1 Introduction

Primary liver cancer is the third most common cause of cancer mortality worldwide, accounting for 6% of all malignancies [1]. Hepatocellular carcinoma (HCC) is the main type of liver cancer with high morbidity and mortality. HCC is often detected at a late stage and is prone to intrahepatic and extrahepatic metastasis and recurrence after surgery [2, 3]. Therefore, it is critical to investigate the molecules involved in HCC development and prognosis.

Environmental variables have been shown to be linked to the pathogenesis of HCC, and abnormal epigenetic alterations lead to deregulation of specific gene expression, ultimately leading to the occurrence and progression of HCC [4]. Abnormal methylation is an initial occurrence at the onset of many cancers [5]. Several studies have indicated that DNA methylation is involved in HCC progression, which is important for early detection and prognosis [6, 7].

HCC is a highly vascular tumor, and angiogenesis occurs at various stages of HCC development and can help malignant cells invade blood vessels and metastasize [8]. Angiogenesis exhibits a complicated pattern that involves multiple processes such as migration, proliferation, and differentiation of endothelial cells. [9]. Collagen triple helix repeat containing-1 (CTHRC1) is an extracellular matrix (ECM) protein [10] connected to angiogenesis. CTHRC1 promotes vascular remodeling by facilitating cell migration [11]. Its dysregulation in many cancers suggests an association with the invasion, metastasis, and angiogenesis of cancer tissue [12]. CTHRC1 is overexpressed in liver cancer tissues and cell lines, correlating with bad prognosis [13, 14]. Thus, CTHRC1 may be used as a prognostic biomarker for HCC.

Our previous public database analysis showed that CTHRC1 is regulated by DNA methylation and is significantly associated with the overall survival of HCC, which may be a prognostic gene controlled by methylation [15]. Nevertheless, the precise mechanism of CTHRC1 in HCC remains unclear. In the present study, we aimed to investigate how CTHRC1 participates in regulating the development of HCC at the cellular level. We examined the methylation status of CTHRC1 and its effects on the biological progression of HCC cells. To better understand the molecular mechanisms of CTHRC1 affecting HCC, high-throughput sequencing analysis was employed to explore the association between angiogenesis, alternative splicing (AS), fusion genes, and CTHRC1.

2 Materials and methods

2.1 Cell culture and protocols

Human normal hepatic stellate cell line (HSC) LX-2 and three HCC cell lines HepG2, Huh-7, and Hep3B2.1, were grown at 37 °C under 5% CO2. LX-2 and Huh-7 cells were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 0.2 mg/mL G418, and 1% P/S (Meilune, China). MEM medium (Boster, China), supplemented with 10% FBS and 1% P/S, was utilized for the cultivation of HepG2 and Hep3B2.1 cells. Additionally, HepG2, Huh-7, and Hep3B2.1 were treated with 5 μM 5‐Aza‐DC for 72 h to explore the effects of methylation on CTHRC1.

2.2 Agena MassARRAY® methylation analysis

Epidesigner (http://www.epidesigner.com, Sequenom, USA) was employed to generate the primers. To optimize PCR conditions, a 10mer tag was added to the 5ʹ end of the forward primer, and a T7-Promoter sequence was inserted at the 5 ʹ end of the reverse primer for in vitro transcription (IVT). Designed primers were sent to bio-company for synthesis. Genomic DNA was isolated through QIAamp DNA Mini Kit (QIAGEN, Germany) and adjusted to a concentration of 75 ng/μL. The DNA was then sulfite-treated with the EZ DNA Methylation-Gold Kit (ZYMO, USA). Treated DNA was used as a template to perform PCR using PCR Accessory Set (Sequenom). The PCR products were sequentially subjected to shrimp alkaline phosphatase (SAP) treatment, IVT, and RNase digestion using a MassCLEAVE Kit, Sequenom). The products were purified using a Spectro CHIP® Arrays and Clean Resin Kit (Sequenom). All steps adhered to the manufacturer's instructions. The purified products were put onto 384 format SpectroCHIP chips using the MassARRAY Nanodispenser RS1000 (Sequenom). Chips were placed into a MassARRAY Compact System for mass spectrometry detection. The data were analyzed by the EpiTYPER software (Sequenom).

