LncRNA TP73-AS1 promotes oxidized low-density lipoprotein-induced apoptosis of endothelial cells in atherosclerosis by targeting the miR-654-3p/AKT3 axis

Although lncRNA TP73-AS1 has been shown to play important roles in various human diseases, its function in atherosclerosis (AS) remains unclear. Human aortic endothelial cells (HAECs) were treated with 50 μg/ml oxidized low-density lipoprotein (ox-LDL) to establish an atherosclerotic cell model. The expression of TP73-AS1, miR-654-3p and AKT3 was detected by qRT-PCR. Cell functions were evaluated CCK-8 assay and flow cytometry. The protein levels of apoptosis-related proteins were evaluated by western blot. The binding relationship among TP73-AS1, miR-654-3p and AKT3 was determined by bioinformatics analysis and luciferase reporter assay. TP73-AS1 was upregulated and miR-654-3p was downregulated in ox-LDL treated HAECs. TP73-AS1 silencing and miR-654-3p mimics decreased the viability and inhibited apoptosis of ox-LDL treated HAECs, decreased the expression levels of c-caspase-9, c-caspase-3 and Bax, and increased Bcl-2 expression. In addition, miR-654-3p inhibitor significantly reversed the inhibitory effects of si-TP73-AS1 on cell viability and apoptosis. TP73-AS1 could positively regulate AKT3 through directly sponging miR-654-3p. TP73-AS1 promoted apoptosis of ox-LDL stimulated endothelial cells by targeting the miR-654-3p/AKT3 axis, suggesting that TP73-AS1 might be a potential target for AS treatment.

endothelial cells' (HAECs) growth and apoptosis may contribute to developing novel therapies to prevent AS development. Long noncoding RNAs (lncRNAs) with a length of approximately 200 nucleotides play essential regulatory functions in AS progression [4]. For example, H19 expression is upregulated during AS development, and H19 inhibition effectively enhances proliferation and inhibits apoptosis of HAECs [5]. NEAT1 knockdown suppresses the proliferation and promotes apoptosis of HAECs [6]. TP73-AS1, a newly identified lncRNA involved in development of various tumors, is highly expressed in ovarian cancer cell lines, and TP73-AS1 downregulation inhibits proliferation, invasion, and migration of the ovarian cancer cell line SKOV3 in vitro [7]. In addition, TP73-AS1 is upregulated in several types of human cancer, and TP73-AS1 knockdown effectively attenuates the progression of ovarian cancer [7], hepatocellular carcinoma [8], and bladder cancer [9]. Based on the crucial roles of TP73-AS1 in human cancers, it might have a potential function in other diseases, such as AS. Thus, this study aimed to explore its potential functions in AS.
MicroRNAs (miRNAs) are small noncoding RNAs (ncRNAs) with a length of 22 nt, which regulate gene expression at the post-transcriptional level [10]. MiRNAs could control a series of AS pathological processes, including lipoprotein metabolism, endothelial cell growth and apoptosis, as well as immune responses [11]. For example, miR-654-3p suppresses tumor proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma [12], colon cancer [13], and gastric cancer [14]. MiR-654-3p was closely involved in the inflammatory responses through modulating RAB22A [15] in AS, indicating a potential role of miR-654-3p in AS.
AKT serine/threonine kinase 3 (AKT3) plays essential functions in many cellular processes, including cell growth, differentiation, proliferation, and apoptosis [16]. AKT3 promotes apoptosis of different types of tumor cells such as gastric cancer [17], endometrial carcinoma [18] and breast cancer [19]. AKT3 overexpression promotes the progression of esophageal squamous cell carcinoma and downregulates the expression level of apoptosisrelated proteins including c-caspase-3, c-caspase-9, Bax and Bcl-2 [20]. AKT3 is highly expressed during AS progression and AKT3 kinase inhibits the pinocytosis of oxidized low-density lipoprotein (ox-LDL) in macrophages [21]. These studies indicate that targeting AKT3 might be a potential treatment strategy for preventing human diseases, including AS.
This study established an atherosclerotic cell model using 50 μg/ml ox-LDL treatment as previously reported [22][23][24] and found that TP73-AS1 was highly expressed in 50 μg/ml ox-LDL treated HAECs. In addition, TP73-AS1 knockdown and miR-654-3p overexpression effectively enhanced viability and inhibited apoptosis of ox-LDL treated HAECs. Furthermore, our results revealed a new regulatory network involved in AS development, i.e. silencing TP73-3P attenuated AS progression via inhibiting aortic endothelial cell apoptosis by targeting the miR-654-3p/AKT3 axis. We believe that our results provided a potential target of AS treatment.

Cell culture
Human aortic endothelial cells (HAECs) were purchased from Platts Life Technology Co., Ltd. (Wuhan, China), and cultured in endothelial cell culture medium (Gibco, USA) supplemented with endothelial cell growth factor and 5% FBS at 37 °C with 5% CO 2 . When needed, 50 μg/ml ox-LDL was added for 24 h.

