Dual impacts of lncRNA XIST and lncRNA SNHG5 on inflammatory reaction and apoptosis of endothelial cells via regulating miR‐155/CARHSP1 axis

Abstract Considering the significance of lncRNA/miRNA axis in explaining atherosclerosis (AS) progression, this investigation was intended to clarify whether lncRNAs XIST/SNHG5 would regulate AS aetiology by sponging miR‐155, an AS‐promoting molecule. We altogether recruited 367 patients who were examined by coronary angiography, and meanwhile, human coronary artery endothelial cells (HCAECs) were purchased to establish cells models via ox‐LDL treatment. The study results indicated that lowly expressed XIST/SNHG5 and highly expressed miR‐155 were frequently detectable among AS patients who showed severe stenosis and possessed high triglyceride (TG), low‐density lipoprotein cholesterol (LDL‐C) and high‐sensitivity C‐reactive protein (hs‐CRP) levels. Besides, HCAECs treated by ox‐LDL released large amounts of inflammatory cytokines, and their apoptosis rate was also raised. Moreover, expressions of XIST and SNHG5 declined markedly within ox‐LDL‐treated HCAECs, whereas miR‐155 expression significantly ascended. Transfection of pcDNA‐XIST and pcDNA‐SNHG5 both reduced the expression of TNF‐α, IL‐6, IL‐8 and IL‐1β within HCAECs and also dampened the apoptotic tendency of HCAECs. Co‐treatment of pcDNA‐XIST and pcDNA‐SNHG5 produced a larger effect on HCAEC activity than pcDNA‐XIST or pcDNA‐SNHG5 alone. Furthermore, miR‐155, modified by XIST and SNHG5, was capable of reversing the impacts of XIST and SNHG5 on HCAEC activity. Eventually, CARHSP1 was activated by XIST and SNHG5, and its overexpression dwindled impacts of miR‐155 mimic on proliferation and inflammation response of HCAECs. In conclusion, targeting XIST and SNHG5 might be an ideal alternative in delaying AS progression, allowing for their repression of downstream miR‐155.


| INTRODUC TI ON
Atherosclerosis (AS), a common disorder that seriously endangers human health, is considered as the chief pathological basis of ischaemic cardiovascular and cerebrovascular disorders, such as coronary heart disease, cerebrovascular disease and thromboembolic disease. 1 The mortality triggered by cardiovascular diseases was estimated to achieve 36% of global death by 2020, 2 which stressed the urgency of figuring out corresponding treatments. Mechanically, AS, initiated by malfunction of endothelial cells (ECs), was documented to result from disorders in lipid metabolism and chronic inflammation. 3 More specifically, in the wake of oxidative stress-forced endothelial dysfunction, endothelial permeability changed, and mononuclear macrophages began to aggregate and release inflammatory factors, which ultimately intensified proliferation of vascular smooth muscle cells and prompted development of AS. 4 Hence, capturing the aetiology that explained proper functioning of ECs appeared pivotal to alleviate AS symptoms.
A wide variety of investigations have emphasized the participation of miRNAs, especially miR-155, in modulating function of ECs. 5 For instance, miR-155 was competent in obstructing EC proliferation and repressing neovascularization by negative modification of angiotensin II type-1 receptor (AGTR1). 6,7 Besides, V-Ets-erythroblastosis virus E26 oncogene homologue 1 (ETS-1), also targeted and modified by miR-155, was indispensable to propelling vascular remodelling and controlling inflammation. 8 To sum up, miR-155 mattered in regulating both inflammation and apoptosis of ECs, and its upstream regulator genes might exert profound effects on AS progression owing to their direct control over miR-155.
As put forward by the competitive endogenous RNA (ceRNA) hypothesis, 9 lncRNAs could sponge miRNAs and then prevent miR-NAs from suppressing the action of their downstream genes. As a matter of fact, the lncRNA/miRNA axes have been documented to involve in regulating endothelial function. In particular, lncRNA LOC100129973 served to promote apoptosis of ECs by targeting miR-4707-5p and miR-4757, which was associated with incremental expression of such apoptins as API5 and BCL2LI2. 10 Besides, apoptosis of ECs was also encouraged by lincRNA-p21 sponging miR-130b, as proved by the luciferase reporter gene assay. 11 Notably, lncRNA X inactive specific transcript (XIST), with a full length of 17 kb and situated in the long arm of X chromosome, was up-regulated in oxidized low-density lipoprotein (ox-LDL)-treated ECs, 12 which implied that XIST might participate in modulating EC function and even AS development. Additionally, there existed a sponged regulation between lncRNA XIST and miR-155 within breast cancer cells, 13 which provoked a speculation that lncRNA XIST might be implicated in AS aetiology via its sponging miR-155. Concerning another lncRNA studied here, the lncRNA SNHG5 was also suggested to negatively regulate miR-155 expression within melanoma cells, 14  Abbreviations: hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride.

