Nucleolin regulates the proliferation of vascular smooth muscle cells in atherosclerotic via Aurora B

Vascular smooth muscle cells (VSMCs) play a significant role in atherosclerosis. As a multifunctional protein, nucleolin (NCL) is involved in many important physiological and pathological processes. In this study, we aimed to investigate the role of nucleolin in VSMCs proliferation and cell cycle. The expression of nucleolin increased in VSMCs of mice with aortas advanced plaques. With the left common carotid‐artery ligation‐injury model, immunofluorescence staining revealed that nucleolin and Ki67 expression increased in VSMCs in mice left carotid artery compared with right carotid artery after surgery. POVPC or ox‐LDL up‐regulated nucleolin mRNA and protein expression in a dose‐ and time‐dependent manner in HAVSMCs. POVPC (5μg/ml) or ox‐LDL (50μg/ml) promoted the proliferation of HAVSMCs. Nucleolin ablation relieved the pro‐proliferation role of VSMCs. The cell cycle assay and cell ability results showing that POVPC or ox‐LDL increased the proliferation, but nucleolin ablation inhibited the proliferation of HAVSMCs. And nucleolin ablation can prevent DNA replication at S phase and induce cell cycle arrest in S phase. The bioinformatics database predicts protein‐protein interactions with nucleolin and aurora B. Nucleolin overexpression and ablation affected the expression of aurora B. These findings indicate for the first time that nucleolin actively involved the proliferation of VSMCs via aurora B.

response and microRNA processing. [3][4][5] Latest research found that nucleolin plays mixed roles in tumour growth, virus infection and angio-genesis3. So that nucleolin also a possible target in the development of anti-tumour and anti-viral strategies. 6,7 However, the potential role of nucleolin in VSMCs proliferation and cell cycle is unclear.
Aurora B kinase, a member of the aurora kinase family, which is highly expressed in many cancers, also plays a crucial role in the mitosis. 8 Aurora B is dynamically distributed during mitosis that the peak of expression level is located in the G2-M phase. 9 The nuclear protein Ki67 is expressed in all phases of the cell cycle, but its expression level is strongly up-regulated in the G1 phase, S phase and G2 phase. 10 Ki67 is widely used in pathological as a cell proliferation markers. Aurora B is a member of the Aurora kinases family, as an important kinase to adjust the cell normal mitosis, Studies have found that it is Involved in the normal proliferation of vascular smooth muscle cells. 11 In this study, we aim to investigate the relationship between nucleolin and Aurora B in atherosclerosis plaque and cell lines and elucidate the potential molecular mechanisms of atherosclerotic smooth muscle cells and ameliorate the therapeutic strategies for the treatment of atherosclerosis.

| Animals and treatment
Male ApoE−/-mice on a C57BL/6 background were procured from the Animal Center of Central South University (Changsha, China).
All animal experiments were conducted in accordance with the Guidelines of Animal Experimentation, Medical Ethics Committee of Xiangya Hospital, Central South University (No.201402027).
Twenty 6-to 8-week-old Apo E-/-mice were haphazardly divided into two groups: the control group and the HFD group. The HFD group mice (n = 10 per group) were fed for 12 weeks on a high-fat diet (HFD), which containing 21% fat and 0.15% cholesterol, the control group mice were fed a normal diet.
Eight-week-old C57BL/6 mice (n = 10) underwent surgical operation to induce carotid artery plaque formation, with a collar placed around the left carotid artery and the right carotid as control. After the surgical operation, mice were fed a high-fat diet and killed 12 weeks later. Then take samples of the left and right carotid arteries.
The mice were killed with isoflurane at the end of the experiment. The aorta and carotid artery were immobilized in 4% paraformaldehyde, and paraffin sections were prepared for haematoxylin-eosin (HE) staining, immunohistochemical analysis and Immunofluorescence analysis.

| Cell culture
The human aortic smooth muscle cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences.

| Cell transfection experiments
After the vascular smooth muscle cells reached 70%-80% confluence, that were transfected with pcDNA3.

