lncRNA MIR503HG Targets miR-191-5p/PLCD1 Axis and Negatively Modulates Apoptosis, Extracellular Matrix Disruption, and Inflammation in Abdominal Aortic Aneurysm

As the most prevalent subtype of aortic aneurysm, abdominal aortic aneurysm (AAA) features the apoptosis, extracellular matrix (ECM) disruption, and inflammation response of vascular smooth muscle cells (VSMCs). Noncoding RNAs (ncRNAs) are crucial factors in AAA progression, while the investigations have not been fully explained. miR-191-5p upregulation is found in aortic aneurysm. However, its role in AAA has not been addressed. This research purposed to excavate the possible and associated molecular axis of miR-191-5p in AAA. In our study, miR-191-5p level was detected to be high in the tissues from AAA patients in comparison with the control group. After miR-191-5p expression was enhanced, cell viability was repressed, cell apoptosis was boosted, and ECM disruption and the inflammation response were fortified. Furthermore, the relationship among MIR503HG, miR-191-5p, and phospholipase C delta 1 (PLCD1) in VSMCs was disclosed via mechanism assays. Decreased MIR503HG lacked the inhibition on miR-191-5p targeting PLCD1, resulting in downregulation of PLCD1, which facilitated the progression of AAA. Thus, targeting MIR503HG/miR-191-5p/PLCD1 pathway will provide an additional method for the cure of AAA patients.


Introduction
Focal dilatations of abdominal aorta with about 50% bigger than proximal normal segment or thickness more than 30 mm in diameter are defined as abdominal aortic aneurysm (AAA) [1,2]. According to our knowledge, AAA is life-threatening due to its high morbidity and mortality among adults, especially among the older [3][4][5]. Unfortunately, the regulatory mechanisms underlying AAA pathogenesis are not well-illustrated, and therefore, effective therapeutic targets are lacking for AAA treatment. Increasing evidence has supported that vascular smooth muscle cells (VSMCs) are able to secrete elastin, an essential element of collagen synthesis and AAA wall remodeling [6,7]. Therefore, the exploration of the cellular activities in VSMCs may be beneficial to the treatment of AAA.
lncRNA MIR503HG is a newly identified repressor in several cancers [21][22][23]. In this study, we also aimed to figure out whether MIR503HG was involved in the regulatory network of miR-191-5p in AAA.
Herein, AAA patients' tissues and VSMCs were acquired for evaluating gene expressions. Cell viability, cell apoptosis, extracellular matrix (ECM) disruption, and inflammation responses were tested to analyze the functional influence of miR-191-5p. Additionally, mechanism assays were designed for detecting relations between genes.

Patients and Cell
Lines. 43 AAA patients and controls were recruited for collecting AAA and normal aortic tissues which were resected and snap-frozen at -80°C in liquid nitrogen immediately. Clinical analysis was conducted with the ethical approval from the Research Ethics Committee of Henan Provincial People's Hospital. Informed consent was provided by all participants. Primary human aortic VSMCs were preserved in RPMI-1640 Glutamax with 1% antibiotic solution (10,000 U/mL streptomycin sulphate and 10,000 U/mL penicillin G) and 10% FBS. Additionally, HEK-293T cells were procured from Procell (Wuhan, China) and incubated in DMEM+10% FBS+1% penicillin/ streptomycin. The environment was kept at 37°C with 5% CO 2 .

Cell Transfection.
The transfection procedures were in line with previous study [25]. VSMCs were cultivated until they reached 60-70% confluence. Subsequently, cells (80,000-120,000) in a 6-well culture plates with serum-free DMEM were subjected to transfection with various plasmids at the concentration of 10 nM using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The miR-191-5p mimics and miR-NC, pcDNA3.1/PLCD1 (PLCD1), and empty vector were provided by GenePharma (Shanghai, China). The miR-191-5p inhibitor and miR-NC, as well as the shRNAs specific to MIR503HG (shMIR503HG), PLCD1 (shPLCD1), and control shRNA (shNC), were also synthesized by GenePharma for gene silencing. 48 h later, VSMCs were harvested. The efficiency was measured by qRT-PCR. 2.6. Flow Cytometry of Cell Apoptosis. VSMCs were transfected and collected to the 6-well plates (3 × 10 3 /well), followed by double-stained with Annexin V/PI Kit (BD Biosciences, San Jose, CA, USA). Apoptosis of VSMCs was analyzed by FACSCalibur flow cytometer (BD Biosciences). 2.13. Statistical Analyses. The data were shown as mean ± SD with at least three replications. Two-tailed Student's t-test or ANOVA by use of SPSS version 19.0 (SPSS, Chicago, IL, USA) was utilized for statistical analyses. A P value less than 0.05 was considered as the threshold value. Spearman's correlation analysis analyzed the correlation between every two genes.

