LncRNA HCG18 affects aortic dissection through the miR-103a-3p/HMGA2 axis by modulating proliferation and apoptosis of vascular smoothing muscle cells

Graphical abstract Image, graphical abstract


Introduction
2][3][4] Acute Stanford Type A Aortic Dissection (TAAD) has the characteristics of sudden onset, rapid progression, and high mortality. 5n recent years, the use of advanced imaging technology has made TAAD clear diagnosis is no longer difficult, but there are still many questions about its pathogenesis and treatment options.Studies found that AD is a comprehensive pathological change process caused by pathological changes involving multiple blood vessel constituents, such as human aortic Smooth Muscle Cells (VSMCs) and extracellular matrix. 6,7Surgical operation is the only approach for curing TAAD; however, the surgery is invasive and costly, and mortality rates are still high, even after treatment.Therefore, understanding the pathogenesis of AD and searching for biomarkers may reveal new therapeutic strategies to reduce clinical mortality.
Long non-coding RNAs (lncRNAs) are a category of non-coding transcripts with longer than 200 nucleotides, which have attracted more and more attention of researchers.][10] A member of the HLA complex, lncRNA HCG18 presents an abnormal expression profile in gastric cancer, 11 melanoma, 12 osteosarcoma, 13 and nasopharyngeal carcinoma. 14HCG18 is overexpressed in intervertebral disc degeneration and it can promote the degradation of extracellular matrix in nucleus pulposus cells. 15Additionally, HCG18 inhibits proliferation and induces apoptosis of VSMCs, 16 indicating that HCG18 may be related to AD.However, the regulatory mechanism of HCG18 in AD progression remains unclear.
MicroRNAs (miRNAs) are a family of small non-coding RNAs with a length of 18-22 nucleotides.Evidence suggests that during normal physiological and pathological conditions, lncRNAs act as competing endogenous RNAs that competitively bind and sequester their target miRNAs, thereby counteracting miRNA-mediated repression of targeted mRNAs, indirectly regulating the expression of the miRNA target genes. 17,18In addition, in this study, the authors found that there are target binding sites with HCG18 with miR-103a-3p and miR-103a-3p with HMGA2.Therefore, the aim of this study was to investigate the role and potential mechanism of HCG18/miR-103a-3p/HMGA2 axis in the development of AD and to provide new targets for targeted therapy of AD.

Surgical indications
Patients presenting with signs and symptoms of TAAD were always treated on an emergency basis as soon as the diagnosis was confirmed by transthoracic 2D echo and/or angio-computed tomography.Potential contraindications were progressively reduced, current indications including patients with systemic malperfusion, with irreversible neurological damage, and advanced age.The surgical strategy was mainly dictated by the preoperative imaging and the intraoperative findings.Graft replacement of the ascending aorta, always extended to include the hemiarch, was considered as a limited repair, performed when the entry tear was found only in the ascending aorta; dilatation of the aortic arch or presence of arch tears, evidenced by routinely performing an open distal anastomosis, represented an indication to replace both the ascending aorta and arch.Management of the aortic root depended upon its size, morphology and function, and involvement of the aortic valve. 19

Clinical sample
In all, 30 patients admitted to the emergency department of the Ningde Municipal Hospital of Ningde Normal University who met the diagnostic criteria of TAAD were collected.Aortic wall tissues of ascending aorta of patients with TAAD were obtained during surgical operations and the aortic tissues of ascending aorta from donors for heart transplants were selected as the control group.Patients in the control group were excluded from Marfan syndrome, Ehlers-Danlos syndrome, familial thoracic abdominal aortic dissection, hyperkinetic inflammation, and other aortic diseases.A detailed description of the clinical characteristics of the study population is presented in Table 1.This study was approved by the Medical Ethics Committee of Ningde Municipal Hospital of Ningde Normal University (n°201906F23).All subjects received informed consent.Aortic tissue was fixed with neutral formalin and stored at −80 °C in EP tubes for further analysis.

Cell culture
Rat aorta VSMCs (Chinese Academy of Sciences) were cultured at 37 °C and 5 % CO 2 in DMEM medium (Invitrogen) containing 10 % FBS (Invitrogen) and necessary antibiotics.

Colony formation experiment
Cell proliferation was detected by colony formation assay.Specifically, VSMCs were inoculated in 6-well plates (700-well) for 14d After being fixed in 10 % formaldehyde, VSMCs were stained with 1 % crystal violet (Beyotime, China) and imaged under a microscope (Leica, Germany) to count colonies (≥ 50 cells).

Flow cytometry
Apoptosis was detected by flow cytometry.Specifically, VSMCs were digested with trypsin, followed by staining with Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) and sorting on the FACScan machine (BD Biosciences).Cell apoptosis rate was determined by Cell-Quest software (BD Biosciences).

