
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12626-6
10.1016/j.heliyon.2024.e36595
e36595
Research Article
CREB1 promotes cholangiocarcinoma metastasis through transcriptional regulation of the LAYN-mediated TLN1/β1 integrin axis
CREB1 promotes cholangiocarcinoma metastasis through regulating LAYN/TLN1/β1 integrin axis
Liang Shuai a
Zhou Shuhua b
Tang Yangshuo c
Xiao Moyan d
Ye Ke 404479@csu.edu.cn
d⁎
a Department of Pancreatic Surgery, Xiangya Hospital, Central South University, Changsha, 410008, China
b Comprehensive Surgery, Xiangya Boai Rehabilitation Hospital, Changsha, 410100, China
c Department of Ultrasound Medicine, Xiangya Hospital, Central South University, Changsha, 410008, China
d Department of Hepatology, Xiangya Hospital, Central South University, Changsha, 410008, China
⁎ Corresponding author. 404479@csu.edu.cn
22 8 2024
15 9 2024
22 8 2024
10 17 e3659520 4 2024
19 8 2024
19 8 2024
© 2024 Published by Elsevier Ltd.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Background

Layilin (LAYN) plays an important role in tumor progression, invasion, and metastasis; however, its role in cholangiocarcinoma (CHOL) has not been elucidated.

Methods

We utilized the GEPIA, STRING, and hTFtarget databases for bioinformatics analysis. Overexpression or knockdown cell lines were constructed by transfecting the cells with different plasmids. Western blot (WB) was performed to detect LAYN, TLN1, and CREB1 expression. Cell proliferation, migration, and invasiveness were assessed using CCK-8 and Transwell assays. Immunofluorescence and WB were used to detect epithelial-mesenchymal transition (EMT) markers. The CHOL metastasis model was established by injecting RBE cells into the tail veins of nude mice. Metastatic lesions were identified using hematoxylin and eosin staining. Co-immunoprecipitation and Chromatin immunoprecipitation were used to validate the interactions.

Results

LAYN was highly expressed in the CHOL cells. Knockdown of LAYN significantly inhibited proliferation, migration, invasion, and EMT in both QBC-939 and RBE human CHOL cells, while overexpression of LAYN had the opposite effect. Furthermore, in a CHOL metastasis model using nude mice, knocking down LAYN expression markedly suppressed CHOL liver and lung metastases. LAYN interacts with TLN1, and CREB1 binds to the LAYN promoter, with all three showing a positive correlation. Additionally, bioinformatics analysis revealed high expression of both TLN1 and CREB1 in CHOL. Knockdown of TLN1 or CREB1 in QBC-939 and RBE cells inhibited cell proliferation, migration, invasion, and EMT, reversing the effects of LAYN overexpression. Moreover, knockdown of TLN1 or CREB1 also suppressed the expression of ITGB1 and the phosphorylation levels of c-Jun, p38 MAPK, and ERK, further reversing the effects of LAYN overexpression.

Conclusion

Our results suggest that CREB1 promotes CHOL metastasis through transcriptional regulation of the LAYN-mediated TLN1/β1 integrin axis.

Keywords

Cholangiocarcinoma (CHOL)
LAYN
TLN1
β1 integrin
Metastasis
==== Body
pmc1 Introduction

Cholangiocarcinoma (CHOL), a rare and aggressive tumor originating from bile duct cells, presents significant challenges in clinical management due to its high heterogeneity and poor prognosis [1,2]. Its pathogenesis involves the dysregulation of the oncogenes, epigenome, metabolism, microenvironment, and immune evasion. Surgical resection is only feasible for a small subset of early-stage cases with high recurrence and metastasis rates. Limited treatment options are available for patients with unresectable or advanced metastatic disease [[2], [3], [4]]. Therefore, there is an urgent need to elucidate the mechanisms underlying CHOL metastasis and identify novel therapeutic targets.