TA B L E 1 Comparison of clinical features between atherosclerosis patients and healthy controls
regulation also worked in ECs remained uncertain. Furthermore, the SNHG5 seemed adept at modifying cell proliferation and apoptosis, such as withstanding oxaliplatin-triggered apoptosis of colorectal cancer cells 15 and evoking inordinate proliferation of melanocytes. 16 Nonetheless, few investigations have been carried out to elucidate the possibility of SNHG5 in regulating activities of ECs.
Taken together, lncRNAs XIST and SNHG5 were potentially capable of disturbing EC function by acting upon miR-155, and this investigation was intended to clarify whether this regulation actually worked, which might conduce to developing AS treatments in future.

| Collection of AS patients
Totally 367 AS patients, identified by coronary angiography, were modified Gensini scoring method. 17 On the other hand, the healthy group was made up of 219 non-AS individuals who were also confirmed by coronary angiography. Furthermore, participants of this investigation were excluded if they were plagued by severe liver/

| MTT assay
HCAECs grown to monolayer confluence were de-adhered by addition of 0.25% trypsin (Gibco, USA), and the HCAECs were then diluted into single-cell suspension with supplementation of 10% on a microplate reader to obtain their absorbance value at the wavelength of 570 nm. All these experiments were repeated for ≥ 3 times.

| Plate cloning assay
HCAECs of logarithmic growth phase were digested to a concentration of 100 per well, and then they were incubated in 5% CO 2 at 37°C for 2 weeks. The culture was terminated when colonies appeared macroscopically. After rinsage with water, 500 μL methanol crystal violet solution (0.1%) was added to each well to stain the cells, and then cell colonies were counted. All these experiments were repeated for ≥ 3 times.

| Cell apoptosis assay
The digested HCAECs were centrifuged at 1000 r/min for 5 minutes, and then pre-cooled phosphate buffer solution (PBS) was added to re-suspend cells at 4°C. The cells were centrifuged again at 1000 r/ min for 5 minutes, and the sediments were blended by 400 μL binding buffer (eBioscience, USA) to re-suspend cells. Subsequently, the cells were mixed by 5 μL Annexin V/FITC (eBioscience, USA), followed by 15-min incubation in the dark. After that, the cells were treated by 10 μL PI (eBioscience, USA), and the resultant samples were gathered to conduct flow cytometry analysis (model: FACSVerse flow detector, BD Biosciences, USA). All these experiments were repeated for ≥ 3 times.  Ultimately, expression levels of target genes were calculated according to 2-△△Ct method. All these experiments were repeated for ≥ 3 times.

| Western blotting
The HCAECs digested by 0.25% trypsin were dissociated after ad- 3 times, was mixed with goat anti-rabbit IgG (H&L) antibodies (1:3000, Catalogue: ab6721) for another 1-h incubation at room temperature. Next, luminescent solution was dropped onto the membrane, which was then placed in a chemi-luminescence imager for exposure and development. The relative expression of proteins was calculated by ImageJ software, with GAPDH as the internal reference. All these experiments were repeated for ≥ 3 times.