| RNA isolation and quantitative Real-Time PCR analysis
Total RNA was isolated from vascular smooth muscle cells using

| Western blotting, and cell viability
Western blotting and cell viability measurements were performed as previously specified. 12,13 Anti-Nucleolin rabbit antibody was obtained from Sigma (1:1000, N2661), anti-Aurora B rabbit antibody

| Cell cycle analysis
HAVSMCs were cultured in DMEM (Thermo Fisher Scientific) containing 10% FBS overnight and then were treated with ox-LDL or POVPC. HAVSMCs were exposed to 25, 50 or 100 µg/mL concentrations of ox-LDL or were exposed to 2.5, 5.0, 10 or 20 µg/mL concentrations of POVPC. And according to the nucleolin protein expression, we choose the 50 µg/mL concentrations of ox-LDL and 5 µg/mL concentrations of POVPC for the next experiment. Cell suspensions corresponding to 2 × 10 5 to 1 × 10 6 cells were collected. The cells were pelleted by centrifugation. Fixed with 70% ethanol, and propidium iodide (Thermo Fisher Scientific) staining for the cell cycle analysed by flow cytometry. All tests were repeated at least 3 times.

| EdU assay
VSMCs were seeded in a 96-well plate and were cultured overnight

| Statistical analysis
All data were expressed as mean ± SEM. Significant differences were analysed using the Student's t test or one-way analysis of variance (ANOVA). A value of P < .05 indicated a statistically significant difference.

| Nucleolin is up-regulated during atherosclerosis development
Nucleolin plays a very important role in cell proliferation; however, its specific role in atherosclerosis is largely unknown. Therefore, we first harvested the normal and high-fat diet feed mice artery and analysed the vascular expression patterns of nucleolin. We observed the expression of nucleolin in the artery by immunohistochemistry. Immunohistochemical assay demonstrated that the protein levels of nucleolin increased in atherosclerosis compared to the sham group ( Figure 1A). As shown in Figure 1A, the expression of nucleolin in arterial plaque of mice from HFD group was higher than that in control group (1.825 ± 0.287 vs. 39.86 ± 1.429, P < .01, n = 3). By double immunofluorescence staining for nucleolin and SMA, we examined nucleolin expression in the artery.
We found that nucleolin was expressed in vascular smooth muscle cells of the thickened intima. Compared with the control group, the expression of nucleolin in the HFD group was significantly increased.
We also checked the result in another model that underwent surgical interventions to induce carotid plaque formation, and a collar around the left carotid artery and the right carotid as control.
Furthermore, immunofluorescence staining revealed that nucleolin expression highly increased in VSMCs in the area of intimal thickening and in the medial layer in mice left carotid artery (PCCP) compared with right carotid artery after surgery. Moreover, we checked Ki67 expression by immunohistochemistry in the control group and in the PCCP group. Immunohistochemical assay demonstrated that the protein levels of Ki67 were increased in PCCP group compared with sham group. These data suggest that nucleolin plays a key role in vascular smooth muscle cells of atherosclerosis.

| POVPC or ox-LDL up-regulated the mRNA and protein expression of nucleolin in HAVSMCs
Atherosclerosis is an arterial disease associated with dyslipidemia and

| POVPC or Ox-LDL induced the cell cycle changes in vascular smooth muscle cells
To elucidate the effect of nucleolin on cell cycle regulation, flow cytometry was used to evaluate the cell cycle distribution of ox-LDL-induced or POVPC-induced VSMCs. As shown in Figure 3A assay. We found that the two groups, as 5.0 µg/mL POVPC group and 50 µg/mL ox-LDL group have the best cell ability ( Figure 3C-D). The results of the EdU assay showed that POVPC or ox-LDL treatment increased VSMCs proliferation ( Figure 3E). As shown in Figure 3F-G, the expression of Ki67 and PCNA in vascular smooth muscle cells treated with POVPC or ox-LDL was higher than that of control group.
Our data indicated that POVPC or ox-LDL treatment significantly increased vascular smooth muscle cell viability.