2.3 Quantitative real-time (RT-qPCR)

The total RNA of four cell lines was extracted using TRIzon Reagent (cwbio, China) and reverse transcribed to cDNA. The mRNA levels of CTHRC1 and angiogenesis-related genes were assessed by RT-qPCR using SYBR® Green Premix Pro Taq HS qPCR Kit and GADPH was considered as internal reference genes. The reaction system was programmed as follows: incubation at 95 ℃ for 30 s repeated 2 times, followed by 40 cycles for 5 s at 95 ℃, and 30 s at 60 ℃ repeated 3 times. The data were analyzed by the 2−ΔΔct method. The specific primers are presented in Table S1.

2.4 Western blot analysis

Cells were washed twice with PBS and lysed with RIPA (Boster) containing protease inhibitor for 3–5 min. The extracted protein was evaluated by BCA Protein Assay Kit (Beyotime, China). Subsequently, 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed to transfer protein to PVDF membranes (Millipore, USA). After blocking with 5% skim milk at room temperature for 1 h, the membranes were incubated overnight at 4 ℃ with the anti-CTHRC1 antibody (Sigma, China). Subsequently, they were exposed to a secondary antibody at room temperature for 30 min. The protein was quantitated using ImageJ (https://imagej.nih.gov/ij/). GADPH (Abclonal, USA) normalized the data of CTHRC1 genes.

2.5 Construction of CTHRC1 knockdown in Hep3B2.1 cells and cell transfection

The stable cell line with CTHRC1 knockdown was constructed by transfection of Hep3B2.1 cells with CTHRC1 short hairpin RNA (shRNA, inhibitor) or negative control plasmids (NC) (Biomed, China). The CTHRC1 inhibitor or NC was diluted with 250 μL Opti-Medium to a concentration of 50 nM. 6 μL Lipofectamine™ 2000 was mixed with 250 μL Opti-Medium and gently swirled. The diluted buffers were gently mixed and allowed to stand for 20 min. The mixture was added to a 6-well plate at 500 μL/well. Finally, the inhibited effect of CTHRC1 in the stable strain was tested.

2.6 Methyl thiazolyl tetrazolium (MTT) assay

The CTHRC1 inhibitor group or NC group was plated in 96-well plates (2000/well) and cultured for 24 h, 48 h, 72 h, and 96 h, respectively. For 4 h before culture termination, 20 μL of 5 mg/mL MTT was applied to the wells. After 4 h the medium was taken out. DMSO was added to the plated and vortexed for 5 min. Cell samples were detected at 490/570 nm using enzyme labeling equipment.

2.7 Cell scratch assay

Two straight lines were marked on the bottom of 12-well plates as a guide. Cells were seeded in the plates (1.5 × 105/well) and grown overnight. A scratch was made along the lines. After washing with PBS, 0.5% FBS was applied. Cells were cultured for 0 h, 24 h, and 48 h, and photographed. The migration rate was calculated based on the width of the scratch.

2.8 Cell invasion assay

Transwell PC membrane inserts (Corning, USA) were utilized for cell invasion assays. Cells were harvested and deposited in the top chamber, while 500 μL complete medium with 10% FBS was added to the below chamber. Then they were incubated at 37 °C for 48 h. Cells that did not pass through the chamber were removed. Cells were fixed with 4% paraformaldehyde for 30 min and the top chamber was inverted and air-dried for 30 min. Cells were stained with 500 μL 0.1% crystal violet (Sangon Biotech, China) for 30 min. After washing, the number of invading cells was counted from three fields in a single chamber.

