Journal of Data Mining in Genomics & Proteomics

Down-Regulation of lncRNA XIST Ameliorates Lipoprotein (A) Induced Endothelial Progenitor Cell Damage via Modulating miR-126/PLK2 Axis

Xiaolei Zhang^*, Guoying Xu, Shizhen Wang, Pei Chen and Shanshan Chen Department of Nursing, Jiangsu College, Jiangsu, China
^*Correspondence: Xiaolei Zhang, Department of Nursing, Jiangsu College, Jiangsu, China, Tel: 86-18761017551, Email:

Received: 10-Nov-2021 Published: 01-Dec-2021

Introduction

Endothelial Progenitor Cells (EPCs) were discovered and named by Asahara et al., as the precursor cells of mature ECs. In fact, EPCs are a group of cells with complete differentiation grade and a collection of progenitors at different differentiation stages, with heterogeneity [1,2]. Currently, it is believed that EPCs, etc. express hematopoietic progenitor cell markers CD34 and CD133, and have specific homing to injured or ischemic sites, being involved in angiogenesis and re-endothelialization. Recent studies have shown that angiogenesis is an important compensatory response to chronic ischemic disease [3]. The number and function of EPCs in the peripheral blood circulation determine the extent of endothelial repair of damaged vessels. Thus, the number of circulating EPCs in patients with AS and diabetes is reduced, and EPC function is impaired [4,5]. Studies have demonstrated that ox-LDL can further induce EPC apoptosis by increasing oxidative stress and inflammatory response, thus weakening its vascular repair ability [6]. Therefore, elucidating the molecular mechanism of ox-LDLmediated EPCs is of great value in exploring the occurrence and development of AS and developing effective therapeutic drugs for AS.

Recent studies have discovered that lncRNAs regulate Lipo Protein (LP) (a)-mediated EPC damage through sponging miRNAs [7]. For example, lncRNA GUSBP5-AS is up-regulated in EPCs in patients with Deep Vein Thrombosis (DVT). LncRNA GUSBP5-AS regulates the expression of FGF2 and MMP2/9 by targeting the miR- 223-3p/FOXO1/Akt pathway, thus modulating the angiogenesis, proliferation and homing capacity of EPCs [8]. In contrast, Guo et al. reported that lncRNA TUG1 exerts an essential role in regulating migration and differentiation of EPCs. LncRNA TUG1 serves as the competitive endogenous RNA of miR-6321, and miR- 6321 inhibits EPC migration and differentiation via its target ATF2 [9]. In addition, lncRNA GAS5, lncRNA-MALAT1, lncRNAAK131850 and other lncRNAs affect the expression of miRNAs to contribute to regulating EPC functions [10-12]. LncRNA XIST, as a common oncogenic non-coding RNA, plays an oncogenic role in Non-Small Cell Lung Cancer (NSCLC), chordoma, osteosarcoma and other tumors [13-15]. Besides, XIST gene knockout increases the activity of IL-1β-treated chondrocytes and attenuates apoptosis and extracellular matrix protein degradation in Osteo Arthritis (OA). What’s more, XIST gene knockout inhibits the occurrence and development of OA through the miR-149-5P/DNMT3A axis [16]. Thus, the XIST gene knockout alleviates inflammatory response and cell damage. However, the role of lncRNA-XIST in regulating EPCs remains elusive.

MiR-126, as a common miRNA, has been reported in multiple tumor literature. For instance, Li et al. believed that the miR-126 expression is enhanced in Esophageal Squamous Cell Carcinoma (ESCC) cells and tissues. Down-regulation of miR-126 reduces ESCC cell survival and promotes its death by regulating autophagic proteins LC3B and P62 [17]. Nevertheless, studies in bladder cancer have revealed that the expression of miR-126 is increased, which is closely related to tumor grade [18]. Therefore, the role of miR-126 in tumors is diversified, and it may play different roles in different tumors. More importantly, relevant studies have also shown miR- 126 plays a role in regulating EC function. For example, Lin et al. reported that miR-126 is down-regulated in OGD-induced HBMECs, and overexpressing miR-126 activates the PI3K/Akt pathway, thereby affecting the proliferation and apoptosis of HBMECs [19]. Hence, it can be speculated that miR-126may also contribute to regulating EC function.