Layilin (LAYN), a 55 kDa transmembrane protein similar to C-type lectins, is widely expressed in various cell types and organs. As a surface receptor for hyaluronic acid (HA), LAYN is crucial for cell adhesion, motility, and cell spread and migration [5,6]. Research has linked LAYN to tumor necrosis factor-α (TNF-α)-induced epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells [7]. Notably, low levels of LAYN have been associated with reduced cell invasion and lymph node metastasis in lung cancer cells [8], indicating its significance in tumor progression, invasion, and metastasis. However, the role of LAYN has not been extensively studied in CHOL cells, and reported studies on LAYN in CHOL are limited.

Talin1 (TLN1) is a cytoskeletal regulatory protein that plays a crucial role in various cellular processes. This protein contributes to the final stage of integrin activation and has been shown to activate integrin β1 (ITGB1), β2, and β3 [9]. TLN1 phosphorylation activates ITGB1 to promote prostate cancer bone metastasis [10]. Furthermore, in melanoma, LAYN is highly expressed and enhances integrin activation-mediated cell adhesion [11]. However, whether LAYN regulates ITGB1-mediated CHOL metastasis via TLN1 has not been reported. cAMP-responsive element (CRE)-binding protein 1 (CREB1), a key transcription factor, functions as a proto-oncogene and plays a pivotal role in various cancers, with dysregulated activity linked to oncogenesis [12]. Inhibition of CREB1 inhibits esophageal cancer metastasis [13]. Prostaglandin E2 (PGE2) activates MMP2 expression in human CHOL cells through the PGE2 receptor EP1/CREB signaling pathway, thereby regulating tumor invasion [14]. However, the interactive relationship and mechanism of action of TLN1 and CREB1 in CHOL have not been reported.

In this study, we used the STRING and hTFtarget databases to predict the upstream and downstream genes of LAYN. Furthermore, we analyzed the expression of LAYN, TLN1, and CREB1 in CHOL and their correlations using the GEPIA database. This study aimed to investigate whether CREB1, LAYN, and TLN1 can serve as novel oncogenes or prognostic factors for CHOL and to explore the potential interactions between them. These findings have important implications for the development of novel targeted therapeutic agents against CHOL.

2 Materials and methods

2.1 Bioinformatics analysis

The GEPIA database (http://gepia.cancer-pku.cn) was used to analyze the mRNA expression levels of LAYN, TLN1, and CREB1 in CHOL and normal tissues. The original images downloaded from the GEPIA database were log-transformed. The association between LAYN and TLN1 and between CREB1 and LAYN and TLN1 was characterized using the GEPIA database. The STRING database (http://string-db.org/) was used to construct Protein-Protein Interaction (PPI) networks. To mine the possible PPI relationships, we entered FADS2 into the STRING database and identified the gene with the highest connectivity as the central gene. Additionally, the hTFtarget (https://ngdc.cncb.ac.cn) and JASPAR databases (http://jaspar.genereg.net) were used to predict the binding site of CREB1 in the LAYN promoter.

2.2 Cell culture and treatment

Human normal bile duct cells HIBEpiC (CP–H175, Pricella), human CHOL cells QBC939 (BES-2522HC, BIOESN), and RBE (CL-0191, Pricella) were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS, AWC0219a, Abiowell) and 1 % penicillin/streptomycin (AWH0529a, Abiowell).

To explore the effects of LAYN on CHOL cells, we constructed the following groups: sh-NC (QBC-939 and RBE were transfected with sh-NC for 12 h, respectively), sh-LAYN (QBC-939 and RBE were transfected with sh-LAYN for 12 h, respectively), oe-NC (QBC-939 and RBE were transfected with oe-NC for 12 h, respectively), oe-LAYN (QBC-939 and RBE were transfected with oe-LAYN for 12 h, respectively).

To investigate whether TLN1 mediates the effects of LAYN on CHOL cells, we constructed the following groups: oe-NC + si-NC (QBC-939 and RBE were transfected with oe-NC and si-NC for 12 h, respectively), oe-LAYN + si-NC (QBC-939 and RBE were transfected with oe-LAYN and si-NC for 12 h, respectively), oe-NC + si-TLN1 (QBC-939 and RBE were transfected with oe-NC and si-TLN1 for 12 h, respectively), oe-LAYN + si-TLN1 (QBC-939 and RBE were transfected with oe-LAYN and si-TLN1 for 12 h, respectively).

