Serum aberrant expression of LINC00963 and its diagnostic value in carotid artery stenosis

Introduction

Carotid artery stenosis (CAS) is defined as an increased intimal thickness of the carotid artery in the neck, accompanied by a reduction in the lumen and an obstruction of blood flow [1]. The progression of CAS will lead to intracranial ischemia, which manifests clinically as a range of symptoms, including amaurosis, blurred vision, hemiplegia, and aphasia [2]. Approximately 30 % of ischemic strokes are attributable to CAS resulting from atherosclerosis, and stroke has long been a leading contributor to disability in clinics with a mortality rate among the top three fatal diseases [2]. The inability of CAS patients developing stroke to care for themselves has a significant impact on their quality of life and long-term prognosis, as well as imposing a considerable social and economic burden [3]. Several risk factors including hypertension, high cholesterol, smoking, and obesity have been identified as contributing to the development of CAS [4], 5]. CAS predominantly manifests at the carotid bifurcation, particularly within the carotid bulb. The primary pathological alteration is the thinning of the vascular endothelium. Additionally, evidence of cellular damage, inflammatory cell infiltration, anomalous lipid deposition, foam cell formation, and atheromatous plaque development was observed [6]. At present, the diagnosis of CAS is primarily based on Doppler ultrasound examination and angiography [7]. Nevertheless, it should be noted that the results of Doppler ultrasound are susceptible to several variables, including the level of expertise of the operating technician. Additionally, it is important to be aware of the potential limitations of angiography, particularly the possibility of loss of flow signals due to slow or complex blood flow, and the possibility of allergic reactions occurring during the procedure should also be taken into consideration. It is therefore imperative to identify a safe and effective biomarker for the diagnosis of CAS.

In recent studies, long non-coding RNAs (lncRNAs) have gradually emerged as a prominent area of research, given their diverse range of functions. LncRNA is a class of non-coding RNA with a length of more than 200 nt that is devoid of protein-coding functionality. It primarily regulates gene expression through several different mechanisms, including chromosome inactivation, transcriptional regulation, post-transcriptional regulation, the miRNA sponge effect, and nucleosome remodeling [8]. LINC00963 has been identified as a potential diagnostic biomarker for various diseases [9]. A reduction of LINC00963 expression was observed in clear cell renal cell carcinoma (ccRCC), and abnormal LINC00963 expression is of great significance for the monitoring and prognosis prediction of the ccRCC [10]. Chang et al. proposed that elevated LINC00963 expression may indicate an unfavorable prognosis in cervical cancer [11]. Furthermore, LINC00963 was identified as being overexpressed in gastric cancer (GC) and could potentially predict the overall survival of GC patients [12].

According to available studies on LINC00963, no direct association between LINC00963 and CAS was revealed. However, dysregulated LINC00963 expression was observed in several diseases closely related to CAS. Ellis’ study demonstrated that DR could potentially contribute to the advancement of atherosclerosis and display similarities in pathological processes with atherosclerosis, which represents the primary pathological factor in the development of CAS [13]. Zhang also found that DR patients were more likely to exhibit an elevated incidence of carotid plaque [14]. LINC00963 was considered as the potential therapy target of DR and downregulation of LINC00963 could suppress cell proliferation and migration of human retinal endothelial cells induced by high glucose, indicating a potential promotion effect of LINC00963 on diabetic retinopathy (DR) [15]. Furthermore, Moyamoya Disease (MD) is also a cerebral vascular disease caused by CAS and a web-based database screening of abnormally expressed lncRNAs associated with MD revealed that LINC00963 was dysregulated [16]. The aforementioned information indicates that the aberrant expression of LINC00963 has been identified in pathologies closely associated with CAS, thereby substantiating the indisputable correlation between LINC00963 and CAS. To further confirm the association between LINC0093 and CAS and explore the clinical role of LINC00963 in CAS, a retrospective study was conducted to analyze the expression level of LINC00963 in CAS patients and proceeded to assess its functions in the diagnosis and prognosis prediction of CAS.