Additionally, PLK2, belongs to the Polo-Like Kinase (PLK) family, mainly contributes to regulating cell cycle or mitosis [20]. PLK2 is known to exert a vital function in inhibiting cancer progression by regulating centriolar replication and being activated by spindle checkups p53-dependently. The study of Liu Ying et al. in gastric cancer confirmed that the tumor suppressor gene PLK2 is directly modulated by miR-126 in SGC-7901 cells. MiR-126 overexpression not only abates SGC-7901 cell growth and colony formation, but also induces apoptosis in vitro, while miR-126 does not affect the cell cycle of gastric cancer [21]. Thus, miR-126 targets PLK2 to regulate the proliferation of cancer cells. More importantly, Lili et al. reported that miR-126 overexpression enhances cell proliferation and migration, while dampening miR-126 has the reverse effect. This study found that PLK2 was the target gene of miR-126 and could inhibit keratinocytes’ survival, colony formation and migration [22]. However, few studies have reported that miR-126 overexpression does not decrease the PLK2 level in NSCLC cells, which may be closely related to the cell-type-specific inhibition of specific miRNAs on target genes [23]. However, the role and specific mechanism of PLK2 in regulating EPC function are still unclear.

Overall, this study speculated that down-regulating XIST improved LP (a)-induced EPC damage by regulating the miR-126/PLK2 axis, and the XIST/miR-126/PLK2 axis exerted an essential role in regulating EPC function.

Materials and Methods

Isolation, culture and identification of EPCs

Human umbilical cord blood was collected, and mononuclear cells were isolated by density gradient centrifugation. The cells were inoculated (4 × 106) and cultured in EC medium (ECM) for 4 days, and the non-adherent cells were washed away with PBS. Then, the medium was altered, and the cells were cultured until 7 days. Subsequently, the cells were inoculated into 6-well plates, and the supernatant was removed. The expression of CD34+ and CD133+ was determined by flow cytometry.

Transfection and treatment of XIST and miR-126

After trypsinization, EPCs were re-suspended and the cell density was adjusted to 5 × 107 cells/mL. After preheating with the electrophoresis apparatus for 15 min, 100 μL EPCs were taken and added into the EP tube. Then, XIST knockdown sequences (si- XIST#1, si-XIST#2), XIST overexpression sequence (XIST), miR- 126 mimics or inhibitors or negative controls were supplemented and mixed. Afterward, the cells and the mixture were gently transferred to the rotor and electroporated (180 V, 25 ms) after precooling on ice for 10 min. After the electroporation was completed, the rotor was removed and balanced at Room Temperature (RT) for 10 min. After the transfected cells were cultured for 24 hours, the transfection validity of miR-126 was verified by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). EPCs were treated with 100 ng/mL ox-LDL for 6 hours to simulate cell injury.

Proliferation analysis of EPCs

MTT was conducted to calculate the number of cell proliferation. EPCs proliferated in vitro were trypsinized with 0.25% trypsin and counted. Then, 200 μL EPCs (1 × 107-1 × 108/L) were uniformly inoculated onto 96-well gelatin-coated plates and cultured for 28 hours. Subsequently, each well was supplemented with 10 μL MTT, and the plates were further incubated at 37℃ with 5% CO2 for 4 hours. Afterward, the supernatant was discarded, and dimethyl sulfoxide was added (150 μL/well). Finally, the plates were oscillated for 5 min with an oscillator, and placed in a microplate reader to examine the Optical Density (OD) at the wavelengths of 490 nm.

EPC apoptosis

Apoptosis was examined with the annexin V-FITC/PI staining kit (BD Biosciences, New Jersey, USA). Stably transfected cells were washed with cold PBS, re-suspended in binding buffer (100 mmol/L NaCl, 25 mmol/L CaCl2, 100 mmol/L HEPES, pH 7.4) and stained with V-FITC/PI at RT for 15 min in the dark. Finally, FACS flow cytometry (BD FACS Calibur, Becton, Dickinson and Company Biosciences, San Jose, USA) was employed to assess the apoptosis rate. Each experiment was repeated three times.