To investigate whether CREB1 can affect CHOL cells by regulating LAYN, we constructed the following groups: oe-NC + si–NC–1 (QBC-939 and RBE were transfected with oe-NC and si–NC–1 for 12 h, respectively), oe-LAYN + si–NC–1 (QBC-939 and RBE were transfected with oe-LAYN and si–NC–1 for 12 h, respectively), oe-NC + si-CREB1 (QBC-939 and RBE were transfected with oe-NC and si-CREB1 for 12 h, respectively), oe-LAYN + si-TLN1 (QBC-939 and RBE were transfected with oe-LAYN and si-CREB1 for 12 h, respectively).

sh-LAYN (HG-SHC025407), oe-LAYN (HG-HOC025407), si-TLN1 (HG-Hi006289), and si-CREB1 (HG-SH134442) plasmids were purchased from Honorgene. The specific sequences of the plasmids are provided in the Supplementary Material 1.

2.3 Western blot (WB)

Total protein was extracted from the cells using a radioimmunoprecipitation assay buffer (AWB0136, Abiowell). After quantification using a BCA kit (AWB0104, Abiowell), total protein was separated by SDS-PAGE and adsorbed onto nitrocellulose (NC) membranes by gel electrophoresis. Primary antibodies including LAYN (1:1000, ab192610, Abcam), N-cadherin (1:1000, ab76011, Abcam), Snai1 (1:1000, 13099-1-AP, Proteintech), E-cadherin (1:1000, ab231303, Abcam), TLN1 (1:1000, ab71333, Abcam), p-TLN1 (1:1000, 13589, Cell signaling), ITGB1 (1:1000, 12594-1-AP, Proteintech), p-c-Jun (1:1000, 28891-1-AP, Proteintech), c-Jun (1:1000, 24909-1-AP, Proteintech), p-ERK (1:1000, 28733-1-AP, Proteintech), ERK (1:1000, 11257-1-AP, Proteintech), p-P38 (1:1000, 28796-1-AP, Proteintech), P38 (1:1000, 14064-1-AP, Proteintech), CREB1 (1:1000, ab32515, Abcam), and β-actin (1:5000, ab8226, Abcam) were incubated at 4 °C overnight. The HRP-conjugated secondary antibody was incubated for 90 min. β-actin was used as an internal reference.

2.4 Cell counting kit 8 (CCK-8) assay

Human CHOL cells (5 × 103 cells/100 μL/well) were seeded into 96-well plates, and CCK-8 (10 μL/well, AWC0114a, Abiowell) was added subsequently. After the cell plates were transferred to a 37 °C incubator and incubated for 4 h, cell viability was assessed by measuring absorbance at 450 nm using a microplate reader (MB-530, HEALES).

2.5 Transwell assay

To conduct the cell migration assay, the lower chamber of the transwell was filled with 500 μL of Complete Medium supplemented with 10 % FBS. Meningioma cells detached using trypsin were resuspended in a serum-free medium at a concentration of 2 × 106 cells/mL. Subsequently, 100 μL of the cell suspension was loaded into each well of the upper chamber. The cells were then incubated at 37 °C for 48 h to enable cell migration. After incubation, the upper chamber was gently removed and transferred to a new well filled with PBS. After staining, the membrane was washed five times with water and photographed under a microscope (DSZ2000X, Cnmicro) to visualize and analyze migrated cells.

To detect cell invasive ability, a sterile gun tip, EP tube, Matrigel (matrix gel), and Transwell, were required to be placed in a pre-cooled environment overnight at 4 °C, one day in advance. The cells were then incubated for 30 min at 37 °C. The final concentration was 200 μg Matrigel per well. Matrigel was diluted by adding 100 μL of ice-cold, serum-free special medium per well to a final concentration of 200 μg of Matrigel per well. The mixture was incubated for 30 min at 37 °C, and the supernatant was aspirated. The remaining procedure was performed as previously described.