Subjects and methods

Results

The LINC00963 expression levels and the ROC curve

The expression level of LINC00963 in the subjects was revealed in Figure 1A. An upregulation of LINC00963 expression was observed in the CAS group compared with the HC group (p<0.0001). According to the ROC curve (Figure 1B), the AUC value of LINC00963 on diagnosing CAS was 0.866 at the cut-off value of 1.20, showing a significant diagnostic value on CAS with a sensitivity of 85.32 % and specificity of 70.11 % (p<0.0001).

Figure 1:

The expression level of LINC00963 and ROC curve. (A) The expression level of LINC00963 in CAS was upregulated compared with healthy individuals. (B) LINC00963 showed a significant diagnostic value on CAS according to the ROC curve. Compared with the HC group, ****p<0.0001.

The association between LINC00963 expression and the prognosis of CAS

The event-free survival rate of LINC00963 on CAS was shown in Figure 2. CAS subjects in the high LINC00963 expression group showed a lower event-free survival rate compared with CAS subjects in the low LINC00963 expression group (p=0.001). Additionally, LINC00963 (HR=2.842, 95 % CI: 1.110–7.281, p=0.029), SBP (HR=3.813, 95 % CI: 1.048–13.876, p=0.042), LDL-C (HR=2.890, 95 % CI: 1.039–8.041, p=0.042), and CAS degree (HR=2.390, 95 % CI: 1.051–5.433, p=0.038) were identified as the independent prognostic factors for CAS and could predict the occurrence of the CIE (Table 3). Higher LINC00963 expression, SBP, LDL-C levels, and CAS degree will predict a high risk of the incidence of CIE in CAS subjects.

Figure 2:

The Kaplan-Meier survival analysis of LINC00963 on the occurrence of CIE in CAS.

Table 3:

Multivariate COX regression analysis on the independent prognosis factors of CAS.

Indicators	HR	95 % CI	p-Value
LINC000963	2.842	1.110–7.281	0.029
Age	1.953	0.802–4.759	0.141
Gender	1.470	0.614–3.521	0.387
BMI	2.189	0.929–5.155	0.073
SBP	3.813	1.048–13.876	0.042
DBP	1.523	0.630–3.683	0.351
TC	1.703	0.755–3.839	0.199
TG	1.323	0.570–3.071	0.514
HDL-C	0.987	0.411–2.367	0.976
LDL-C	2.890	1.039–8.041	0.042
FBG	1.648	0.607–4.480	0.327
CAS degree	2.390	1.051–5.433	0.038

1. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; FBG, fasting blood glucose; CAS, carotid artery stenosis. Bold font: significant difference.

Discussion

The onset of CAS is insidious, and patients are often not aware of its development until clinical symptoms manifest. Without timely intervention, CAS can result in aphasia, stroke, hemiplegia, and even death. Therefore, effective biomarkers and progression prevention are of great significance for the quantity and survival rate of CAS patients. The potential of lncRNAs in diagnosing disease occurrence and predicting disease progression has been extensively explored, and multiple lncRNAs have been identified to show considerable diagnostic value in diagnosing CAS. It was proposed by Wang et al. that lncRNA THRIL could serve as an effective biomarker for the diagnosis of CAS and was upregulated in CAS patients [18]. Besides, lncRNA RMST [19], LncRNA HOTAIR [20], and LncRNA PCA3 [21] were all reported to show significance in CAS diagnosis with a high sensitivity and specificity. Based on previous studies [13], 15], 16], the potential of LINC00963 on CAS attracted our attention. To confirm the clinical significance of LINC00963 in CAS, the expression level of LINC00963 was analyzed and the diagnostic and prognostic value of LINC00963 in CAS was also identified in this study.