EPC migration

The migration of EPCs was tested by Boyden transwell assay. First, EPCs were trypsinized with trypsin/EDTA. Then, EPCs were seeded in the upper chamber of 24-well plates with polycarbonate membranes (4 × 104/well) and cultured in the serum-free endothelial growth medium, while the medium containing VEGF (50 ng/mL) was placed in the lower chamber. After 24 hours of culture, the membranes were washed with PBS, and then fixed with 4% paraformaldehyde. The upper side of the membranes was gently wiped with cotton balls, and the membranes were then stained with hematoxylin. The migration of EPCs was calculated by counting cells in each well under the microscope.

EPC adhesion assay

The in vitro proliferated EPCs were trypsinized and counted. Then, 2 mL suspension of EPCs (1 × 107/L) was inoculated in six-well plates and incubated for 48 hours. The same number of EPCs were trypsinized and inoculated to the gelatin-coated cover glass, and then incubated at 37℃ with 5% CO₂ for 30 min. The nonadherent cells were washed with PBS, and the adherent cells were counted at random from 3 microscope fields (20 ×), and the mean number was obtained.

Detection of EPC colony formation

The cells that were not adherent within 48 hours were inoculated in hybridoma dishes at a density of 107/mL. After further culture for a week, EPC-CFUs with typical morphology in each hybridoma dish were counted under a microscope by two people. Namely, a cluster of cells was centered around, and free round cells, spindle cells and needle-like cells around were chosen. Each group was counted twice and the mean was taken. The primary EPC-CFUs from the bone marrow of each mouse was treated with 0.25% trypsin and dispersed, and then inoculated in gelatin-coated six- well plates with 103-104/mL. The number of EPC-CFUs (9 wells/ group) within a week was counted, and the colony unit size and cell density were observed.

In vitro angiogenesis analysis

In vitro angiogenesis kit was used to test the angiogenesis of EPCs. The ECMatrixTM glue solution and ECM 10 × diluent was placed in a refrigerator at 4℃ overnight for freezing-thawing. Then, 100 μL ECM 10 × diluent was added to every 900 μL ECMatrixTM solution and mixed. Afterward, the mixture was added to 96-well plates (50 μL/well) and incubated at 37℃ for 1 hour for gelatinization. EPCs were obtained by trypsinizing the adherent cells, and then they were re-suspended in the culture medium with the cell density adjusted to 5 104/mL. Subsequently, the EPCs were inoculated into ECMatrixTM glue (5000 ~ 10000/well). The canaliculization (angiogenesis) was observed under a 200-fold inverted microscope after 24 hours of culture at 37℃. Canaliculization was recognized when the cells were elongated and deformed to a length of more than 4 times the width. The total length of the vasculature was measured and calculated.

Dual-luciferase reporter assay

Luciferase report vectors (XIST-WT/XIST-MT; PLK2-WT/PLK2- MT) were constructed by GenePharma (Shanghai, China). EPCs were seeded in 48-well plates and cultured to 70% confluence. Then, XIST-WT/XIST-MT and PLK2-WT/PLK2-MT were co-transfected with miR-126 mimics of negative controls (miR-NC) into EPCs according to the Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection, the luciferase activity was validated as per the manufacturer's instructions. All experiments were done in triplicate.

RNA Immune-Precipitation (RIP)

First, 2 × 107 MPCs were collected, and the RIP lysates of the same volume were added. Then, the cells were centrifuged (12,000 rpm, with a diameter of 420 mm, 10 min), and the supernatant was obtained. Next, 900 μL RIP buffer (including RNase inhibitor, protease inhibitor, DNase) and 100 μL cell lysate was added to the EP tube containing magnetic beads, and the IgG antibodies or AgO₂antibodies were supplemented for incubation at 4℃ overnight. After that, the cells were centrifuged (12,000 rpm, with a diameter of 420 mm, 10 min), and the supernatant was removed. After washing with 500 μL RIP wash buffer for 6 times, RNA purification was carried out immediately. Finally, the purified RNA was dissolved in 15 μL DEPC water for quantitative RT-PCR.