2.6 Immunofluorescence (IF)

The cell slides were fixed with 4 % paraformaldehyde for 30 min, followed by treatment with 0.3 % Trilatone at 37 °C for 30 min. The slides were blocked with 5 % bovine serum albumin (BSA) for 60 min. Subsequently, the slides were incubated with primary antibodies against Snail (AWA42085, Abiowell) overnight at 4 °C. This was followed by incubation with goat anti-rabbit IgG (H + L) secondary antibody (AWS0005c, Abiowell) at 37 °C for 60 min. The nuclei were stained with DAPI at 37 °C for 10 min.

2.7 Animal model and experimental treatment protocol

Four-week-old male BALB/c nude mice (Hunan SJA Laboratory Animal Co. Ltd.) were used. After one week of acclimatization, the mice were injected with RBE cells in the tail vein at 5 × 105 cells/50 μL/per mouse [13]. The nude mice were randomly divided into two groups of six mice each: sh-NC (RBE cells transfected with sh-NC) and sh-LAYN (RBE cells transfected with sh-LAYN). During the observation period, the body weights of the tumor-bearing mice were measured once per week. After 35 days of tumor seeding, the mice were injected intraperitoneally with 7 mg/100 g excess pentobarbital solution and euthanized. Liver and lung tissues were collected for subsequent testing.

2.8 Hematoxylin-eosin (H&E) staining

The tissues were collected and fixed in 4 % paraformaldehyde and were subsequently fixed and embedded in paraffin. Tissue sections were prepared and stained sequentially with hematoxylin (AWI0001a, Abiowell) and eosin (AWI0029a, Abiowell) for 5 min. The stained sections were covered with a layer of neutral gum and examined under a light microscope (BA210T, Motic).

2.9 Co-immunoprecipitation (Co-IP)

Proteins were isolated from cell lysates using a cell lysis buffer (AWB0144, Abiowell) and incubated with normal rabbit IgG (B900610, Proteintech) or LAYN antibody (AWA50354, Abiowell) at 4 °C overnight. To achieve protein specificity, the protein complexes were incubated with protein A/G agarose beads for 2 h at 4 °C. Subsequently, the immunocomplexes underwent heat treatment at 95 °C for 5 min before being analyzed using WB.

2.10 Chromatin immunoprecipitation (ChIP)

ChIP experiments were performed using a ChIP kit (ab500; Abcam) according to the manufacturer's protocol. The pull-down step involved the use of specific antibodies and quantities: a positive control (ab1791, Abcam), a negative control (beads only), and a CREB1 antibody (AWA01868, Abiowell). Following DNA purification, 2 μL of DNA was used for quantitative PCR (qPCR) with the LAYN primers: forward 5′-TGAAACAGACCCTCAAATGCCAACT-3′ and reverse 5′-TAACAGCGGTTGTGGCAGGA3.′.

2.11 Statistical analysis

Statistical analyses were conducted using GraphPad Prism 8.0 software. The experimental data were presented as mean ± standard deviation (SD). Student's t-test was used to evaluate the differences between two groups, while one-way ANOVA and two-way ANOVA were used to analyze multiple groups. Statistical significance was set at p < 0.05.