| Double luciferase reporter gene assay
The miR-155-binding sites located within XIST, SNHG5 and CARHSP1 were predicted using StarBase v2.0, and then corresponding sequences of XIST, SNHG5 and CARHSP1 were amplified through RT-PCR. After undergoing agarose gel electrophoresis (AGE), the PCR products (0.5 pmol/L, 1 μL) were purified via usage of gel extraction kit (TaKaRa, Japan) and were then connected to 0.5 μL pGL3 vector (0.2 pmol/L) (Promega, USA). After that, every 10 μL connection product was mixed by 50 μL DH5α E. coli competent cells (TaKaRa, Japan), and penbritin was added to screen drug-resistance cells.
The connection plasmid and empty plasmid were then extracted to experience double-enzyme (ie XhoI and Hind III) restriction, and sequences of the products were determined. After all these procedures, pGL3-XIST-Wt, pGL3-SNHG5-Wt and pGL3-CARHSP1-Wt reporter vectors were successfully established. In a similar way, the miR-155-binding sites in XIST, SNHG5 and CARHSP1 were mutated, and relevant mutant reporter vectors, namely pGL3-XIST-Mut, pGL3-SNHG5-Mut and pGL3-CARHSP1-Mut were constructed.

| RNA immunoprecipitation (RIP) assay
HCAECs were disintegrated by RIP lysate following instructions of EZ-Magna RIP kit (Millipore, USA), and the products were centrifuged at 12 000 r/min for 10 minutes to collect supernatants. Subsequently, IgG antibody or Ago2 antibody was supplemented into magnetic bead-containing EP tubes which were added by 900 μL RIP buffer, including RNase inhibitor, proteinase inhibitor and DNase, and 100 μL supernatant of HCAEC in advance. After incubation at 4°C for overnight, the mixture was centrifuged at the speed of 12 000 r/min for 10 minutes, and supernatants were discarded. Afterwards, the samples were rinsed by 500 μL RIP wash buffer, and RNAs were purified by 15 μL DEPC and conserved at −80°C. HCAECs with addition of IgG antibody and Ago2 antibody were separately included into anti-IgG group and anti-Ago2 group, while HCAECs without any treatment were set as positive control group (ie Input group).

| Statistical analyses
All the statistical analyses were performed using SPSS 16.0 software. Measurement data in the form of mean ± standard deviation (SD) were analysed by Student's t test and one-way analysis of variance (ANOVA). On the other hand, the count data (n or %) were analysed by chi-square test. And P < .05 was considered as statistically significant.

| Correlation between XIST/SNHG5/miR-155 expressions and clinical characteristics of AS patients
Comparing the baseline characteristics of 367 AS patients with 219 healthy controls, there revealed a higher proportion of smokers among the AS patients than among the healthy crowd (P = .003).
More than that, the AS patients were associated with higher levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-sensitivity C-reactive protein (hs-CRP) than healthy controls (P < .05) ( Table 1). However, hardly any statistical differences were observable between the two groups, with respect to mean age, gender ratio and drinking status (P > .05).

| ox-LDL promoted inflammatory response and apoptosis of HCAECs
Besides, with the incremental concentration of ox-LDL, expressions of XIST and SNHG5 declined markedly in HCAECs, whereas miR-155 expression took on a gradual ascent (P < .05) ( Figure 2E).
Additionally, driven by ox-LDL, growth of the HCAECs was held back, while apoptosis of the HCAECs was promoted (P < .05) ( Figure 2F-H).
On the contrary, viability and proliferative capacity of the HCAECs were largely reinforced by pcDNA-XIST and pcDNA-SNHG5 (P < .05),

| XIST and SNHG5 sponged miR-155 to downregulate its expression
The luciferase activity of HCAECs in the pGL3-XIST-Wt + miR-155 mimic group was significantly decreased, as compared with pGL3-XIST-Mut + miR-155 mimic group and pGL3 + miR-155 mimic group (P < .05) ( Figure 5A). Moreover, RIP results ( Figure 5B) suggested that XIST and miR-155 were concentrated in both anti-Ago2 group and Input group as compared with anti-lgG group (P < .05), and levels of XIST and miR-155 were higher in Input group than in anti-Ago2 group (P < .05). In addition, miR-155 expression was restrained by overexpressed XIST and was elevated in case of under-expressed XIST (P < .05) ( Figure 5C). However, XIST expression revealed no significance difference under treatments of miR-155 mimic and miR-155 inhibitor (P > .05) ( Figure 5D).