Rat model of AD
β-Aminopropionitrile (BAPN) can inhibit the crosslinking of collagen and elastin fibers, leading to AD. 20,21 AD animal models were established [22,23 as described previously.AD rats (3-weeks of age) were fed a normal diet with 0.25 % BAPN (Sigma) orally until 7-weeks of age.
The sham rats were treated with the same amount of normal saline.Then, at 6-weeks of age, AD rats have injected with either si-HCG18 or Table 1 The clinical characteristics of TAAD and control groups.

HE-staining
The fixed aorta tissue was embedded in paraffin and sectioned into 4 μm.Tissue sections were dewaxed in xylene and rehydrated in graded ethanol.Staining experiments were performed using the HE-kit (C0105, Beyotime).After the sections were dehydrated, cleared, and sealed, the histopathological changes in the aortic tissue were observed under a microscope (Olympus, Japan).

RT-qPCR
Total RNA was isolated using TRIzol reagent (Invitrogen) and then reverse-transcribed into cDNA using the PrimeScript RT Master Mix Kit (Takara).Real-time qPCR analysis was performed using SYBR Green Realtime PCR Master Mix (ToyoBo) in the 7900HT PCR System (ABI).All primers (Table 2) for RT-qPCR were synthesized by Sangon.GAPDH and U6 were considered internal controls for lncRNA/mRNA and miRNA analysis, respectively.

Statistical analysis
All data were processed with GraphPad Prism 6.0 software.Values were expressed as mean ± standard deviation and assessed by t-test (two groups) or one-way ANOVA (multiple groups); p < 0.05 indicated statistical significance.

Downregulating HCG18 improves the pathological injury of the aorta in AD rats
Finally, an AD animal model was induced to verify the regulatory effect of HCG18.HCG18 was upregulated in AD rats (p < 0.05; Fig. 6A).si-NC or si-HCG18 lentivirus was injected into the tail vein of AD rats, and successful lentivirus injection was verified by RT-qPCR (p < 0.05; Fig. 6B).HE-staining observed that the aortic wall of AD rats contained broken elastic fibers, and blood cells entered the aortic wall, leading to tearing and stripping; down-regulation of HCG18 could improve the above pathological changes (Fig. 6C).

Discussion
It is known that HCG18 exerts adjustive effects on VSMCs. 16This work studied the function of HCG18 in AD and explored its related downstream mechanism.Abnormal regulation of lncRNAs is often related to pathological processes, including coronary heart disease. 24,25ncRNAs have also been reported to be involved in regulating cardiovascular diseases such as heart failure, hypertension, and aneurysm. 26,279][30] The current work identified that HCG18 was upregulated in the aortic tissues of TAAD patients, and down-regulated HCG18 protected VSMCs regarding cell proliferation and apoptosis.In addition, down-regulated HCG18 could improve the pathological injury of aorta in AD rats.
2][33] It is noted that HCG18 aggravates diabetic peripheral neuropathy through modulating miR-146a. 34t has been proposed that HCG18 accelerates lung adenocarcinoma development by targeting miR-34a-5p. 35The present study found that HCG18 was competitively bound to miR-103a-3p.7][38] As suggested, targeting HMGA2 is a useful method in miR-26b inhibiting Stanford Type A AD. 39 The current work identified HMGA2 as the target gene of miR-103a-3p, and HMGA2 was upregulated in the aortic tissues of AD patients.HMGA2 promotion could impair HCG18 downregulation or miR-103a-3p upregulation in mediating VSMC proliferation and apoptosis.
Considering the limitations of this study, a limited sample may not fully confirm the accuracy of the results.Secondly, the relationship between HCG18 and other potential targeted miRNAs needs further attention and research.In addition, only TAAD patients were included in this study, and the experimental results cannot be generalized to all AD patients for the time being.

Conclusion
In summary, this study for the first time found that HCG18 promotes AD progression by competing with miR-103a-3p to target HMGA2 expression.This provides a theoretical basis for gene therapy for aortic coarctation, and surgical treatment assisted by gene therapy may be a new therapeutic strategy for patients with TAAD.

2 Z.
Yang et al.Clinics 79 (2024) 100400 Negative Controls (si-NC) lentivirus (1 × 10 11 PFU) through the caudal vein.Finally, the aorta tissues were collected from each euthanized rat and fixed in 4 % paraformaldehyde (P0099, Beyotime).Animal experiments complied with the ARRIVE guidelines and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.The experiments were approved by the Institutional Animal Care and Use Committee of Ningde Municipal Hospital of Ningde Normal University (n°201908J11).