LncRNA LINC01605 Regulates Smooth Muscle Cell Functions and Participates in the Development of Aortic Dissection Through Regulating SGK1

Human aortic tissues were collected from 30 aortic dissection (AD) patients and 30 healthy controls (HC) with informed consent. The study was approved by the Ethics Committee of the Fujian Medical University Union Hospital (Approval No. 2024KY178). Vascular smooth muscle cells (VSMCs) were isolated from tissues using collagenase digestion and identified by α‐SMA immunofluorescence staining [13].

Differentially expressed lncRNAs in AD were screened from two GEO datasets (GSE107844 and GSE147026). The GSE107844 dataset included 3 normal control (NC) samples and 3 AD samples, while the GSE147026 dataset included 4 NC samples and 4 AD samples. Raw count data from these datasets were preprocessed and analysed using the DESeq2 package in R. Differentially expressed genes (DEGs) were identified with the following criteria: adjusted p‐value (padj) < 0.05 and |log2FoldChange| ≥ 0.5 for volcano plot analysis. To specifically identify differentially expressed lncRNAs, a stricter threshold of p‐value (padj) < 0.05 and |log2FoldChange| ≥ 1 was applied. Venn analysis was performed to identify common differentially expressed lncRNAs across the two datasets, and LINC01605 was identified as a significantly upregulated lncRNA in both datasets.

Target genes of LINC01605 were predicted using the ENCORI database (https://rnasysu.com/encori/↗). To validate the predicted targets, we intersected them with AD‐related differentially expressed mRNAs identified from the GSE107844 dataset and the Genecards database. This intersection analysis helped prioritise biologically relevant target genes for further investigation.

Raw count data from the GSE107844 and GSE147026 datasets were preprocessed by filtering low‐expression genes (retaining genes with at least 10 counts in at least one group). The data were then normalised using the DESeq2 package, which estimates size factors to correct for sequencing depth differences and dispersion parameters for differential expression analysis. Multiple‐testing correction was performed using the Benjamini‐Hochberg procedure to control the false discovery rate (FDR) at a threshold of < 0.05.

Human aortic vascular smooth muscle cells (VSMCs, CP‐H116) were purchased from Procell Life Science & Technology Co. Ltd., Wuhan, China. The cells were cultured in Ham's F‐12 K medium (PM150910) supplemented with 0.05 mg/mL vitamin C, 0.01 mg/mL insulin, 0.01 mg/mL transferrin, 10 ng/mL sodium selenite, 0.03 mg/mL ECGs, 10% fetal bovine serum (FBS), 10 mM HEPES, 10 mM TES and 1% penicillin–streptomycin (P/S), all of which were obtained from Procell. HA‐VSMCs were used for experiments at passages 3–5. For genetic manipulation, lentiviral vectors targeting LINC01605 (overexpression: OE‐LINC01605; knockdown: sh‐LINC01605) and SGK1 (OE‐SGK1 and sh‐SGK1) were purchased from Genechem Inc. (Shanghai, China). Scrambled shRNA (sh‐NC) and empty vector (OE‐NC) served as negative controls.

VSMCs were seeded in six‐well plates (5 × 10⁴ cells/well) and transfected 24 h later with lentivirus at a multiplicity of infection (MOI) of 20 (1 × 10⁸ TU/mL, 2 μL/well). The blank control group received an equivalent volume of fresh culture medium. After 72 h of transfection, cells were treated with Angiotensin II (Ang II, Sigma, St. Louis, MO, USA) at a concentration of 1000 nM (dissolved in sterile PBS) for 24 h to induce pathological stress.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First‐strand cDNA was synthesised from 1 μg total RNA using the PrimeScript RT Master Mix (Takara, Shiga, Japan) following the manufacturer's protocol. qRT‐PCR was performed on an ABI 7500 Real‐Time PCR System (Applied Biosystems, USA) with SYBR Green PCR Master Mix (Toyobo, Osaka, Japan). Each 20 μL reaction contained 10 μL SYBR Green mix, 1 μL cDNA template, 0.5 μM forward and reverse primers (Table ) and 8.5 μL nuclease‐free water. Cycling conditions: 95°C for 10 min (initial denaturation), followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. β‐actin was the housekeeping gene. Relative expression levels were calculated using themethod. All reactions were performed in triplicate (technical replicates) for each biological sample. S1 2 − ∆ ∆ C t

After fixation with 4% formaldehyde, cellular membranes of VSMCs were permeabilised using 0.3% Triton X‐100, followed by blocking with 5% bovine serum albumin (BSA) to minimise non‐specific binding. FITC‐labelled LINC01605 probes and Cy3‐labelled SGK1 probes (RiboBio, Guangzhou, China) were hybridised to VSMCs overnight at 37°C. After washing with PBS, nuclei were counterstained with DAPI (Sigma, 1 μg/mL) for 5 min. Images were captured using a Zeiss LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany) and analysed with ImageJ (NIH, MD, USA).

