Sustained expression of MCP‐1 induced low wall shear stress loading in conjunction with turbulent flow on endothelial cells of intracranial aneurysm

Shear stress was reported to regulate the expression of AC007362, but its underlying mechanisms remain to be explored. In this study, to isolate endothelial cells of blood vessels, unruptured and ruptured intracranial aneurysm (IA) tissues were collected from IA patients. Subsequently, quantitative real‐time PCR (qRT‐PCR), Western blot and luciferase assay were performed to investigate the relationships between AC007362, miRNAs‐493 and monocyte chemoattractant protein‐1 (MCP‐1) in human umbilical vein endothelial cells (HUVECs) exposed to shear stress. Reduced representation bisulphite sequencing (RRBS) was performed to assess the level of DNA methylation in AC007362 promoter. Accordingly, AC007362 and MCP‐1 were significantly up‐regulated while miR‐493 was significantly down‐regulated in HUVECs exposed to shear stress. AC007362 could suppress the miR‐493 expression and elevate the MCP‐1 expression, and miR‐493 was shown to respectively target AC007362 and MCP‐1. Moreover, shear stress in HUVECs led to the down‐regulated DNA methyltransferase 1 (DNMT1), as well as the decreased DNA methylation level of AC007362 promoter. Similar results were also observed in ruptured IA tissues when compared with unruptured IA tissues. In conclusion, this study presented a deep insight into the operation of the regulatory network of AC007362, miR‐493 and MCP‐1 upon shear stress. Under shear stress, the expression of AC007362 was enhanced by the inhibited promoter DNA methylation, while the expression of MCP‐1 was enhanced by sponging the expression of miR‐493.


| INTRODUC TI ON
Intracranial aneurysm (IA) is a type of cerebrovascular disorder featured by intracranial artery dilatation induced by endothelial layer defects. 1,2 The rupture of an IA leads to subarachnoid haemorrhage (SAH), a disease featured by high mortality as well as significant disability. The global incidence of IA is about 3%, although most patients with IA will not experience IA rupture during their life time. [3][4][5] Correspondingly, it is of a significant value to accurately predict the chance of IA rupture. Haemodynamic characteristics play an essential role in the prognosis of IA. In particular, the shear stress applied to an IA is a key factor determining the physiological features of the arteries in the IA. 6 In addition, the degeneration of IA wall starts from the apical surface of the IA, which is also the most common location of IA rupture. Moreover, the value of wall shear stress (WSS) is the minimum near the apex point of an IA. 7 Furthermore, an IA with a narrow neck is more likely to generate a slow flow around the fundus region accompanied with a low and fluctuating WSS vector, which in turn can significantly change shear rate while accelerating the degeneration of IA wall. 8,9 A reduce blood flow velocity tends to cause thrombus while further expanding the dome of the IA. 10,11 Furthermore, a ruptured IA also tends to have a larger area of the aneurysm exposed to low WSS as compared to an unruptured IA. 12 Long non-coding RNAs (lncRNAs) are a class of non-coding RNA with >200 nucleotides in size. [13][14][15] In recent years, lncRNAs have been implicated in various cellular processes such as apoptosis, proliferation, invasion, and inflammation, which contributes to the pathogenesis of many disorders. [16][17][18] Furthermore, emerging evidence suggests that lncRNAs play a critical role in the development of cardiovascular diseases. [19][20][21] MCP-1 is an inflammatory chemokine constitutively expressed by microglia, neurons, astrocytes, and microvascular endothelial cells. The expression of MCP-1 is up-regulated in traumatic brain damage, cerebral infarcts, as well as in many cognitive disorders. [22][23][24][25] It has been suggested that most of the MCP-1 content in the body is synthesized by astrocytes. Upon binding to its receptor, MCP-1 expressed in microglia can trigger their activation while promoting neuro-inflammation, which in turn leads to cognitive disorders. 25 In patients with IA, MCP-1 can recruit and subsequently activate macrophages to attack blood vessel wall. 26 Then, these macrophages will secret various pathological factors including proteinases as well as cytokines. 27,28 The role of MCP-1 in IA pathogenesis has also been demonstrated in mice with MCP-1 knock-out. 27,29 It has been shown that AC007362 is deregulated upon shear stress and miR-493 is a competing endogenous RNA of AC007362. 30,31 Furthermore, MCP-1 is a direct target gene of miR-493. In addition, shear stress may suppress the methylation of promoters of many genes. 32 Therefore, we hypothesized that shear stress may promote the expression of AC007362 by suppressing its methylation while enhancing the expression of MCP-1 by sponging the expression of miR-493. To test this hypothesis, we set up a cellular model of shear stress and studied the effect of shear stress on the expression of genes involved in the signalling pathway of AC007362/miR-493/MCP-1. We also collected tissue samples from patients with ruptured small IA and unruptured big IA to compare their expression of AC007362/miR-493/MCP-1.