2.9 Cell cycle

Adherent cells were grown to ~ 80% confluence, and collected in the centrifuge tubes for centrifugation. Cells were washed with cold D-Hanks (pH = 7.2–7.4, Meilune). The sample was centrifuged and then fixed with 75% ethanol for at least 1 h. After centrifugation, it was washed with D-Hanks. Cells were stained with cell staining buffer (the ratio of 40 × PI, 100 × RNase, and 1 × D-Hanks: 25, 10, and 1000) and detected.

2.10 Cell apoptosis assay

1 × binding buffer was used to resuspend the collected cells. 100 μL resuspended cells were incubated in the dark for 15 min with 5 μL Annexin V-PE and 5 μL 7-AAD (Meilune). The samples were mixed with 400 μL 1 × Binding Buffer and detected by flow cytometry (NovoCyte, China).

2.11 RNA sequencing and data preprocessing

The CTHRC1 inhibitor group and NC group (at least three replicates per group) were subjected to perform RNA sequencing. Total RNA was isolated from cells using the TRIzol reagent. DNBseq platform (PE150 strategy) was employed to perform RNA sequencing. Low-quality sequences, such as adapter sequences, those with a quality score < 20, and sequences with an N base rate of raw reads > 10%, as well as those less than 25 bp, were deleted to generate clean reads. The mapped data was obtained by aligning clean data and human reference genome using HISAT2 (version 2.1.0).

2.12 Identification of differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs), and functional annotation

DEmRNAs and DElncRNAs were obtained through DEseq2 with p < 0.05 and |log2foldchange|> 1. Correlation analysis was performed between DEmRNAs, DElncRNAs, and CTHRC1. GeneCodis4.0 (https://genecodis.genyo.es/) was employed to perform GO classification (p-val adj < 0.05) for DEmRNAs [16]. We used the hallmark gene set (h.all.v2022.1.Hs.symbols.gmt) obtained from MSigDB (https://www.gseamsigdb.org/gsea/msigdb/) to conduct gene set enrichment analysis (GSEA) with a significance level of P < 0.05.

2.13 Angiogenesis analysis

A total of 36 mRNAs related to angiogenesis were acquired from the MSigDB database [17]. CXCL6 was obtained from the intersection of 36 angiogenesis-related mRNAs, and CTHRC1-related DEmRNAs. Furthermore, intersecting lncRNAs were obtained overlapping the CTHRC1-related DElncRNAs with the CXCL6-related DElncRNAs.

2.14 AS analysis

AS is essential for gene expression in eukaryotes. During the process of converting mRNA precursors to mature RNAs, accurate excision of introns/non-coding sequences and combination of exons occurs through different splicing modes, which increase transcriptome and proteome diversity [18]. AS analysis was performed from RNA sequencing data with FDR < 0.05 and LncLevel Difference > 0.05 using rMATS (http://rnaseq-mats.sourceforge.net/index.html). The AS events identified by rMATS were classified into the following five categories: skipped exon (SE), alternative 5ʹsplice site (A5SS), alternative 3ʹsplice site (A3SS), mutually exclusion exon (MXE), retained intron (RI).

2.15 Gene fusion analysis

We used Arriba to analyze NGS RNA fastq data from the NC and CTHRC1 inhibitor samples to obtain nRMA fusion genes [19]. The common fusion gene events were obtained by intersecting the fusion genes from the three replicate samples of the NC and CTHRC1 groups, respectively. The common fusion gene events from the NC and CTHRC1 groups were intersected to investigate the changes in gene fusion events after CTHRC1 inhibition.

2.16 Statistical analysis

The statistical software SPSS 20.0 (Chicago, USA) was used to conduct the analyses. The mean ± standard deviation (SD) was displayed for the data. Two samples were compared using the t-test. GraphPad Prism version 8.3.0 (USA, www.graphpad.com) was used to conduct a one-way ANOVA on more than two samples. P values < 0.05 were considered statistically significant.