VSMCs (5 × 10³ cells/well) were seeded in 96‐well plates. At 24, 48 and 72 h post transfection, 10 μL CCK‐8 reagent (Dojindo, Kumamoto, Japan) was added to each well. After 2 h of incubation, absorbance at 450 nm was measured using a BioTek Synergy H1 microplate reader (BioTek Instruments, VT, USA).

Cells were incubated with 10 μM EdU (Beyotime, Shanghai, China) for 2 h, fixed with 4% PFA, and stained using the Click‐iT EdU Alexa Fluor 488 Kit (Invitrogen). Fluorescence images were acquired with an Olympus IX73 microscope (Olympus, Tokyo, Japan). Proliferation rates were quantified as the ratio of EdU‐positive cells to total DAPI‐stained nuclei.

VSMCs (1 × 10⁵ cells) in serum‐free DMEM were added to the upper chamber of Transwell inserts (8 μm pores, Corning, NY, USA). The lower chamber contained DMEM with 10% FBS. After 24 h, migrated cells were fixed with methanol, stained with 0.1% crystal violet (Beyotime) and counted under a Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan).

Confluent VSMC monolayers were scratched with a 200 μL sterile pipette tip. Wound closure was monitored at 0 and 24 h using a phase‐contrast microscope (Nikon). Migration distance was measured using ImageJ.

Male ApoE^−/− mice (8–10 weeks old, C57BL/6 background) were housed under standard SPF conditions and randomised into four groups (n = 8–10/group): Sham (ApoE^−/− mice infused with saline via subcutaneous osmotic minipumps [Alzet 2004] for 28 days), AD (mice receiving angiotensin II [Ang II, 1000 ng/kg/min] via osmotic pump to induce aortic dissection over 4 weeks), AD + sh‐NC (AD mice injected intravenously with scrambled shRNA lentivirus [1 × 10⁸ transduction units (TU), every 7 days]) and AD + sh‐LINC01605 (AD mice treated with LINC01605‐specific shRNA lentivirus [same dose/frequency]). Lentiviral delivery commenced 3 days prior to Ang II infusion to ensure target gene knockdown during AD progression, as adapted from published protocols [15]. Lentiviral particles (in 0.1 mL PBS) were administered via the tail vein 3 days before minipump implantation to ensure preemptive gene knockdown. Mice were anaesthetised with 2% isoflurane (Vet One), and osmotic pumps were subcutaneously implanted after a dorsal skin incision. Ang II/saline infusion continued for 28 days. At endpoint, mice were euthanised with pentobarbital sodium (100 mg/kg), and aortas were harvested. AD was confirmed by histopathology (H&E staining) showing medial rupture, thrombosis or elastic fibre fragmentation. All procedures complied with NIH guidelines and were approved by the Fujian Medical University Union Hospital Ethics Committee (Approval No. IACUC FJMU 2024–0277).

Proteins were extracted with RIPA lysis buffer (Beyotime) containing protease inhibitors. Lysates (30 μg/lane) were separated on 10% SDS‐PAGE gels and transferred to PVDF membranes (Millipore, MA, USA). Membranes were blocked with 5% nonfat milk and probed overnight at 4°C with primary antibodies [α‐SMA (14395‐1‐AP, 1:6000, Proteintech), SM22α (ab14106, 1:1000, Abcam), MMP‐2 (10373‐2‐AP, 1:600, Proteintech), MMP‐9 (ab137867, 1:1000, Abcam), p62 (ab155686, 1:500, Abcam), LC3B (ab222776, 1:200, Abcam), SGK1 (28454‐1‐AP, 1:600, Proteintech), GAPDH (ab181603, 1:10,000, Abcam)]. HRP‐conjugated secondary antibodies (SA00001‐2, 1:5000, Proteintech) and ECL substrate (Millipore) were used for detection. Band intensities were quantified with ImageLab (Bio‐Rad, CA, USA).