| Human subjects and sample collection
A total of 40 IA patients were recruited in our study. Then, the IA tissues from each patient were collected to isolate endothelial cells of blood vessels. Endothelial cells were isolated by utilizing a previously described protocol. 33 In addition, depending on where the IA in each patient was ruptured or not, these patients were divided into two groups, that is a group of unruptured IA, in which the maximal diameter of IA is >4 cm, and a group of ruptured IA. In the next step, the demographic and clinicopathological characteristics of all participants, such as their age, sex, current status and past history of smoking, and comorbidities (such as hypertension, heart disease, high cholesterol, history of stroke, diabetes and osteoarthritis), were summarized and compared between the two groups using

| Exposure of cultured endothelial cells to shear stress
The endothelial cells isolated from the blood vessels of IA tissues collected from the patients in both groups of unruptured IA and ruptured IA were seeded into tissue culture treated Petri dishes with a 0.1% gelatin surface coating. After the cells were 70%-80% confluent, a flow apparatus (BD Bioscience) was employed to apply shear stress on the cells in accordance with the following protocol: On the day prior to the application of experimental shear stress, the cells were pre-conditioned for 24 hours under 12 dyne/cm 2 of a stable shear stress. Then, the value of shear stress was elevated to 35 dynes/cm 2 generated by a non-reversed flow to create a haemodynamic environment.

| Reduced representation bisulphite sequencing (RRBS)
An RRBS analysis was carried out to determine the status of methylation of AC007362 promoter. In brief, HUVECs were divided into two groups, that is a control group and a shear stress group. Then, the cells in the shear stress group was subjected to the shear stress generated using the method described above, while the cells in the control group were not subjected to the stress. Similarly, the endothelial cells isolated from the blood vessels of IA tissues collected from the patients in groups of unruptured IA and ruptured IA were also exposed to the same shear stress, and the status of methylation of AC007362 promoter in these cells was analysed by utilizing a previously described protocol. 33

| RNA isolation and real-time PCR
Total RNA in cultured HUVECs, as well as in the endothelial cells isolated from the blood vessels of IA tissues collected from the patients in both groups of unruptured IA and ruptured IA, was extracted following the conventional TRIzol method (Invitrogen).
Then, the RNA isolated from each sample was converted to cDNA using reverse transcription carried out with an RT assay kit (Promega). In the next step, real-time PCR was carried out using a TaqMan qPCR assay kit (Applied Biosystems) on a Light Cycler

| Cell culture and transfection
HUVECs were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 5% dextran (Sigma-Aldrich), 2 mmol/L l-glutamine, 8% heat inactivated foetal bovine serum, 100 μg/mL streptomycin and 100 U/mL penicillin (Gibco, Thermo Fisher Scientific). The tissue culture operation was carried out in a standard 37°C tissue culture incubator containing 5% CO 2 and 95% air. When the cells reached about 80% confluence, they were randomly divided into 3 groups, that is an empty vector group, in which the cells were transfected with an empty pcDNA3.1 (+) vector, a AC007362 group, in which the cells were transfected with AC007362 mimics, and an anti-miR-493 group, in which the cells were transfected with miR-493 siRNA. The transfection was carried out using Lipofectamine 3000 (Invitrogen) in accordance with the protocol of the manufacturer.

