Circular RNA hsa_circ_0000690 as a potential biomarker for diagnosis and prognosis of intracranial aneurysm: Closely relating to the volume of hemorrhage

This study aimed to explore circular RNA (circRNA) hsa_circ_0000690 as a potential biomarker for diagnosis and prognosis of intracranial aneurysm (IA) and its relationship with clinical factors and complications of IA.


INTRODUCTION
Intracranial aneurysm (IA) is a cerebrovascular disease caused by high blood flow or congenital weakness of the cerebral artery wall (Marbacher et al., 2020), which seriously threatens human life and health. Currently, the diagnosis of IA mainly depends on digital subtraction angiography (DSA) and computed tomography angiography (CTA). Although brain imaging techniques and surgery strategies have been improved in past decades, the prognosis of subarachnoid hemorrhage (SAH) has not sufficiently improved (Algra et al., 2019). IA in most patients is asymptomatic until the aneurysm ruptures and pain appears and leads to SAH, which could be diagnosed and treated before rupture. Thus, early diagnosis and surgical treatment before rupture could significantly improve the prognosis of IAs.
Circular RNA (circRNA) is a type of novel nucleic acid molecule that exists in the form of a covalent loop, without a characteristic 5′cap end and 3′poly (A) end (Rybak-Wolf et al., 2015). Present studies have shown that circRNAs have many functions, such as miRNA sponges, gene regulation, and close relation with cell functions and diseases.
A large number of subsequent studies have confirmed that circRNAs involve many important pathological processes such as inflammation, SMC phenotype transformation, and extracellular matrix, which play important roles in epigenetic regulation in vascular diseases (Ashraf et al., 2021;Li et al., 2022). So, circRNAs have rapidly become popular in cerebrovascular disease research. Although DSA and CTA are dominating clinical tools for diagnosing IA, these methods are invasive and radioactive, which makes them unsuitable as a method of population screening. Peripheral blood is easy to collect, detecting that circulating circRNAs could be an efficient and effective method for the screening.
Meanwhile the noninvasive and disease-specific pattern is an essential feature of a biomarker. Since that, circRNAs have been deeply explored as a rising biological marker and therapeutic target of IA (Wu et al., 2021) and other diseases.
Based on our previous high-throughput sequencing results , we first validated that hsa_circ_0000690 downregulated significantly in IA patients. Hsa_circ_0000690 locates at chr16:30495147-30495584 in circBase with its associated-gene symbol as integrin subunit αL (ITGAL). In this study, we explored the role of hsa_circ_0000690 in the diagnosis and prognosis of IA and its relationship with clinical factors and complications.

cDNA synthesis and real-time PCR
The cDNA was synthesized using the High-Capacity cDNA Reverse

Admission CT risk factors
A total of 216 brain CT data on admission were acquired in Digital Imaging and Communications in Medicine format (DICOM). All DICOM data were performed using a GE Discovery 750 CT scanner (General Electric Company, Fairfield, CT, USA,) with a slice thickness of 1.25 mm and an increment of 0.5 mm.
Based on these DICOM data, the modified Fisher scale (mFS) was computed for each patient according to Frontera et al. (2006). For a quantitative measure of aSAH and IVH volume, the DICOM data were transferred to a standard personal computer (Intel Core i5-8250U CPU, 1.6 × 1.8 GHz, 8GB RAM) and then assessed by 3D Slicer (version 4.11.20200930) independently by two physicians. The Threshold Effect tool in Segment Editor module was applied to automatically mark the aSAH and the IVH. Manual drawing was then performed with the Paint Effect tool to prune or modify the colored structures slice by slice. Then a 3D model was constructed, and the volume was given by accumulating volume of all the pixels. We defined the result of aSAH and IVH volume as 'Slicer Volume' henceforth. For subsequent statistical analyses, the average of the two physicians' Slicer Volume was obtained.

Non-CT risk factors
Other non-CT risk factors included demographic factors (gender, age), past history (smoking, drinking, hypertension, cardiac disease, and diabetes), admission clinical features (Hunt and Hess level (Ghosh et al., 2012), Glasgow Coma Scale (Avezaat et al., 1977)), admission radiological features (multiple aneurysms or not, position, and size), and surgical type. The position of the aneurysm was classified as anterior circulation or posterior circulation aneurysm. Size of the aneurysm was defined as small when diameter <5 mm, normal with diameter ≥5 and <15 mm, large with diameter ≥15 and <25 mm, giant with diameter ≥25 mm.
Surgical type was divided into aneurysm clipping or embolism.

Definition of hydrocephalus and delayed cerebral ischemia (DCI)
Hydrocephalus was defined as ventricular enlargement, Evans' index ≥.3 (Lee et al., 2021), and ventriculoperitoneal shunt was performed.
When potential causes of clinical deterioration, such as rebleeding, or seizures, were rigorously excluded, DCI was defined as (1) unexplained clinical deterioration (i.e., a new focal deficit, decrease in level of consciousness, or both) or (2) a new infarct on CT that was not visible on the admission or immediate postoperative scan, or both. DCI was diagnosed by the treating neurologist and confirmed in a retrospective review of each subject's clinical course by two physicians. Evidence of arterial spasm by transcranial doppler sonography was generally used to support the diagnosis but was not mandatory.

Prognosis and follow-up
The prognosis after surgery for 3 months was determined by modified Rankin Scales (mRS). The follow-up was from the hospital discharge time after the patient was treated to April 2022. For survival patients at the end of the follow-up visit, the follow-up data were the last contact state. For patients lost follow-up, the follow-up data were the last census state. The survival time was expressed by survival months.

