox-LDL-treated HUVECs, which was reversed by the transfection of ABLIM1 siRNA ( Fig. 3D ).

The treatment of ox-LDL induced decreased proliferation ( Fig. 4A ), migration ( Fig. 4B ), and invasion ( Fig. 4C ) of HUVECs. While silencing miR-2467-3p alleviated the inhibition of cell growth ( Fig. 4A ) and motility ( Fig. 4B and C ), which was reversed by ABLIM1 knockdown. Additionally, ox-LDL also induced significant inflammation ( Fig. 4D ) and oxidative stress ( Fig. 4E ) in HUVECs, which was also attenuated by the knockdown of miR-2467-3p. Silencing ABLIM1 could reverse the effect of miR-2467-3p on HUVECs inflammatory response ( Fig. 4D ) and oxidative stress ( Fig. 4E ).

Fig. 4.

ox-LDL inhibited the proliferation ( A ), migration ( B ), and invasion ( C ) of HUVECs and induced increasing inflammation ( D ), and oxidative stress ( E ). Silencing miR-2467-3p could alleviate HUVECs injury induced by ox-LDL, which was reversed by ABLIM1 knockdown. ^ns p  > 0.05, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001. HUVEC, human vascular endothelial cell; ox-LDL, oxidative low-density lipoprotein.

Discussion

Vein thrombosis has become a global burden and a major vascular disease following acute myocardial infarction and cerebral apoplexy. With the increasing aging of the population and the increasing incidence of obesity and complications related with thrombosis, the occurrence of vein thrombosis, especially DVT, has gradually increased. Inferior vena cava stenosis is a common method for DVT modeling, which could keep a certain degree of blood flow and conforms to the pathogenesis of DVT in patients with long-term bed rest after surgical treatments. ^{22 23} In this study, DVT rat models were established by the ligation of the inferior vena cava, and significant formation of thrombosis was observed. Inflammation and oxidative stress have been demonstrated to possess close association with the occurrence and development of DVT. Coagulation activation and inflammation shared similar cellular and plasma-related factors, such as proinflammatory cytokines. ^{24 25} Therefore, the onset of DVT is also accompanied by the inflammatory response. The increasing levels of MDA and ROS during oxidative stress could induce endothelium dysfunction and activate platelets and leukocytes, influencing coagulation function and therefore promoting the formation of thrombosis. ²⁶ Increasing levels of inflammation factors and generation of MDA and ROS were observed in DVT rats, indicating the occurrence of inflammatory response and oxidative stress. ABLIM1 was previously revealed to be correlated with vein thrombosis formation in osteomyelitis. ¹⁰ Herein, ABLIM1 was found to be downregulated in DVT rats, and overexpressing ABLIM1 significantly suppressed the formation of thrombosis and alleviated inflammation and oxidative stress in DVT rats, indicating its involvement in the occurrence and development of DVT.

Previous studies have also observed the abnormal expression of ABLIM1 in various human diseases, which was revealed to regulate tumor progression, and cellular processes, and predict disease development. ^{11 14 27 28} The functional role of ABLIM1 was also suggested to be related to the modulation by upstream ncRNAs, such as lncRNA ZNF667-AS1, miR-6165, and miR-129-3p. ^{13 29 30} The regulatory effect of ABLIM1 on DVT development was further investigated to disclose the potential regulatory mechanism. With the help of online databases, miR-2467-3p was predicted as a direct upstream miRNA of ABLIM1. miR-2467-3p has also been illustrated as a functional ncRNA in regulating cancer progression and mediating the function of lncRNAs and circular RNA (circRNAs). For instance, downregulated miR-2467-3p in type 1 diabetes was found to promote renal fibrosis and kidney dysfunction via negatively regulating Twist 1. ³¹ Serving as the competing endogenous RNA (ceRNA) of circ_0089153, miR-2467-3p mediates the promoted cell growth and metastasis of breast cancer by circ_0089153. ¹⁷ Here, miR-2467-3p was found to negatively regulate the luciferase activity and expression of ABLIM1 in HUVECs. HUVECs injury would induce vascular wall injury, which could lead to the asymmetry of membrane and the loss of anticoagulant membrane component and therefore improve the risk of DVT. ^{32 33 34} ox-LDL could stimulate inflammation in various cells in the inner wall of blood vessels and has been demonstrated to participate in the development of coronary atherosclerosis. ^{35 36 37} In the present study, ox-LDL was employed to induce HUVECs injury, which resulted in reduced cell proliferation, migration, and invasion. Consistent with DVT rat models, ox-LDL-induced HUVECs also showed significant inflammation and oxidative stress. In ox-LDL-induced HUVECs, significant upregulation of miR-2467-3p and downregulation of ABLIM1 were observed. Silencing miR-2467-3p could alleviate the inhibitory and proinflammatory effect of ox-LDL on HUVECs, which was reversed by ABLIM1. Hence, the miR-2467-3p/ABLIM1 axis regulates ox-LDL-induced HUVECs injury, which was considered the mechanism underlying the suppression of DVT development by ABLIM1.

In mechanism, the regulation of vascular endothelial growth factor (VEGF) and angiogenesis also plays crucial roles in DVT development, which has not been revealed in the present study. Additionally, previous studies have reported that Phosphatidylinositol-3-kinase/Protein Kinase B (PI3K/Akt) signaling pathway regulated the secretion of VEGF and HUVECs viability and was involved in the formation and progression of DVT. ^{38 39 40 41} Hence, future investigations should pay attention to the deep molecular mechanism underlying the miR-2467-3p/ABLIM1 axis in DVT.

According to the above findings, downregulated ABLIM1 could regulate the formation of vein thrombosis, inflammation, and oxidative stress in DVT. The miR-2467-3p/ABLIM1 axis could regulate the growth, motility, inflammation, and oxidative of ox-LDL-induced HUVECs. These findings demonstrated the potential of ABLIM1 as therapeutic target for DVT. Previously, there were also several lncRNAs identified as biomarkers for the diagnosis and prognosis of DVT based on clinical data. Hence, further clinical investigations would be performed by collecting clinical samples and data, aiming to declaim the clinical significance of ABLIM1 and to evaluate its potential in clinical application.

Funding Statement

Funding This study was funded by Shanghai 2020 “Science and Technology Innovation Action Plan” Biomedical Science and Technology Support Special Project (20S31902000) and the National Natural Science Foundation of China (81971710).