| D ISCUSS I ON
The onset of AS was partly acknowledged to result from endothelial dysfunction, specifically embodied as enhancive inflammatory cytokines released by impaired ECs. 18 Notably, ox-LDL was insinuated as a primary cause of vascular endothelial injury. In particular, the receptor structure of ox-LDL was different from that of LDL, which made ox-LDL no longer identifiable by macrophages. As a consequence, the ox-LDL was readily devoured by macrophages, which led to incremental production of inflammatory cytokines (eg TNF-α and IL-8). For another, ox-LDL could trigger apoptosis of ECs, which also motivated endothelial malfunction 19 and thereby AS onset. [20][21][22] Allowing for the considerable part of ox-LDL underlying AS pathogenesis, here ox-LDL was utilized to build cell models, and molecules that were involved in ox-LDL-induced AS onset were explored.
Robust With regard to SNHG5, the mature spliceosome of U50, 27 a majority of existing studies mainly emphasized its role in tumorigenesis, such as colorectal cancer, gastric cancer, breast cancer and melanoma. 15,16,28 Nevertheless, finite researches were launched to correlate it with EC function. The current investigation might, to some extent, fill this gap, which demonstrated that overexpressed SNHG5 could restrain EC apoptosis ( Figure 4D) and inflammation ( Figure 3E). Actually, the apoptosis-restraining role of SNHG5 was also detectable in certain tumours. For instance, overexpressed SNHG5 could antagonize oxaliplatin-induced apoptosis of colorectal cancer cells, 15 and elevating serum SNHG5 level was conjectured to urge metastasis of melanoma. 16 Contrary to evidences as mentioned above, Zhao et al documented that SNHG5 was lowly expressed in gastric cancer, and its recruiting metastasis associated 1 family member 2 (MTA2) was involved in suppressing proliferation and metastasis of gastric cancer cells. 29 It was thus conjectured that SNHG5 might function distinctly within various disorders, and specific accounts for SNHG5' curbing EC apoptosis entailed thorough inquiries.
Taken together, it might be owing to these molecular mechanisms that serum levels of XIST and SNHG5 were clinically indicative of AS onset and prognosis ( Figure 1C). There were implications that non-coding RNAs in serum were possibly secreted by necrotic or apoptotic cells (eg HUVECs), 30 or were derived from exosomes in blood. 31 However, we failed to show how XIST and SNHG5 in HCAECs were released into serum of AS patients, because of shortfalls on technology. Huge efforts would be contributed to remedying this part in future. To further account for the role of XIST and SNHG5 in AS, here we proposed that the couple of lncRNAs might interfere with EC activity by sponging downstream miR-155 ( Figure 5A,E), just as revealed by the ceRNA theory. 32 As a matter of fact, XIST and SNHG5 were previously documented to act upon miRNAs in other disorders. To be specific, XIST accelerated migration of human skin fibroblasts (HSFs) by sponging miR-29a and restraining its expression. 33 Besides, SNHG5 could sponge miR-32 to elevate expression of Kruppellike factor 4 (KLF4), which altogether urged metastasis of gastric cancer cells. 28 Herein it might be due to sponging miR-155, as proved by dual luciferase reporter gene assay ( Figure 5A,E) and RIP assay ( Figure 5B,F) Figure 8A). The CARHSP1 was reported to enhance stability of TNF-α mRNA, which could induce injury of vascular ECs, 39 and depressing CARHSP1 expression could inhibit release of TNF-α by foam cells in AS. 40 Apart from the above, this investigation gained knowledge that CARHSP1 also mediated the role of miR-155 in regulating inflammation and viability of HUVECs ( Figure 9). In effect, the miR-155 has been documented to regulate disease progression after being sponged by lncRNAs, 41

ACK N OWLED G EM ENT
None.

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