3 Results

3.1 Expression of CTHRC1 correlates with DNA methylation levels in HCC cell lines

Our previous study identified five differentially methylated sites (DMSs) in the promoter region of the CTHRC1 [15]. Three HCC cell lines (HepG2, Huh-7, and Hep3B2.1) and one HSC cell line (LX-2) were included to explore the DNA methylation level of CTHRC1. Methylation assay indicated lower methylation levels in these DMSs in HCC cell lines (Fig. 1A). Additionally, the levels of CTHRC1 were significantly increased in HCC cell lines compared with LX-2 cells (Fig. 1B, C, Figs. S1-2).

Fig. 1
figure 1

Expression of CTHRC1 correlates with DNA methylation levels in HCC cell lines. A The methylation levels of CTHRC1 in the promoter region were detected by Agena MassARRAY® Methylation in HSC cell line LX-2 and three HCC cell lines HepG2, Huh-7, and Hep3B2.1 (left), and presented in hot map (right). B RT-qPCR and C western bolt were used to detect the mRNA and protein levels of CTHRC1 in LX-2, HepG2, Huh-7, and Hep3B2.1 cell lines. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. LX-2 group. D RT-qPCR was used to detect the mRNA levels of CTHRC1 in LX-2, HepG2, Huh-7, and Hep3B2.1 cell lines treated with 5-Aza-DC. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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5-Aza-DC is a frequently employed agent for DNA demethylation. The mRNA levels of CTHRC1 were significantly decreased in HepG2, Huh-7 and Hep3B2.1 cell lines after treatment with 5-Aza-DC (Fig. 1D). These results suggested a tight correlation between the expression of CTHRC1 and its methylation level in HCC cell lines. Thus, further investigation was conducted into the functions of CTHRC1 in HCC cell lines.

3.2 Downregulation of CTHRC1 inhibited cell proliferation, migration, and invasion, blocked cells in the G1/S phase, and promoted apoptosis

To clarify the role of CTHRC1 in HCC, we constructed a CTHRC1 knockdown cell model using shRNA-induced suppression in Hep3B2.1 cells. As shown in Fig. 2A, CTHRC1 knockdown was successfully constructed in Hep3B2.1 cells. MTT and scratch assays indicated that after CTHRC1 downregulation, both the proliferation and migration were notably reduced in a time-dependent manner (Fig. 2B, C). We used a transwell assay to evaluate the effect of CTHRC1 on cell invasion. The results showed that after CTHRC1 downregulation, the invasion was significantly decreased in Hep3B2.1 cells (Fig. 2D).

Fig. 2
figure 2

Effects of CTHRC1 inhibition on HCC cell lines. CTHRC1 inhibitor Hep3B2.1 cells were constructed. A Validation of CTHRC1 expression. B Cell proliferation was evaluated for 24 h, 48 h, 72 h, and 96 h using MTT assay on 490 nm. ***P < 0.001 vs. NC group. C Cell migration at 0 h, 48 h, and 72 h were evaluated by cell scratch assay. D Cell invasion was detected at 48 h. E Cell cycle and F cell apoptosis were assessed using flow cytometry. **P < 0.01, ***P < 0.001, ****P < 0.0001

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Since the cell cycle and apoptosis are the main factors affecting cell proliferation, we investigated whether CTHRC1-mediated inhibition of HCC cell growth was related to apoptosis and cell cycle. The results revealed a significant increase in cell counts in the G1 phase following the downregulation of CTHRC1, with no notable difference observed in the S phase (Fig. 2E). The results suggested that downregulation of CTHRC1 might arrest the transition from G1 to S phase. Meanwhile, apoptosis detection showed that downregulation of CTHRC1 significantly promoted the early apoptosis of Hep3B2.1 cells (Fig. 2F). Taken together, those results suggested that CTHRC1 may play a crucial role in the regulation of HCC.