RT-PCR

The total RNA in brain tissues and cells was extracted with Trizol (Tiangen, China) according to the instructions. After the RNA purity and concentration were determined, RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) was adopted for reverse transcription. The specific amplification primers of quantitative PCR were shown in Table 1. U6 was used as internal reference of miR-126, and GAPDH was for internal reference of XIST and PLK2. PCR was conducted according to the guidelines of the SYBR^® Premix Ex Taqand kit (Takara Biotech Corporation, Dalian, China) with Bio-Rad CFX96 (Bio-Rad, USA). Each sample was tested three times, and the results obtained were analyzed with the 2 -ΔΔCT method (Table 1).

Table 1: PCR primer sequences.

Statistical analysis

The data were expressed as mean ± standard deviation, statistical software Prism GraphPad 7.0 (GraphPadSoftware, USA) was adopted for statistical analysis, and differences between groups were compared by one-way ANOVA or t test. P<0.05 indicated significant difference.

Results

Knocking down lncRNA XIST significantly reduced the injury of LP(a)-mediated EPCs

PCR results confirmed that the XIST expression was distinctly up-regulated after EPCs were treated with 100 ng/mL LP (a) for 6 hours. Apoptosis was examined by flow cytometry and cell proliferation was detected by MTT. The results confirmed that after LP(a) treatment, EPC apoptosis was increased and the cell proliferation was inhibited. More importantly, knocking down XIST significantly reduced apoptosis and enhanced proliferation of EPCs. In addition, the results of migration, adhesion assay and colony formation experiment showed that the migration, adhesion and colony formation of EPC cells were attenuated after LP(a) treatment, while knocking down XIST exerted the reversed effects. Moreover, the angiogenesis experiment manifested that after LP(a) treatment, the angiogenesis of EPCs decreased, while knocking down XIST facilitated the angiogenesis. The above results demonstrated that knocking down lncRNA XIST notably reduced the injury of the LP(a)-mediated EPCs (Figure 1).

Figure 1: Knocking down lncRNA XIST significantly reduced the injury of LP (a)-mediated EPCs. A: PCR was carried out to test the effect of knocking down XIST in EPCs; B-C: EPCs were treated with LP(a) at a dose of 100 ng/mL for 6 hours, and EPC apoptosis was verified by flow cytometry (B), while cell proliferation was examined by MTT (C); D: Transwell assay was performed to determine cell migration. E: Cell adhesion assay was conducted to monitor the changes in cell adhesion. F: Colony formation assay was implemented to examine the colony formation of EPCs; G: Angiogenesis assay detected the angiogenesis of EPC ^*p＜0.05, ^** p＜0.01, ^*** p＜0.001. N=3.

Overexpressing miR-126 mitigated the injury of LP(a)- mediated EPCs

EPCs overexpressing miR-126 were induced, and EPCs were treated with LP(a) at a dose of 100 ng/mL for 6 hours. Then, EPC apoptosis was verified by flow cytometry, and cell proliferation was determined by MTT. As a result, overexpressing miR-126 dampened apoptosis and enhanced proliferation of EPCs. Besides, the results of migration assay, adhesion assay and colony formation experiment indicated that the migration, adhesion and colony formation of EPCs were all weakened after LP(a) treatment, while they were all elevated after overexpressing miR-126. Furthermore, the angiogenesis experiment proved that the angiogenesis of EPCs decreased after LP(a) treatment, while overexpressing miR-126 notably improved EPC angiogenesis. These results demonstrated that overexpressing miR-126 mitigated the injury of LP(a)-treated EPCs (Figure 2).

Figure 2: Overexpressing miR-126 notably mitigated the injury of LP(a)-mediated EPCs. A: The effect of miR-126 overexpression in EPCs was determined by PCR; B-C: EPCs were treated with LP(a) at a dose of 100 ng/mL for 6 hours. Apoptosis was examined by flow cytometry (B), and cell proliferation was testified by MTT (C); D: Cell migration was monitored by Transwell assay. E: Adhesion assay was implemented to monitor the changes in cell adhesion. F: Colony formation experiment was carried out to verify the colony formation of EPCs; G: Angiogenesis assay was employed to determine EPC angiogenesis. ^*P<0.05, ^** P<0.01, ^*** P<0.001. N=3.

LncRNA XIST targeted miR-126

Moreover, we searched the targeted gene of XIST on the bioinformatics database Starbase (http://starbase.sysu.edu.cn/ index.php) to further clarify the association between XIST and miR- 126, and discovered that XIST targeted miR-126. Furthermore, the dual-luciferase reporter assay and RIP experiment were carried out to determine the targeted binding between XIST and miR-126.