3 Results

3.1 LAYN promotes CHOL cell proliferation, migration, invasion, and EMT in vitro

After analyzing LAYN expression levels in CHOL using the GEPIA database, we observed significantly elevated expression compared to that in the Normal group (Fig. 1A, p < 0.01). WB results indicated that LAYN expression was increased by 96.8 % and 193.6 % in human CHOL cell lines QBC-939 and RBE, respectively, compared with human normal bile duct cells HIBEpiC (Fig. 1B). To further explore the role of LAYN in CHOL, we knocked down or overexpressed LAYN respectively. WB confirmed the successful construction of the above cell lines (Fig. 1C). Subsequent evaluations (Fig. 1D–F) indicated that knocking down LAYN in QBC939 cells resulted in a 40.3 % decrease in cell proliferation at 72 h, and reductions of 33.5 % and 33.2 % in migration and invasion abilities, respectively. In RBE cells, knocking down LAYN led to a 29.2 % decrease in cell proliferation at 72 h, and decreases of 38.3 % and 46.2 % in migration and invasion abilities, respectively. Furthermore, overexpressing LAYN in QBC939 cells increased cell proliferation in CHOL cells by 26.6 % at 72 h, with increases of 27.5 % and 29.7 % in migration and invasion abilities, respectively. In RBE cells, overexpressing LAYN resulted in a 25.0 % increase in cell proliferation at 72 h, and enhancements of 18.8 % and 39.5 % in migration and invasion abilities, respectively. Additionally, changes in EMT-related molecular markers were observed. Specifically, LAYN knockdown led to increased E-cadherin expression and suppressed N-cadherin and Snail expression (Fig. 1G–H). This further demonstrates that LAYN knockdown inhibits EMT in CHOL cells. Collectively, these findings indicate that LAYN promotes cell proliferation, migration, invasion, and EMT in vitro.Fig. 1 LAYN promotes CHOL cell proliferation, migration, invasion, and EMT in vitro. A. Expression box diagram of LAYN. *p < 0.01. B. WB assay for LAYN expression. C. WB assay for detecting LAYN expression. D. CCK-8 assay for detecting cell proliferation. E. Transwell assay for detecting cell migration. F. Transwell assay for detecting cell invasion. Scale bar = 100 μm. G. IF assays for detecting Snail expression. Scale bar = 50 μm. H. WB assay for detecting E-cadherin, N-cadherin, and Snail expression. **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 3. Non-adjusted images of blots are shown in Supplementary Fig. 2.

Fig. 1

3.2 Knockdown of LAYN inhibits the metastatic ability of RBE cells in vivo

To further investigate the in vivo effects of LAYN on CHOL, we constructed a nude mouse model of CHOL using RBE cells transfected with sh-NC or sh-LAYN injected into the tail veins of nude mice. Nude mice in the sh-LAYN group weighed more, indicating that LAYN knockdown attenuated systemic metastasis of CHOL (Fig. 2A). We also measured the liver and lung weights in both groups of model mice, showing that the knockdown of LAYN in nude mice increased liver and lung weights by 31.6 % and 49 %, respectively. The liver-to-body weight ratio and lung to-body weight ratio showed similar trends (Fig. 2B). We then evaluated the metastatic nodules on the lung and liver surfaces and confirmed them using H&E staining. As shown in Fig. 2C–F, the knockdown of LAYN reduced the number of liver metastatic foci by 66.7 % and lung metastatic foci by 69.2 %, suggesting an important role for LAYN in regulating CHOL metastasis.Fig. 2 Knockdown of LAYN inhibits the metastatic ability of RBE cells in vivo. A. Weight of nude mice. B. Weights of liver and lungs of nude mice. C. Diagram showing specimens of metastatic lesions in liver and lung tissue. D. H&E staining of specimens of metastatic lesions from liver and lung tissue. Scale bar = 100 μm, and 25 μm. E-F. Statistical analysis of the number of liver and lung metastatic nodules. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 6.