Western blot
Total protein was extracted using RIPA buffer. Approximately equal amounts of proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad

Apoptosis analysis
Cells apoptosis was examined by an Annexin V FITC/PI apoptosis detection kit purchased from Vazyme Biotech Co., Ltd. Briefly, HAECs were re-suspended in 1 × binding buffer (100 µl), and incubated with 5 µl of PI and 5 µl of Annexin V FITC in darkness for 10-20 min. The apoptotic rate was detected using flow cytometry (Jiyuan, Guangzhou, China).

Luciferase activity assay
Luciferase reporter assay was performed as previously reported [25]. TP73-AS1 wildtype (WT) or mutant (MUT) or AKT3 containing the putative miR-654-3p binding site was synthesized and cloned into luciferase reporter pGL3 Basic vector (Promega). MiR-654-3p mimics or miR-NC was co-transfected with recombinant luciferase reporter plasmids into HAECs using Lipofectamine 2000. After 48 h for transfection, the relative luciferase activity was detected.

Statistical analysis
Data were presented as the mean ± standard deviation (SD). Differences were determined using Student's t-test (between two groups) or one-way analysis of variance (ANOVA) (among multiple groups). p < 0.05 was considered as the significant threshold.

Discussion
In the last decades, the chronic inflammatory disease AS has become a global clinical problem [26]. Therefore, there is an urgent need to explore the specific pathogenic mechanisms in AS progression to help develop effective therapeutic targets and relieve patients' pain. Here, we found that TP73-AS1 was upregulated in ox-LDL treated HAECs. Our results revealed a complete mechanism of TP73-AS1 in AS development. Specially, TP73-AS1 promoted apoptosis of ox-LDL treated HAECs by upregulating AKT3 expression via directly sponging miR-654-3p.
Recently, a number of lncRNAs have been identified to be closely involved in cell proliferation, apoptosis, and the immune response during AS occurrence and might be considered as potential diagnostic and therapeutic biomarkers. For example, FA2H-2 knockdown activated inflammation and inhibited autophagy flux, leading to aggravation of ox-LDL induced inflammatory responses in human aorta vascular smooth muscle cells [27]. MALAT1 was robustly expressed in the macrophages of rats with diabetic atherosclerosis, and low-dose sinapic acid abated the pyroptosis of macrophages through inhibiting MALAT1 expression [28]. TUG1 was upregulated during AS, and its knockdown significantly inhibited the inflammatory response and attenuated atherosclerotic lesions in a mouse model [29]. MALAT1 is downregulated in ox-LDL treated vascular endothelial cells, and its downregulation promoted dendritic cell maturation in AS development [30]. lncRNA-ATB promoted viability, migration and angiogenesis of human microvascular endothelial cells, and might be considered as a potential biomarker for AS [31]. Although many lncRNAs have been revealed to be associated with AS development, more effective and specific molecular targets are still necessary. In this study, we found that TP73-AS1 downregulation effectively inhibited AS progression in vitro, suggesting that TP73-AS1 might be another potential target for AS treatment. LncRNAs always function as sponges of miRNAs to regulate biological processes in the development of human cardiovascular diseases [32,33]. Silencing H19 attenuated the inflammatory response by sponging miR-130b in ox-LDL-treated Raw264.7 cells [34]. LEF1-AS1 modulated the proliferation of vascular smooth muscle cells through targeting miR-544a [35]. MALAT1 suppression protected endothelium against ox-LDL induced inflammation by inhibiting miR-181b expression [36]. LncRNA ZFAS1 enhanced inflammatory responses in AS by directly sponging miR-654-3p to upregulate ADAM10 and RAB22A [15]. Hence, we hypothesized that TP73-AS1 might directly sponge miR-654-3p to affect AS progression. Both luciferase reporter and RIP assays confirmed their relationship. Rescue experiments demonstrated that co-transfection of miR-654-3p inhibitor and si-TP73-AS1 attenuated si-TP73-AS1 induced inhibition on cell apoptosis. These data indicated that the effect of TP73-AS1 in AS was partially mediated by miR-654-3p.
Previous studies demonstrated that AKT3 could promote tumor development in different human cancers [37]. Importantly, AKT3 was also highly expressed in AS, and loss of AKT3 led to inhibition of AS development [38]. Consistent with these reports, our results confirmed that AKT3 was significantly upregulated in ox-LDL treated HAECs. Interestingly, a previous study reported AKT3 as a target of miR-654-3p, and the miR-654-3p/AKT3 axis was closely involved in the proliferation and invasion of ovarian cancer cells [39]. In the present study, rescue experiments confirmed the role of the miR-654-3p/AKT3 axis in AS.
High-fat diet-induced animal models have been a widely used strategy to determine the function of lncRNAs/miRNAs in AS development [22,40]. The impact of TP73-AS1 in AS progression should be confirmed using these models in the future.

Conclusion
In summary, we investigated the role of TP73-AS1 in the apoptosis of HAECs and revealed that TP73-AS1 downregulation effectively inhibited apoptosis of ox-LDL treated HAECs through regulating the miR-654-3p/AKT3 axis. Our study suggested that TP73-AS1 might be a potential target for AS treatment.