Interestingly, miR-126 mimics weakened the luciferase activity of XIST-WT, while ha`d no effect on that of XIST-MUT (P<0.05). Meanwhile, RIP manifested that XIST was up-regulated in AgO2 after supplementing with miR-126 mimics (P<0.05). Meanwhile, miR-126 was attenuated in LP(a) treatment, and was enhanced in EPCs with XIST overexpression plasmids. The above results suggested that XIST targeted miR-126 (Figure 3).

Figure 3: LncRNA XIST targeted miR-126. A: The association between XIST and miR-126 was determined by the Starbase; B-C: Dual-luciferase reporter assay (B) and RIP experiment (C) confirmed the targeted relationship between XIST and miR-126-3p; D: PCR was employed to verify the changes of miR-126.^*p＜0.05, ^** p＜0.01, ^*** p＜0.001. N=3.

lncRNA XIST targeted miR-126 to regulate LP(a)-mediated EPC damage

XIST overexpression plasmids were supplemented in EPCs overexpressing miR-126 for investigating whether XIST in exosomes targeted miR-126 to regulate EPC function (P<0.05). The results of apoptosis and proliferation assay testified that overexpressing miR- 126 reduced the apoptosis, and promoted the proliferation of EPCs. However, XIST supplementation on this basis partially offset the regulatory effects of overexpressing miR-126 on EPC proliferation and apoptosis. Moreover, the results of migration, adhesion and colony formation experiments confirmed that overexpressing miR- 126 improved the migration, adhesion and colony formation of EPCs treated with LP(a), while supplementation of XIST on the basis of miR-126 overexpression abated the migration, adhesion and colony formation of EPCs. Furthermore, the angiogenesis experiment proved that supplementing XIST repressed the angiogenesis capacity of EPCs. These findings testified that XIST targeted miR-126, which regulated the EPC damage mediated by LP(a) (Figure 4).

Figure 4: lncRNA XIST targeted miR-126 to modulate the injury of LP(a)-treated EPCs. A: PCR was employed to verify the changes of miR-126 after supplementing XIST overexpressed plasmid in EPCs overexpressing miR-126; B-C: LP(a) at a dose of 100 ng/mL was applied to EPCs for 6 hours after overexpressing miR-126 and adding XIST overexpression plasmid. EPC apoptosis was monitored by flow cytometry (B), and cell proliferation was determined by MTT (C); D: Cell migration was detected by Transwell; E: Cell adhesion assay was employed to monitor the change of cell adhesion ability; F: Colony formation experiment was implemented to determine the colony formation of EPCs; G: angiogenesis assay was adopted to verify angiogenesis of EPC ^*p＜0.05, ^** p＜0.01, ^*** p＜0.001. N=3.

miR-126 targeted PLK2

Besides, we searched the targeted gene of miR-126 with the Starbase to further clarify the association between PLK2 and miR-126, and found that miR-126 targeted PLK2. Furthermore, dual-luciferase reporter assay and RIP were implemented to clarify the targeted relationship between PLK2 and miR-126. Interestingly, miR-126 mimics markedly dampened the luciferase activity of PLK2- WT, while had no effect on that of PLK2-MUT (P<0.05). RIP experiment proved that PLK2 expression was facilitated in AgO₂ after supplementing miR-126 mimics. These results confirmed that miR-126 targeted PLK2 (Figure 5).

Figure 5: miR-126 targeted PLK2. A: The Starbase was adopted to clarify the association between PLK2 and miR-126; B-C: Dual-luciferase reporter assay (B) and RIP experiment (C) were implemented to determine the targeting relationship between PLK2 and miR-126 ^*p＜0.05, ^** p＜0.01, ^*** p＜0.001. N=3.