Fig. 2

3.3 LAYN promotes CHOL cell proliferation, migration, invasion, and EMT through the TLN1/ITGB1 axis

We explored the potential mechanisms underlying the role of LAYN in CHOL. Using STRING database prediction, we found that LAYN might bind to TLN1 (Fig. 3A). TLN1 phosphorylation activates ITGB1 to promote bone metastasis in prostate cancer [10]. However, whether LAYN regulates ITGB1-mediated CHOL metastasis via TLN1 has not been reported. The GEPIA database revealed a significantly elevated TLN1 expression in CHOL (Fig. 3B, p < 0.01). Furthermore, Fig. 3C shows a positive correlation between TLN1 and LAYN expression. Co-IP results showed that LAYN interacts with TLN1 (Fig. 3D). Together, these results confirm the relationship between LAYN and TLN1 in CHOL. To verify whether TLN1 mediates the role of LAYN in CHOL metastasis, we constructed a TLN1-knockdown CHOL cell line. The WB results showed that the expression of TLN1 was reduced in the si-TLN1 group, indicating that the knockdown of TLN1 expression was successful. The knockdown efficiency of si-TLN1#2 was the highest for subsequent experiments (Fig. 3E). Fig. 3F–H showed that knocking down TLN1 in QBC939 cells resulted in a 38.2 % decrease in cell proliferation at 72 h, and reductions of 60.2 % and 52.1 % in migration and invasion abilities, respectively. In RBE cells, knocking down TLN1 led to a 29.9 % decrease in cell proliferation at 72 h, and decreases of 59.2 % and 62.7 % in migration and invasion abilities, respectively. This downregulation effectively reversed the enhanced proliferation, migration, and invasion induced by LAYN overexpression. Furthermore, we assessed the expression of EMT-related molecular markers. Knockdown of TLN1 expression resulted in decreased levels of N-cadherin and Snail and increased E-cadherin expression. These effects were reversed by LAYN overexpression (Fig. 3I–J). We further examined the regulatory relationship between LAYN and TLN1 expression. WB results showed that LAYN overexpression increased the p-TLN1/TLN1 ratio, thereby promoting TLN1 phosphorylation (Fig. 3K). We also analyzed the effects of the LAYN/TLN1 axis on the phosphorylation of c-Jun, p38 MAPK, and ERK. As shown in Fig. 3L, LAYN overexpression upregulated the expression of ITGB1 and the phosphorylation levels of c-Jun, p38 MAPK, and ERK, whereas TLN1 knockdown reversed this effect. These results suggest that LAYN promotes CHOL cell proliferation, migration, invasion, and EMT via TLN1/ITGB1 axis.Fig. 3 LAYN promotes CHOL cell proliferation, migration, invasion, and EMT through the TLN1/ITGB1 axis. A. STRING database for predicting proteins that may interact with LAYN. B. Expression box diagram of TLN1. *p < 0.01. C. Correlation analysis between LAYN and TLN1. D. Co-IP assay for validating the interaction between LAYN and TLN1. E. WB assay for detecting TLN1 expression. F. CCK-8 assay for detecting cell proliferation. G. Transwell assay for detecting cell migration. H. Transwell assay for detecting cell invasion. Scale bar = 100 μm. I. IF assays for detecting Snail expression. Scale bar = 50 μm. J. WB assay for detecting E-cadherin, N-cadherin, and Snail expression. K. WB assay for detecting LAYN, TLN1, and p-TLN1 expression. L. WB assay for detecting ITGB1, c-Jun, ERK, p38, p-c-Jun, p-ERK and p-p38 expression. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 3. Non-adjusted images of blots are shown in Supplementary Fig. 3.

Fig. 3

3.4 CREB1 may be an upstream target of LAYN

We further explored the regulatory mechanisms of LAYN in CHOL. Using the hTFtarget database prediction, we found that the transcription factor CREB1 might bind to the promoter of LAYN. A previous report showed that the inhibition of CREB1 suppressed esophageal cancer metastasis [15]. However, the role of CREB1 has not been extensively studied in CHOL cells. We found significantly elevated CREB1 expression in CHOL as predicted by the GEPIA database (Fig. 4A, p < 0.01). CREB1 positively correlated with the expression of LAYN and TLN1 (Fig. 4B). Further, we predicted the motifs and the promoter sequences potentially targeted by CREB1 using the JASPAR database (Fig. 4C). Subsequently, ChIP experiments confirmed CREB1 binding to the LAYN promoter (Fig. 4D). These results indicate that CREB1 upregulates LAYN expression by binding to its promoter.Fig. 4 CREB1 may be an upstream target of LAYN. A. Expression box diagram of CREB1. *p < 0.01. B. Correlation analysis of CREB1 with LAYN and TLN1. C. JASPAR database for prediction of CREB1 motifs and promoter sequences for possible targeting. D. ChIP assay for validating the binding of CREB1 to the LAYN promoter. *p < 0.05, **p < 0.01. n = 3.