Cells were transfected with a plasmid encoding tandem fluorescent‐tagged LC3 (GFP‐mRFP‐LC3) using Lipofectamine 2000 according to the manufacturer's instructions. After 24 h, cells were treated with sh‐LINC01605, Ang II or CQ as described above. Confocal microscopy was used to visualise the localization of GFP‐mRFP‐LC3 puncta. Images were captured using a confocal microscope (model name), and the fluorescence intensity was quantified using ImageJ software. The number of GFP − mRFP+ and GFP + mRFP+ puncta per cell was counted and averaged across multiple fields of view.

All experiments were performed in triplicate (n = 3). The experimental outcomes were processed and analysed utilising SPSS 13 (Statistical Package for the Social Sciences). The data derived from measurements are presented as the mean ± standard deviation (SD). To evaluate the differences in means between two distinct groups, an independent samples t‐test was employed. For comparisons involving more than two groups, a one‐way analysis of variance (ANOVA) was conducted. Statistical significance was established at a threshold of p < 0.05.

We obtained the datasets GSE107844 and GSE147026 from the GEO database and analysed the differentially expressed long noncoding RNAs (lncRNAs) in the aortic wall tissue of TAD patients and the normal aortic wall tissue of healthy donors from these datasets. GSE107844 identified a total of 2164 differentially expressed genes (DEGs), including 878 upregulated and 1286 downregulated genes (Figure 1A). GSE147026 identified a total of 2738 DEGs, including 1545 upregulated and 1193 downregulated genes (Figure 1B). A total of 261 differentially expressed lncRNAs were identified in GSE107844, including 105 upregulated and 156 downregulated ones. In GSE147026, 87 differentially expressed lncRNAs were obtained, with 35 upregulated and 52 downregulated. By taking the intersection of the differentially expressed lncRNAs in GSE107844 and GSE147026, 11 overlapping genes were identified through Venn analysis (Figure 1C). Among these genes, LINC01605 caught our attention. Through RT‐PCR detection, we found that it was abnormally highly expressed in the aortic wall tissue of patients with aortic dissection compared with normal aortic wall tissue (Figure 1D). Further, we isolated vascular smooth muscle cells (VSMCs) from the aortic wall tissue of patients with aortic dissection and normal aortic donors. Similarly, LINC01605 was overexpressed in VSMCs derived from patients with aortic dissection (Figure 1E). Moreover, through fluorescence in situ hybridization (FISH) localization, LINC01605 was found in the cytoplasm and nucleus (Figure 1F).

We explored the role of LINC01605 in VSMCs by knocking down and overexpressing LINC01605. Ang II significantly upregulated the expression of LINC01605 in VSMCs. After transfection, the expression of LINC01605 was significantly downregulated and upregulated, providing a reliable model for subsequent functional studies (Figure 2A,B). In terms of cell proliferation, Ang II treatment significantly enhanced the proliferation ability of VSMCs. Low expression of LINC01605 reversed this effect, while overexpression further enhanced it. In the absence of Ang II stimulation, the effect of LINC01605 on VSMC proliferation was similar to that under Ang II stimulation (Figure 2C–H). In terms of cell migration, Ang II treatment significantly improved the migration ability of VSMCs, and overexpression of LINC01605 further enhanced these effects. Similarly, in the absence of Ang II, overexpression of LINC01605 also significantly promoted the migration of VSMCs (Figure 3A–H). However, the effect of LINC01605 knockdown was opposite to that of LINC01605 overexpression (Figure 3A–H). The results of western blot showed that the expression of LINC01605 was positively correlated with the expression of MMP‐2, MMP‐9 and LC3B, and negatively correlated with the expression of α‐SMA, SM22α and p62 (Figure 4A–P). These results indicate that LINC01605 promotes the phenotypic transformation and autophagy of VSMCs with or without Ang II induction. The results of immunofluorescence further confirmed that LINC01605 promotes the occurrence of autophagy. The autophagic flux assay was employed to further validate that LINC01605 inhibits autophagy. In the Western Blot (WB) experiments, after the addition of chloroquine (CQ), both the sh‐LINC01605 group and the Ang II + sh‐LINC01605 group exhibited a significant increase in LC3B and p62 levels (Figure S1A–C). This indicates the accumulation of autophagosomes and the blockade of autophagic flux. The colocalization results of LC3‐mRFP and LC3‐GFP demonstrated that in the Sh‐LINC01605 group, there were fewer mRFP single‐fluorescence spots (red) and more GFP‐mRFP colocalization spots (yellow). This suggests an impairment in the fusion of autophagosomes with lysosomes (Figure S1D–G). After the addition of CQ, the number of mRFP single‐fluorescence spots (red) slightly increased, while the number of GFP‐mRFP colocalization spots (yellow) further increased. In the Ang II + sh‐LINC01605 group, the number of mRFP single‐fluorescence spots (red) increased, whereas the number of GFP‐mRFP colocalization spots (yellow) decreased, indicating enhanced fusion of autophagosomes with lysosomes. Following the addition of CQ, the number of mRFP single‐fluorescence spots (red) significantly increased, as did the number of GFP‐mRFP colocalization spots (yellow) (Figure S1D–G). These findings suggest that LINC01605 may regulate autophagy levels by inhibiting autophagic flux.