LINC00963 was found to be overexpressed in CAS patients, indicating a potential diagnostic value on CAS. The diagnostic value of LINC00963 on CAS was further confirmed by the ROC curve. The close association between LINC00963 expression and the levels of SBP, TG, LDL-C, and CAS degree in CAS patients was also revealed. Obesity has been identified as a contributing factor to the progression of CAS [22]. But in this study, to circumvent the potential interference of numerous factors on the trend of LINC00963 expression levels, we would endeavor to include subjects with analogous baseline information and this approach may result in the observation of comparable BMI values. It is well-known that both SBP and DBP are diagnostic indicators to determine whether a patient developed hypertension. High levels of TC, TG, and LDL-C may indicate that the patients had an elevated level of blood lipids. Elevated SBP and DBP levels were found in CAS patients and their SBP levels were deviating from the established normal range as SBP values ranging from 130 to 139 mmHg or/and DBP values ranging from 85 to 89 mmHg could be classified as elevated normal blood pressure readings [23], indicating high blood pressure was developed in CAS patients and SBP was also identified as the risk factor for CAS occurrence [24]. The association between blood pressure and CAS was already confirmed by a previous study and as CAS progresses, more patients developed hypertension [25]. TG levels have also been previously reported to be associated with the degree of carotid stenosis in patients with CAS and could serve as the independent risk factor for the progression of CAS [26]. Besides, the correlation between LDL-C level and CAS was also verified by Yi’s study which indicated that CAS patients with more than 50 % stenosis had elevated LDL-C levels [27]. Since LINC00963 was significantly associated with SBP, TG, and LDL-C levels, the correlation between LINC009363 expression and CAS was further confirmed. The degree of carotid stenosis is often used for risk stratification in patients with CAS and a higher CAS degree often results in a significantly increased risk of cerebral ischemia, so timely monitoring of the CAS degree is very crucial for CAS patients [7]. Our study also proved the close association between CAS degree and the LINC00963 expression levels and CAS patients with higher CAS degree showed an elevated LINC00963 expression.

CIE, such as transient ischemic attack, cerebral infarction, stroke, and death, sometimes occurs in the prognosis of CAS [28], [29], [30]. The occurrence of CIE is often caused by uncontrolled living habits and diet of CAS patients after surgery or intimal hyperplasia and thrombosis at the surgical site [31], 32]. To further characterize the role of LINC00963 expression in the poor prognosis of CAS, CAS patients with different LINC00963 expression levels were evaluated by the K-M survival analysis and CAS patients with high LINC00963 expression showed an anabatic rate of developing CIE. Additionally, LINC00963, together with SBP, LDL-C, and CAS degree, was identified as the potential independent prognostic factors for CAS and could predict the occurrence of CIE. In combination with the results of the K-M curve and multivariate COX regression analysis, the predictive value of LINC00963 for the poor prognosis of CAS was confirmed.

However, some limitations still existed in this work. When comparing clinical indicators between CAS patients and healthy individuals, there was no significant difference in TC and TG indicators, which are closely related to the occurrence and development of CAS [26], 33]. The reason for this result may be due to the relatively small sample size. Moreover, the present study exclusively investigated the clinical significance of LINC00963 in CAS. The potential of LINC00963 to serve as an early diagnostic marker for CAS merits further exploration which should be conducted exclusively on CAS patients in the early stages, and the underlying regulatory mechanism of LINC00963 and its downstream genes in CAS development remains to be elucidated by subsequent in vitro cell experiments. As LINC00963 showed a close association with SBP, TG, and LDL-C levels, the possible regulatory mechanism of LINC00963 in the progression of CAS may be vasoconstriction and lipid transport which need further investigation. Despite the above limitations, this study still provided a meaningful biomarker for the clinical diagnosis and prognosis prediction of CAS.

In summary, LINC00963 demonstrated notable diagnostic value on CAS and could serve as the independent prognostic factor for CAS to predict the poor prognosis.