3.3 Identification of DEmRNAs and DElncRNAs

High-throughput transcriptome sequencing analyses were further performed to explore the molecular mechanisms that regulate HCC by CTHRC1. In total, 34 DEmRNAs, including 27 upregulated and 7 downregulated mRNA, and 7 DElncRNAs, including 5 upregulated and 2 downregulated lncRNAs (Fig. 3A, B, Table 1) were screened between the two groups. The correlation analysis between DEmRNAs and CTHRC1 showed that 15 DEmRNAs, including SEMA3D, PCDH7, IFNK, GJB7, TNFRSF19, LRP4, KITLG, LRP1B, PYGM, CXCL6, GCNT1, VN1R48P, SLC22A20P, MEGF10, and SLC2A14, were negatively correlated with CTHRC1, while MYEOV and AC027796.3 were positively correlated with CTHRC1 (Fig. 3C). The correlation analysis between DElncRNAs and CTHRC1 showed that AC020978.8 and AL162231.4 were positively correlated with CTHRC1, while AP001636.3, LCAL1, and LINC02127 were negatively correlated with CTHRC1 (Fig. 3D). Moreover, the correlation analysis between DEmRNAs and the correlation analysis between DEmRNAs and DElncRNAs were acquired (Fig. 3E and F). Among them, CXCL6 significantly correlated with 6 DElncRNAs (Fig. 3F).

Fig. 3
figure 3

Identification of DEmRNAs and DElncRNAs in HCC cell line. RNA-seq was performed in the NC and CTHRC1 inhibitor groups. Identification of A DEmRNAs and B DElncRNAs. Correlation analysis between CTHRC1 and C DEmRNAs or D DElncRNAs. Correlation analysis between E DEmRNAs or F DEmRNAs and DElncRNAs

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Table 1 Downregulation and upregulation of DEmRNAs/DElncRNAs
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3.4 Functional enrichment analysis of DEmRNAs and CTHRC1

GO enrichment analyses were used to evaluate the function of DEmRNAs. GO analysis showed that these DEmRNAs were primarily found in cellular components including the integral component of the membrane, integral alpha–beta 8 complex, extracellular space, receptor complex, etc. They were enriched in various biological processes such as cell adhesion, cell–cell signaling, protein dephosphorylation, endocytosis, and other biological processes (Fig. 4A). Furthermore, GSEA enrichment analysis was used to find the potential biological function of CTHRC1. The results showed that four signature signaling pathways were significantly enriched in the CTHRC1 inhibitor group, including apical junction, KRAS signaling up, Wnt/β-catenin signaling, and IFN-γ response (Fig. 4B).

Fig. 4
figure 4

Functional enrichment analysis of DEmRNAs. A GO and B GESA analysis of DEmRNAs

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3.5 Angiogenesis and CTHRC1

We acquired 36 mRNAs related to angiogenesis from the MSigDB database. These mRNA expressions were upregulated in angiogenesis. CXCL6 was obtained from the intersection of 36 mRNAs related to angiogenesis and 17 CTHRC1-related DEmRNAs (Fig. 5A). Furthermore, 5 CTHRC1-related DElncRNAs and 6 CXCL6-related DElncRNAs were taken to intersect to obtain 4 lncRNAs, including AC020978.8, AL162231.4, AP001636.3, LINC02127 (Fig. 5B). RT-qPCR results showed that CXCL6 and LINC02127 were significantly increased, and AC020978.8 was significantly decreased in Hep3B2.1 cells with CTHRC1 downregulation (Fig. 5C).