Statistical analysis
All statistical analyses were performed with SPSS statistics 25 (IBM

Study population
A total of 216 cases of IA patients included 73 males and 143 females.
The age range was 28-83 years, and the average age was 58 years. The diameter of the aneurysm <5 mm in 82 cases, between 5 and 15 mm in 109 cases, between 15 and 25 mm in 24 cases, more than 25 mm in 1 case; anterior circulation aneurysms in 207 cases, posterior circulation aneurysms in 9 cases; single aneurysm in 188 cases, multiple aneurysms in 28 cases. IA patients and normal controls had the same distribution among gender, age, smoking, drinking, cardiac disease, and diabetes, expect hypertension. Hypertension was significantly higher in IA than that in normal controls (p < .001). See Table S1 for the results.

Down-expression of hsa_circ_0000690 in IA patients
Total RNA of peripheral blood was extracted from 216 IA patients and 186 normal controls and analyzed by quantitative real-time PCR. Our results showed that expression of hsa_circ_0000690 in IA patients was significantly lower than that in normal controls (p < .001) ( Figure 1A).

Relationship between expression of hsa_circ_0000690 and clinical features in IA patients
As shown in Table 1, hsa_circ_0000690 expression was significantly associated with Glasgow Coma Scale (GCS), Slicer Volume, mFS, Hunt-Hess levels, and surgical type (p < .05).

3.5
Relationship between expression of hsa_circ_0000690 and hydrocephalus in IA patients IA patients were grouped according to whether they had hydrocephalus, and the univariate analysis showed that the expressions of

Relationship between expressions of hsa_circ_0000690 and DCI in IA patients
IA patients were grouped according to whether they had DCI, and the univariate analysis showed that the expressions of hsa_circ_0000690, age, diabetes, Slicer Volume, mFS, Hunt-Hess levels, GCS, and surgical type were significantly different between the two groups (p < .05) (Table S3). But through binary logistic regression for significant factors mentioned above (Table 3), DCI was only significantly associated with age, diabetes, mFS, and surgical type, without an association with the expression of hsa_circ_00006 90.

3.7
Relationship between expressions of hsa_circ_0000690 and mRS in IA patients mRS was significantly associated with expression of hsa_circ_0000690, age, Slicer Volume, mFS, Hunt-Hess levels, GCS, aneurysm size, surgi-F I G U R E 1 Quantitative real-time PCR (qRT-PCR) analysis and receiver operating characteristic (ROC) curve for hsa_circ_0000690 in intracranial aneurysm (IA) patients and control group. (A) The relative expression of hsa_circ_0000690 in IA patients is significantly lower than that in control group. (B)The ROC curve of hsa_circ_0000690.  (Table S4). Through ordinal logistic regression for significant factors mentioned above (Table 4), mRS was significantly associated with expression of hsa_circ_0000690, Slicer Volume, mFS, and Hunt-Hess levels.

Results of survival and risk factors
According to Kaplan-Meier curve (

DISCUSSION
circRNAs have good structural stability and been abundant in brain tissue, making the exploration of circRNAs as biological markers widely concerned and deeply studied. circRNAs play roles in different kinds of neurological disease, such as glioma (Sun et al., 2020), brain development diseases (Mehta et al., 2020), neurological traumatic injury (Xu et al., 2021;Zhang et al., 2022), and IA (Wu et al., 2021).
In our previous study   it showed potential diagnostic value for IA through ROC curve that the significant AUC of hsa_circ_0000690 was 0.752, and diagnostic threshold was 0.0449, with 0.780 in specificity and 0.620 in sensitivity, respectively.
In a systematic review and meta-analysis (Wu et al., 2021) about noncoding RNAs as circulating biomarkers for the diagnosis of IA in 2021, most of noncoding RNAs for the diagnosis of IA were micro RNAs. There were only three circRNAs (Huang et al., 2019;Teng et al., 2017) having diagnostic value, which called hsa_circ_0021001, hsa_circ_0072309, and hsa_circ_0008433. Interestingly, the same as three circRNAs mentioned above, hsa_circ_0000690 is also downregulated and involved in inflammation and cell adhesion pathway, which are closely related to vascular strength and integrity. It is known that inflammation is an important factor in the occurrence and development of IA. Meanwhile most dysfunctional circRNAs are mainly involved in inflammation and cell adhesion pathways that are known to be critical for the pathogenesis of IA (Pawlowska et al., 2018).
Inflammatory response (Signorelli et al., 2018;Turjman et al., 2014) and leukocyte infiltration (Kanematsu et al., 2011) are the pathological basis for the development of IA. Infiltration of inflammatory cells is the primary pathological change during the early stages of IA (Pawlowska et al., 2018). Reported in many research studies (Chu et al., 2015;Kim et al., 2014;Rothoerl et al., 2006), intercellular cell

CONCLUSION
In conclusion, we introduced hsa_circ_0000690 as a potential novel biomarker for IA can predict the prognosis of 3 months after operation.
Moreover, this is the first circRNA found to have closely relationship with the volume of hemorrhage when IA is ruptured as we know. We will explore its regulatory mechanism in the formation, development, and rupture of IA through further functional tests in the future, in order to provide a new perspective for the future research of IA noninvasive diagnosis and treatment strategies.

AUTHOR CONTRIBUTIONS
Yanming Huang and Shuanglin Que contributed to the study conception and design. PCR experiment was performed by Huimin Cao.
Clinical material preparation and data collection were performed by

CONFLICT OF INTEREST STATEMENT
The authors declare that they have no known conflict of interests or personal relationships that could have appeared to influence the work reported in this paper.

CONSENT FOR PUBLICATION
The manuscript is not submitted for publication or consideration elsewhere.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this published article.