miR-126 modulated the LP(a)-mediated EPC injury via targeting PLK2

Further clarify whether miR-126 in exosomes targeted PLK2 and regulated EPC function.The results showed that miR-126 mimics attenuated PLK2 expression (P<0.05). Meanwhile, the detection of cell apoptosis and proliferation revealed that overexpressing PLK2 notably increased the apoptosis and abated the proliferation of EPCs. Nevertheless, the supplementation of miR-126 mimics on this basis partially offset the regulatory effects of overexpressing PLK2 on EPC proliferation and apoptosis. In addition, the cell migration, adhesion and colony formation experiments confirmed that overexpressing PLK2 further reduced the LP(a)-treated EPC migration, adhesion and colony formation, which were all strengthened after supplementing miR-126 mimics on the basis of overexpressing PLK2. Furthermore, angiogenesis experiment proved that overexpressing PLK2 further declined the EPC angiogenesis, which was enhanced by miR-126 mimics. These results demonstrated that miR-126 targeted PLK2 and modulated the injury of LP(a)-treated EPCs (Figure 6).

Figure 6: miR-126 targeted PLK2 to regulate injury of LP(a)-mediated EPCs. A: Changes of PLK2 after supplementing miR-126 mimics in EPCs were monitored by RT-PCR and WB; B-C: LP(a) at a dose of 100 ng/mL was applied to EPCs overexpressing PLK2 for 6 hours after supplementing with miR-126 mimics. EPC apoptosis was verified by flow cytometry (B), and cell proliferation was determined by MTT (C); D: Cell migration was examined by Transwell; E: Cell adhesion assay was used to detect the change of cell adhesion; F: Colony formation assay was implemented to test the colony formation of EPCs; G: Angiogenesis assay was carried out to monitor the angiogenesis of EPCs. ^*p＜0.05, ^** p＜0.01, ^*** p＜0.001.N=3.

Discussion

AS is a complex chronic disease and an important cause of various secondary diseases [1,2]. LP(a) is considered to be a major risk for the occurrence and development of AS by promoting foam cell formation, EPC dysfunction, plaque formation, and inflammatory response [24,25]. Therefore, LP(a) was transfected in EPCs in this study to simulate EPC damage in vitro. The results indicated that XIST targets the miR-126-3p/PLK2 axis to regulate the LP(a)- mediated EPC damage.

XIST, as a common lncRNA, has been extensively studied in a variety of diseases. XIST was initially discovered to silence X chromosome genes via cis-acting [26]. Recent studies have suggested that the abnormal expression of XIST and its "ceRNA" effect as a miRNA sponge are responsible for the dysfunctions of many miRs, and plays a regulatory role in various neoplasms. Zhenzhang et al. reported that XIST is highly expressed in esophageal cancer, and inhibiting XIST significantly attenuates the proliferation, migration and invasion of TE-1 and SKGT-4 cells, and induces their apoptosis. The abnormal expression of XIST may be involved in the occurrence and development of esophageal cancer through regulating the JAK2/STAT3 signaling pathway and miR-494/ CDK6 axis [27]. Meanwhile, the carcinogenic effect of XIST has been demonstrated in thyroid cancer, colorectal cancer, laryngeal squamous cell carcinoma and other tumors [28-34]. In recent years, reports on XIST in other non-tumor diseases are also increasing. Tao et al. showed that XIST promotes myocardial infarction by targeting miR-130A-3p [35]. Consistent with the results reported by Tao et al., Xiaoyu et al. found that XIST expression in cartilage specimens of OA patients is distinctly up-regulated, which confirms that XIST regulates the proliferation and apoptosis of OA chondrocytes through the miR-211/CXCR4 axis [36]. Studies in spinal cord injury have revealed that XIST gene knockout limited the neuronal apoptosis in rats, and its protective effect is regulated by the phosphorylation of AKT through competitive binding to miR-494 [37]. Thus, it can be seen that XIST could strengthen cell apoptosis in inflammatory diseases, and knocking down XIST plays a protective role in injury. Above all, the role of XIST in EPC is scarcely reported. This study clarified that knocking down XIST significantly reduces LP(a)-induced EPC damage.