Fig. 5
figure 5

Angiogenesis and CTHRC1 in HCC cell line. A CXCL6 was obtained from the intersection of angiogenesis-related genes and CTHRC1-related DEmRNAs. B The intersection of CTHRC1-related DElncRNAs and CXCL6-related DElncRNAs. C RT-qPCR was used to detect the mRNA levels of CXCL6, LINC02127, and AC020978.8 in CTHRC1 inhibitor Hep3B2.1 cells. *P < 0.05, **P < 0.01

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3.6 AS, gene fusion analysis and CTHRC1

In HCC, abnormal AS events were frequently observed and could affect the functionality of cancer cells [20]. Therefore, we investigated the impact of CTHRC1 on AS. We identified 12 significant differential AS events between the CTHRC1 inhibitor group and NC group, including 2 A3SS, 9 SE, and 1 RI (Table 2). Abnormal transcription or trans-splicing events may generate fusion genes. The fusion events in the three replicate samples of NC group were 58, 66, and 60, and there were 23 common fusion genes in the NC group. The fusion events in the three replicate samples of the CTHRC1 inhibitor group were 49, 77, and 60, and there were 24 common fusion genes in the CTHRC1 inhibitor group. Differential fusion event analysis showed that 3 fusion events appeared in the CTHRC1 inhibitor group compared with the NC group, including PHRF-DRD4, ACSL3-FARSB, STAG2-H3P47 (27352)-AL030996.1 (16126), while 2 fusion events, GNB5-MYO5A and PI4KB-TCHHL1, were missing. The characterization of these fusion genes is listed in Table 3.

Table 2 Difference alternative splicing
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Table 3 The characterization of fusion genes
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4 Discussion

Despite extensive research on HCC markers, effective biomarkers for HCC in clinical practice remain elusive [21, 22]. CTHRC1 is linked to cirrhosis, vascular invasion, HCC tumor size, and stage, suggesting it may serve as a promising biomarker [14]. In the present study, we comprehensively analyzed the mechanisms of CTHRC1 in regulating the development of HCC.

CTHRC1 has been recognized as a protein associated with cancer, and its elevated levels have been linked to the occurrence, proliferation, invasion, and metastasis of a variety of cancers [10, 23]. Knockdown of CTHRC1 can inhibit proliferation in renal cell carcinoma cells [24]. Overexpression of CTHRC1 promotes tumor aggressiveness and is related to poor prognosis [25]. Furthermore, knockdown of CTHRC1 in HepG2, Huh7 and HA22T cells reduced cell proliferation, migration, and invasion [15, 26]. Consistent with these results, we found that downregulation of CTHRC1 expression could influence the functions of Hep3B2.1 cells. Our results supported previous findings and further illustrated the important role of CTHRC1 in HCC.

DNA methylation has received considerable attention in the cancer field due to advances in sequencing technology. DNA methylation has been shown to progress dynamically from healthy individuals to cirrhosis patients to those with HCC [6]. Additionally, DNA methylation patterns are different in HCC based on the associated risk factors, such as hepatitis B Virus (HBV), hepatitis C Virus, alcohol and non-alcoholic fatty liver disease [27]. Our study observed varied methylation patterns of CTHRC1 across three different HCC cell lines, possibly reflecting the tumor's heterogeneity. Regions of low differentiation and invasiveness within HCC tend to express higher levels of CTHRC1 [28]. Tumor aggressiveness may influence the gene's methylation status. HBV infection upregulates CTHRC1 expression, promoting the progression of HBV-associated HCC [29]. HBV X protein influences DNMT activity in HCC cell lines [30]. In glioma, CTHRC1 methylation has shown a negative correlation with tumor grade [31]. Therefore, it is speculated that CTHRC1 methylation status may differ across various pathological grades, etiologies, and invasive states of HCC.

Following this, RNA-seq was performed to explore the molecular mechanisms of CTHRC1 in HCC. The functional enrichment analysis results of DEmRNAs between the NC and CTHRC1 inhibitor groups indicated that they participate in a range of biological functions, including cell–cell signaling, integral components of membrane, cell adhesion, etc. GSEA results indicated that CTHRC1 may regulate HCC via the Wnt/β-catenin, IFN-γ, apical junction, and KRAS signaling pathways. The Wnt/β-catenin pathway is aberrantly activated in HCC and plays a crucial role in regulating HCC migration, invasion, and angiogenesis [32, 33]. A previous study has revealed an important association between CTHRC1 and the Wnt/β-catenin pathway in HCC [34]. IFN-γ is known to exert anti-tumor effects and is linked to immunotherapy in HCC [35]. CTHRC1 took part in the HBV-induced immune response by regulating the IFN pathway [36]. Apical junction proteins could potentially be utilized for the diagnosis and treatment of cancer [37]. Mutations in KRAS are important drivers of HCC tumorigenesis [38]. There are no published reports regarding the potential role of CTHRC1, along with KRAS and apical junction pathways, in the development of cancer.