| Vector construction, mutagenesis and luciferase assay
To determine the regulatory relationship between AC007362 and miR-493 as well as between miR-493 and MCP-1, the sequences of AC007362 promoter and 3′ untranslated region (UTR) of MCP-1 containing miR-493 binding sites were respectively cloned into pmiR-REPORT luciferase vectors (Promega) to create wild-type (WT) luciferase plasmids of AC007362 and MCP-1. Then, site-directed mutagenesis was performed to mutate the miR-493 binding sites in the promoter of AC007362 and 3′ UTR of MCP-1, respectively, and the mutated sequences were also cloned into pmiR-RE-PORT luciferase vectors to create mutant (MUT) luciferase plasmids of AC007362 and MCP-1, respectively. In the next step, HUVECs were plated into 24-well plates and co-transfected with WT or MUT plasmids of AC007362 or MCP-1 in conjunction with miR-493 mimic or a scramble control using Lipofectamine 3000. At 24 hours after transfection, the luciferase activity of transfected HUVECs was assayed using a Dual Luciferase assay kit (Promega) following the protocol suggested by the manufacturer.

| Western blot
Total protein in cultured HUVECs, as well as in the endothelial cells

| Statistical analysis
The levels of DNA methylation of the AC007362 promoter in different groups were compared using Student's t tests. A significance level of P ≤ .05 was used in this study. All continuous variables were compared using two-sided Mann-Whitney U tests, two-sided Wilcoxon tests or two-sided Student's t tests, while all qualitative data were compared using two-sided Fisher's exact tests or twosided chi-square tests. All statistical analyses were carried out with GraphPad Prism 5.0 software (GraphPad, Santa Barbara, CA).

| Shear stress up-regulated AC007362 and FTLP3 in HUVECs
Real-time PCR was performed to check the expression of several lncRNAs in HUVECs exposed to shear stress. As shown in

| Shear stress decreased miR-493 and elevated MCP-1 in HUVECs
MiR-493 and MCP-1 expression was analysed in HUVECs exposed to shear stress and the results showed that miR-493 expression was dramatically down-regulated by shear stress (Figure 2A). On the contrary, the expression of MCP-1 in HUVECs was apparently elevated after the cells were exposed to shear stress ( Figure 2B,C).

| MiR-493 could bind to AC007362 and MCP-1
An online bioinformatic analysis (TargetScan, http://www.targe tscan.org/vert_71/) of potential targets of miR-493 showed the presence of a miR-493 binding site in AC007362 ( Figure 4A). Then, luciferase vectors containing WT (Wild Type) or MT (Mutant) F I G U R E 1 Expression of AC007362, FTLP3 was elevated in HUVECs exposed to shear stress, while no obvious changes were observed in the expression of FTH1P10, RP11-53P19, LINC00341 and XLOC_040241, AF121215, XLOC_035542, RAMP2-AS1 and RP11-690G19, RP11-690G19, RP11-24F7, XLOC_023926, LINC00622 and RAB11B-AS1 (*P value <.05 compared with control group) F I G U R E 2 Shear stress altered the expression of miR-493 and MCP-1 (*P value <.05 compared with control group). (A) MiR-493 expression was inhibited in HUVECs exposed to shear stress. (B) MCP-1 mRNA expression was enhanced in HUVECs exposed to shear stress. (C) Western blot showed that MCP-1 expression was enhanced in HUVECs exposed to shear stress AC007362 were co-transfected to the cells with miR-493 mimics, and the luciferase activity of WT AC007362 was significantly inhibited by the transfection with miR-493 mimics ( Figure 4B). Similarly, the binding of miR-493 to the 3′ UTR of MCP-1 ( Figure 4C) was confirmed by the luciferase assay ( Figure 4D).

| Shear stress reduced DNA methylation of AC007362 promoter in HUVECs
As shear stress was reported to affect DNA methylation, RRBS was performed here to study the effect of shear stress on the level of DNA methylation in the promoters of several genes including AC007362.
In HUVECs, the exposure to shear stress remarkably suppressed the DNA methylation of AC007362 promoter ( Figure 5A). In addition, as shown in Figure 5B, DNMT1 mRNA expression was dramatically decreased after exposure to shear stress and this result was presumably caused by the change in DNA methylation status.