As a classical miR, miR-126 has been widely studied. Alexandru et al. found that miR-126 is up-regulated in plasma EVS and urine, and is positively correlated with tumor grade [38]. Studies in gastric cancer also have confirmed that miR-126 inhibits cell apoptosis and promotes its proliferation by down-regulating the PI3K pathway, such as IGF1R, INSR, PDK1 and AKT1 [39]. Meanwhile, the carcinogenic effect of miR-126 has been proved in glioblastoma, NSCLC, porcine granulosa cells, and other tumors [40-42]. There are vast studies on miR-126 in AS, and most scholars believe that miR-126 plays a protective role in ECs. For instance, Momoka et al. hold that IL-6 reduces the accumulation of miR-126 in EA.hy926 cells, thus increasing the expression of miR-126-targeted genes. Moreover, IL-6 enhances the adhesion of human mononuclear cell line THP-1, accompanied by an increase of intercellular adhesion molecule-1 level [43]. Wen-long et al. confirmed that high glucose reduces the endogenous miR-126 level in HUVECs and increases the expression of DNMT1, thus resulting in the dysfunction of HUVEC migration [44]. In addition,studies in cerebral ischemia have found that overexpression of miR-126 dampens the expression of pro-inflammatory cytokines and adhesion molecules after ischemic stroke, thus alleviating the destruction of blood-brain barrier [45]. Furthermore, miR-126 mimics down-regulate VCAM-1 in the bleeding area and ease the destruction of the blood-brain barrier after cerebral hemorrhage [46]. Thus, studies in both hemorrhagic/ischemic stroke and HUVECs show that miR-126 plays a positive role in protecting the function of HUVECs. The results of this study initially demonstrated the protective effect of miR-126 in LP(a)-mediated EPC damage.

Also, this study confirmed that miR-126 is negatively regulated by XIST. Although there have been vast studies on XIST, only Zhihua et al. reported that XIST regulates the IRS1/PI3K/Akt pathway as the ceRNA of miR-126 in glioma [47]. Consistent with previous reports, this study also proved that XIST gene knockout inhibits the survival, migration, invasion, anti-apoptosis and glucose metabolism of glioblastoma cells. Therefore, this study initially testified through compensation experiments in non-tumor diseases that XIST sponges miR-126 as a ceRNA, thus regulating the function of EPC, which provides a basis for in-depth exploration of the role and mechanism of XIST in EPCs.

By further exploring the downstream mechanism of the XIST/miR- 126 axis, we discovered PLK2 is the target of miR-126. PLK2 also was a cell cycle regulation gene that affects cell cycle progression, cell proliferation and individual bone development [48]. Meanwhile, PLK2 plays a carcinogenic role in multiple tumors, such as glioma, oral cancer and colorectal carcinomas [49-51]. In contrast, downregulating PLK2 inhibits synovial damage in KOA rats, promotes synovial cell apoptosis, dampens angiogenesis, cartilage damage, chondrocyte apoptosis and alleviates the inflammatory damage of synovial tissue and cartilage tissue in inflammatory diseases. PLK2 overexpression reverses the effects of miR-27a up-regulation on synovial angiogenesis and chondrocyte injury in KOA rats [52]. Ischemia/reperfusion (IR) up-regulates PLK2, but decreases the expression of miR-128. This study proved that PLK2 regulated by miR-128 induces apoptosis/death by activating the nuclear factor κB (NF-κB). MiR-128 and PLK2 are novel targets for the prevention of myocardial I/R or oxidative stress-mediated injury [53].

Conclusion

These studies have confirmed that PLK2 is significantly upregulated in injuries. The findings of this study were consistent with the previous researches. Namely, PLK2 is significantly upregulated in damaged EPCs, and overexpressing PLK2 further aggravates the damage of EPC, while compensation of miR-126 mimics alleviates the damage of EPC. In conclusion, our study testified that knocking down XIST ameliorates LP (a)-induced EPC damage. The specific mechanism is that XIST sponges miR-126 as a ceRNA to regulate the expression of PLK2, which provides a new way to explore the treatment of AS.

Authors Contribution

Conceived and designed the experiments: Xiaolei Zhang; Performed the experiments: Xiaolei Zhang, Guoying Xu, Shizhen Wang; Statistical analysis: Pei Chen, Shanshan Chen; Wrote the paper: Xiaolei Zhang. All authors read and approved the final manuscript.

Consent for Publication

Not applicable.

Competing Interests

The authors declare that they have no competing interests.

Ethics Statement

Our study was approved by the Ethics Review Board of Jiangsu College of Nursing.

Data Availability Statement

The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgements

Not applicable.

Funding

This work was supported by Huaian Natural Science Research Plan (No. HAB201844).

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