In the present study, one mRNA (CXCL6) and four lncRNAs (AC020978.8, AL162231.4, AP001636.3, and LINC02127) were identified as potential players in angiogenesis in HCC. CTHRC1 has been reported to be involved in angiogenesis in cancer. For example, CTHRC1 promotes angiogenesis in lung adenocarcinoma via fatty acid metabolism [39]. Additionally, CTHRC1 can mediate cancer cell invasion and angiogenesis through the TGF-β pathway [40]. TGF-β, as a growth factor, plays a critical role in angiogenesis and remodeling processes [41]. In the early stages of tumorigenesis, TGF-β induces cell cycle arrest in late G1, preventing cell proliferation [42]. This may be one of the pathways that block the cell cycle transition from G1 to S phase after inhibiting CTHRC1 expression. CXCL6 is involved in the development and angiogenesis of multiple cancers [43]. It was reported that CXCL6 is regulated by circRNAs and microRNAs, and participates in HCC growth and invasion [44]. Moreover, high expression of CXCL6 is associated with angiogenesis and poor prognosis in HCC [45]. Several lncRNAs have been implicated in HCC progression and angiogenesis, including MYLK-AS1, PAARH, and TGFB2-OT1 [46,47,48]. AL162231.4 was strongly associated with overall survival in HCC [49]. LINC02127 may potentially correlate with airway wall thickening in chronic obstructive pulmonary disease [50]. AC020978.8 and AP001636.3 are novel lncRNAs with no existing reports in HCC.

Studies have shown that AS may serve as prognostic markers for HCC, and targeting AS may regulate the growth and metastasis of HCC [51, 52]. Some genes corresponding to differential AS may be critical in cancer biology. For example, ATXN3 has been implicated in the ubiquitin–proteasome system, which is known to be dysregulated in cancer [51]. CACNB2 is related to calcium channel, which plays a significant role in cancer signaling and drug resistance [53]. Therefore, CTHRC1 may contribute to the malignant phenotype by influencing these differential AS. At the transcriptional level, fusion genes can indicate not only potential genomic rearrangements, but also can originate due to aberrant transcription or trans-splicing events [54]. Many fusion genes have been identified as causative agents in various types of human cancers, including HCC [55, 56]. Among them, STAG2 gene has been associated with chromosomal instability in cancer, and disruptions in its normal function could contribute to oncogenesis [57]. Overall, the effects of CTHRC1 in HCC may be related to alterations in AS and gene fusion events.

The current study has some limitations. The study was conducted only at the cellular level, and future investigations should be employed in animal models to further explore the mechanism of CTHRC1 in HCC development. Moreover, the RNA sequencing conducted in this study was limited to one cell line, which may not fully represent the heterogeneity of HCC. Furthermore, the correlation between CTHRC1-related DNA methylation and angiogenesis should also be studied in detail. Additionally, it is necessary to explore the clinical applications of CTHRC1 in HCC.

5 Conclusion

In the present study, we discovered that high expression of CTHRC1 is associated with hypomethylation and could regulate various biological functions in Hep3B2.1 cells. Additionally, the role of CTHRC1 in HCC development may be linked to 12 AS and 5 gene fusion events. Importantly, CXCL6, AC020978.8, and LINC02127 may be key regulatory genes and lncRNAs associated with CTHRC1, playing crucial roles in angiogenesis. Taken together, this study may provide new insights into the regulatory mechanisms of HCC.