In this study, we predicted potential targets of LINC01605 using the ENCORI database, obtained Alzheimer's disease (AD)‐related genes from Genecards, and identified differentially expressed genes from the GSE107844 dataset. The intersection of genes derived from these three approaches was determined through Venn analysis, leading to the identification of SGK1 as a candidate target (Figure 5A). We hypothesised that LINC01605 might affect the migration, growth and autophagy of VSMCs by targeting the key regulatory factor SGK1. To verify this hypothesis, a RIP assay was performed, and the results showed that LINC01605 could be significantly enriched by SGK1 (Figure 5B). Moreover, SGK1 was overexpressed in the aortic wall tissues and VSMCs of patients with aortic dissection (Figure 5C). SGK1 was overexpressed in VSMCs derived from patients with aortic dissection (Figure 5D–F). FISH colocalization analysis revealed that LINC01605 and SGK1 were coexpressed in the cytoplasm (Figure 5G). We have already conducted functional experiments to validate the role of SGK1 in AD in vitro. These data are provided in the Supporting Information (e.g., Figures S2–, S4). Specifically, our results demonstrate that knockdown or overexpression of SGK1 significantly affects cellular processes relevant to TAD pathogenesis, such as vascular smooth muscle cell (VSMC) migration, proliferation and autophagy. Knockdown of LINC01605 significantly reduced the mRNA and protein levels of SGK1, and overexpression of SGK1 could alleviate this effect (Figure 6A–D). In terms of cell growth, the CCK‐8 assay showed that low expression of LINC01605 significantly inhibited the proliferation of VSMCs, while overexpression of SGK1 attenuated this inhibitory effect (Figure 6E). In terms of cell migration, the scratch assay indicated that knockdown of LINC01605 significantly inhibited the migration ability of VSMCs, while overexpression of SGK1 reversed this effect (Figure 6F,G). In terms of VSMC phenotypic transformation and autophagy regulation, by detecting the expression of phenotype‐related proteins (α‐SMA, SM22α, MMP‐2 and MMP‐9) and autophagy‐related proteins (such as LC3B, p62), we found that inhibition of LINC01605 expression significantly attenuated the phenotypic transformation and autophagic activity of VSMCs, while overexpression of SGK1 inhibited this effect (Figure 7A–H).

EVG and HE staining were utilised to detect pathological changes. The results of EVG staining demonstrated that the sham group exhibited a normal vascular wall structure with well‐organised elastic fibres. In contrast, the AD group showed evident disruption and disarray of elastic fibres. The AD + sh‐NC group did not exhibit significant improvement in the degree of lesion. However, the AD + sh‐LINC01605 group displayed a more orderly elastic fibre arrangement compared to both the AD and AD + sh‐NC groups, suggesting that the knockdown of LINC01605 may contribute to the restoration of the elastic fibre structure in the vascular wall (Figure S5A). The findings of HE staining indicated that the sham group had a normal vascular wall structure without any infiltration of inflammatory cells. The AD group, however, presented with substantial infiltration of inflammatory cells and proliferation of smooth muscle cells. There was no noticeable improvement in the inflammatory response in the AD + sh‐NC group. Conversely, the AD + sh‐LINC01605 group exhibited reduced inflammatory cell infiltration and decreased vascular wall thickness, indicating that the knockdown of LINC01605 aids in mitigating the pathological changes induced by AD (Figure S5B). We found that LINC01605 and SGK1 were overexpressed in the AD mouse model, and the transfection of lentivirus carrying small interfering RNA targeting LINC01605 significantly reduced their expression (Figure 8A–D). Next, we further evaluated the expression of synthetic phenotype‐related and autophagy‐related markers in the mouse aortas. In the model group, the expression of MMP‐2, MMP‐9 and LC3B was significantly increased, while the expression of α‐SMA, SM22α and p62 was significantly decreased. Low expression of sh‐LINC01605 could eliminate this effect (Figure 8E–L).