| The cell proliferation ability changed after interference with the expression of nucleolin
Several studies have found that nucleolin played a significant role in cell was confirmed by Western blotting as shown in Figure 4B. We observed a significant reduction in its expression (P < .01) compared with the cells transfected with control siRNA(siNC).
Then, we used the CCK-8 assay to measure the proliferation of VSMCs. These data show that nucleolin overexpression obviously increased cell viability ( Figure 4C). When knock-down of nucleolin drastically reduced the proliferation of VSMCs ( Figure 4D). The results showed that nucleolin played a vital role in the proliferation of vascular smooth muscle cells.

| Nucleolin overexpression or down expression change the cell cycle progression of VSMCs
We  Figure 5F).

| Aurora B is a direct target of nucleolin in VSMCs
To P-values were determined using the two-tailed Student's t test for comparing two groups and one-way ANOVA for comparing multiple groups analysis. The expression of aurora B in vascular smooth muscle cells treated with POVPC or ox-LDL was lower than the control group in Figure 6B (P < .05). Furthermore, we also analysed the aurora B expression in VSMCs by nucleolin overexpression or nucleolin ablation. VSMCs transfected with pcDNA3.1-NCL plasmid significantly reduced aurora B, in comparison with nontransfected VSMCs or cells transfected with pcDNA3.1(P < .01).
In parallel, nucleolin ablation by siRNA-nucleolin (siNCL) exhibited considerably higher levels of aurora B expression in VSMCs (P < .01) ( Figure 6D). We used the protein immunoprecipitation method to observe the interaction between nucleolin and aurora B in vascular smooth muscle cells under physiological conditions.
As shown in Figure 6E, aurora B interacts with nucleolin under physiological conditions. It is suggested that nucleolin may interact with aurora B to regulate the cycle and proliferation of vascular smooth muscle (Figure 7).

| D ISCUSS I ON
Atherosclerosis is a chronic vascular inflammatory disease charac- It is gradually recognized that ox-LDL plays a vital role in promoting the occurrence, progression and plaque destabilization of atherosclerosis. 15 Multiple studies have designated that ox-LDL can stimulate the VSMCs proliferation. 16,17 Therefore, the prolif- is the basic cytopathological basis for its occurrence and development. Therefore, we believe that nucleolin is pro-atherogenic in F I G U R E 7 Schematic summary of the findings. POVPC or ox-LDL up-regulated nucleolin mRNA and protein expression in HAVSMCs, and then the cell cycle changes. Nucleolin may interact with aurora B to regulate the cell cycle and proliferation of vascular smooth muscle cells VSMCs of atherosclerosis. We know that the occurrence and development of diseases are regulated by many aspects, and atherosclerosis is not only involved in vascular smooth muscle cells. Moreover, it is not clear whether the expression of nucleolin changes at different stages of atherosclerosis, and its potential role and mechanism in the development of atherosclerosis are still unclear.
CDKs and Cyclins are positive regulators that are directly involved in cell proliferation. 22 We notice the Aurora kinases B, 8,9 that is a member of the silk-threonine kinase family, and its coding gene is located in human chromosome 17p13.1, which possesses a conserved catalytic domain and a N-terminal domain. In human cells, Aurora B is mainly nuclear, and it has a function to modulate cell proliferation. Aurora B is dynamically distributed during mitosis and the peak of expression level is located in the G2-M phase. 23 We have found through bioinformatics that Aurora B and nucleolin may have a protein interaction with each other. It was found that when the expression of nucleolin was increased, the Aurora B content decreased compared with the control group. When the expression of nucleolin decreased, Aurora B expression increased significantly. We found that there was an interaction between nucleolin and Aurora B through the protein immunoprecipitation experiments. Therefore, In our study, it was demonstrated that the expressions of nucleolin in atherosclerosis plaque and cell lines were up-regulated compared with that in normal. Restored expression of nucleolin inhibited the cell cycle of cells in vitro. In addition, we also found that Aurora B is a direct target of nucleolin. Therefore, the present result highlighted the importance of nucleolin as a vascular smooth cells proliferation by targeting Aurora B in atherosclerosis.