miR-191-5p
Negatively Regulated the Apoptosis, ECM Degradation, and Inflammation in AAA. miR-191-5p is demonstrated to be elevated in aortic aneurysm, but its function is not explored in detail [20]. Therefore, we first detect the expression of miR-191-5p in clinical samples. As a result, miR-191-5p was significantly elevated in AAA tissues in comparison with controls ( Figure 1(a)). For confirming the participation of miR-191-5p during AAA development, we carried out gain-of-function experiments with VSMCs. In preparation, miR-191-5p expression was elevated in VSMCs ( Figure 1(b)). Then, we detected that when miR-191-5p was enhanced, cell viability and proliferation were dramatically reduced (Figures 1(c) and 1(d)). In addition, experimental results also demonstrated that cell apoptosis was induced under miR-191-5p increment (Figures 1(e)-1(g)). Subsequently, western blot affirmed that Ki67 and Bcl-2 proteins declined, but Total-caspase-3 protein was not changed, and the Bax and Cleaved-caspase-3 proteins were augmented, hinting that cell apoptosis was accelerated ( Figure 1(h)). The impact of miR-191-5p elevation on ECM degradation was then analyzed. From western blot results, augmented miR-191-5p expression enhanced MMP2 and MMP9 protein expressions (enzymes able to degrade various components of ECM proteins [26]) but lessened the expression level of their inhibitor TIMP-1. The protein levels of α-SMA, a marker of contractile VSMCs, were downregulated. The protein levels of OPN, a marker of synthetic VSMCs, were upregulated ( Figure 1(i)). Additionally, ELISA kit detected the augment of TNF-α and IL-6, two proinflammatory mediators (Figures 1(j) and 1(k)). Taken together, miR-191-5p elevation could cause the promotion of apoptosis, ECM degradation, and inflammation.
After conducting a series of assays, we confirmed that cell viability weakened by MIR503HG knockdown was facilitated by miR-191-5p silence or PLCD1 enhancement (Figures 5(b) and 5(c)), while cell apoptosis activated due to MIR503HG repression was impaired with miR-191-5p depletion or PLCD1 increment, as determined by flow   Mediators of Inflammation cytometry, TUNEL, and caspase-3 activity kit (Figures 5(d)-5(f)). We also detected that the reduction in Ki67 and Bcl-2 proteins and increase in Bax and Cleaved-caspase-3 proteins by MIR503HG silencing were abrogated by miR-191-5p inhibitor or PLCD1 ( Figure 5(g)). Additionally, ECM degradation and inflammation boosted by depletion of MIR503HG were offset by knockdown of miR-191-5p or addition of PLCD1. In the shMIR503HG group, protein levels of MMP2, MMP9, and OPN as well as TNF-α and IL-6 levels were augmented, and protein levels of TIMP-1 and α-SMA were lowered compared with the control group.

Discussion
The apoptosis and phenotypic shift of VSMCs and inflammation response are noted features of AAA. More apoptotic and synthetic VSMCs as well as induced inflammation are  Mediators of Inflammation observed in AAA progression. The apoptotic VSMCs result in the numerous loss of contractile cells and thus promoting the expansion of the abdominal aortic wall [27]. Furthermore, it has been revealed that inflammation could secrete MMP, which can further degrade the components of the ECM and cause ECM disruption. The TIMP family has been documented to strictly regulate MMP family members [28]. Also, a synthetic phenotype of VSMCs occurs due to the imbalance of synthetic and contractile phenotype. α-SMA is a biomarker of contractile VSMCs, and OPN is a biomarker of synthetic VSMCs. In this study, all these factors were tested to reflect the functions of genes in AAA.
Past studies have excavated that miR-191-5p was elevated in aortic aneurysm, but its function role in AAA has never been explored [19,20]. Hence, we attempted to explic-itly figure out the role of miR-191-5p in AAA. In this paper, elevated miR-191-5p expression was discovered in AAA tissues. In addition, miR-191-5p promotion repressed cell viability, enhanced cell apoptosis, and enhanced ECM disruption and inflammation in VSMCs. This was the first time that miR-191-5p was demonstrated as a contributor for AAA. From starBase, we obtained four lncRNAs targeting miR-191-5p. MIR503HG was the most possible lncRNA binding to miR-191-5p in VSMCs, which was downregulated in AAA tissues and had negative regulation on miR-191-5p. In consistent with the antioncogenic role of MIR503HG played in tumors [21][22][23], the role of MIR503HG in VSMCs was proven to be suppressive. Then, we continued to probe into the downstream factor that MIR503HG/miR-191-5p axis targeted in VSMCs. Among 13 Mediators of Inflammation the thirteen targets gained from the Venn diagram, pPLCD1 was screened out for its significantly declined luciferase activity under the improvement of miR-191-5p. PLCD1 was previously disclosed to inhibit diseases, airway smooth muscle hypertrophy, colorectal cancer, and breast cancer contained [29][30][31]. PLCD1 as a target of miR-191-5p was affirmed through mechanism experiments. PLCD1 also exerted suppressive function in VSMCs. The functions of miR-191-5p/PLCD1 axis and MIR503HG/miR-191-5p/ PLCD1 axis in VSMCs were verified via rescue assays.
Totally, in AAA progression, the sponge of miR-191-5p by MIR503HG was weakened, and therefore, PLCD1 expression was decreased, resulting in weakened viability, facilitated apoptosis, and enhanced ECM disruption and inflammation. Addition of MIR503HG might be a novel therapeutic strategy for patients with AAA.

Data Availability
The data used to support the research findings are included within this article.