| DNA methylation was increased in the endothelial cells collected from unruptured IA
A group of IA patients were recruited into our study and then divided into unrultured IA group and ruptured IA group according to the presence of IA rupture. As summarized in Table 1, no difference

| MCP-1 and AC007362 were elevated and miR-493 was inhibited in ruptured IA tissues
Furthermore, the expression of MCP-1, AC007362 and miR-493 was examined in the groups of unruptured and ruptured IA. Both plasma MCP-1 and cellular AC007362 expression was notably elevated in the group of ruptured IA (Figures 7 and 8A). However, the expression of cellular miR-493 was obviously decreased in the group of ruptured IA ( Figure 8B).

| D ISCUSS I ON
SAH is a form of stroke which is associated with high mortality and morbidity and the pathogenesis of SAH is primarily caused by the  41,42 In this study, we established a cellular model to evaluate the effect of shear stress on the expression of a group of lncRNAs.
We found that AC007362 was significantly up-regulated in HUVECs exposed to shear stress. Meanwhile, we found that the expression of miR-493 was decreased while the expression of MCP-1 was elevated obviously in HUVECs exposed to shear stress. A few shear-induced events may be responsible for triggering the positive-feedback circle of CCN1/α6β1-NFκB. First, shear-induced deposition of fibronectin occurs soon after the exposure of ECs to turbulent flow. 43 Then, αvβ3 of ECs binds to fibronectin in the provisional matrix to mediate F I G U R E 5 Shear stress reduced the DNA methylation of AC007362 promoter (*P value <.05 compared with control group). (A) DNA methylation of AC007362 promoter was decreased in HUVECs exposed to shear stress.  MCP-1 plays a critical role in neuro-inflammation. 47 In the human brain, microglia are the primary carrier of MCP-1 as well as its receptor MCP-1. 47 While inflammatory reactions can prevent disease-induced tissue damages in certain cases, chronic inflammatory reactions can exert a cytotoxic effect to aggravate the condition of many diseases. An increased level of MCP-1 was found in the brain of patients of alcohol abuse. 48 In addition, the levels of IL-1β, TNF-α, MCP-1, and IL-6 expression in serum were increased in IA patients. 49,50 MCP-1 can also promote the infiltration of macrophages as well as the adhesion of macrophages onto the wall of IA artery. 51 IA rupture can also be induced by Ets-1, which is a regulator of vascular inflammation. 52 In this study, we enrolled a group of IA patients and divided them into groups of unruptured and ruptured IA, respectively. The DNMT1 expression in the group of unruptured IA was higher than that in the group of ruptured IA. As a result, the level of DNA methylation of AC007362 promoter was also higher in the group of unruptured IA. Besides, miR-493 expression was up-regulated while MCP-1 expression was inhibited in the group of unruptured IA.
MCP-1 is a type of chemokine with the ability to attract monocytes as well as to induce the inflammation in gout. In human genome, the promoters of about half of protein-coding genes are hypo-methylated under normal conditions. Nevertheless, the expression of human genes is often impaired by the hypermethylation of their promoters. 53 A previous study demonstrated that the hypo-methylation of the MCP-1 gene increases the risk of gout. 54 In addition, an apparently decreased level of MCP-1 promoter methylation has been found in patients with gout. Thus, it has been speculated that the hypo-methylation of MCP-1 promoter may increase its expression to promote the pathogenesis of gout. 55 However, despite that human subjects were used in our study, the small sample size was one of the limitations of our study. To further explore the molecular mechanisms proposed in our study, larger sample size is necessary in our subsequent study, which is more appropriate for the determination of IA rupture risk among the patient groups.

| CON CLUS ION
The above data showed that low WSS in conjunction with turbulent flow can induce the expression of MCP-1 in endothelial cells, which in turn promotes sustained macrophage infiltration during the progression of IA and increases the risk of IA rupture.

CO N FLI C T O F I NTE R E S T
None.

I N FO R M ED CO N S ENT
Written informed consent was obtained from all patients before the initiation of this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.