Fig. 4

3.5 CREB1 promotes CHOL cell proliferation, migration, invasion, and EMT via LAYN

To explore whether CREB1 is involved in the effects of LAYN on CHOL cells, we constructed CREB1 knockdown CHOL cell lines. The WB results showed that the expression of CREB1 was reduced in the si-CREB1 group, indicating that the knockdown of CREB1 was successful. The knockdown efficiency of si-CREB1#2 was the highest for subsequent experiments (Fig. 5A). Knocking down CREB1 in QBC939 cells resulted in a 47 % decrease in cell proliferation at 72 h, and reductions of 59.2 % and 57.3 % in migration and invasion abilities, respectively. In RBE cells, knocking down CREB1 led to a 32.5 % decrease in cell proliferation at 72 h, and decreases of 57.3 % and 62.4 % in migration and invasion abilities, respectively (Fig. 5B–D). Furthermore, this downregulation reversed LAYN-induced enhancement of proliferation, migration, and invasion. Evaluation of EMT-related molecular markers revealed that decreased CREB1 expression led to reduced N-cadherin and Snail levels and increased E-cadherin expression. These effects were reversed by LAYN overexpression, as shown in Fig. 5E–F. We further examined the regulatory relationships between CREB1, LAYN, and TLN1. WB showed that CREB1 knockdown downregulated LAYN expression and the p-TLN1/TLN1 ratio. It also downregulated the expression of ITGB1 and the phosphorylation of c-Jun, p38 MAPK, and ERK, reversing the effects of LAYN overexpression (Fig. 5G–H). These results suggest that CREB1 promotes CHOL cell proliferation, migration, invasion, and EMT by regulating LAYN.Fig. 5 CREB1 promotes CHOL cell proliferation, migration, invasion, and EMT via LAYN. A. WB assay for detecting CREB1 expression. B. CCK-8 assay for detecting cell proliferation. C. Transwell assay for detecting cell migration. D. Transwell assay for detecting cell invasion. Scale bar = 100 μm. E. IF assays for detecting Snail expression. Scale bar = 50 μm. F. WB assay for detecting E-cadherin, N-cadherin, and Snail expression. G. WB assay for detecting LAYN, TLN1, and p-TLN1 expression. H. WB assay for detecting ITGB1, c-Jun, ERK, p38, p-c-Jun, p-ERK and p-p38 expression. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 3. Non-adjusted images of blots are shown in Supplementary Fig. 4.

Fig. 5

4 Discussion

In various cancer types, LAYN expression levels differ between cancerous and normal tissues [16]. Examination of TCGA data revealed elevated LAYN expression in CHOL, HNSC, and LIHC cells, whereas decreased expression was observed in BLCA, BRCA, and LUAD cells compared to that in adjacent normal tissues [17]. There have been many reports on the correlation between LAYN and cancer development. For example, LAYN, a prognostic biomarker, is correlated with immune infiltration in gastric and colon cancers [18]. Targeting LAYN inhibited hyaluronan oligosaccharide-induced metastasis and tumor-associated macrophage infiltration in colorectal cancer (CRC) [19]. Furthermore, LAYN downregulation inhibits the invasion and lymphatic metastasis of human lung A549 cells [9]. However, the specific role of LAYN in CHOL has not been previously explored, and our research aimed to address this knowledge gap. In this study, we investigated LAYN expression in human normal CHOL cells (HIBEpiCs) and CHOL cell lines (QBC-939 and RBE). Our findings revealed that LAYN expression was significantly higher in CHOL cells than in normal CHOL cells, which was consistent with the analysis of the GEPIA database. Furthermore, through LAYN knockdown and overexpression experiments in CHOL cells, we demonstrated that LAYN promotes cell proliferation, migration, invasion, and EMT in CHOL. However, a study on the role of LAYN in BRCA metastasis indicated that LAYN had no impact on the proliferation of A549 cells in vitro [8]. This may be due to the differences in the downstream targets of LAYN action in CHOL cells compared to other tumor cells. Additionally, our in vivo experiments indicated a significant reduction in the number of metastatic nodules in the liver and lungs of homozygous mice in the sh-LAYN group compared to those in the sh-NC group, suggesting that LAYN knockdown could inhibit the metastasis of CHOL. We also investigated the potential mechanisms underlying the promotional effect of LAYN on CHOL metastasis.

To explore the downstream mechanism of LAYN in CHOL, we used the STRING database to predict that LAYN might bind to TLN1. TLN1 is an isoform of TLNs that plays a crucial role in mediating cell adhesion [20,21]. TLNs play a pivotal role in mediating integrin-dependent cell adhesion to the extracellular matrix (ECM) by serving as cytoplasmic adapter proteins [22]. Additionally, TLNs are essential for activating integrins by linking them to cytoskeletal actin and organizing the recruitment of microtubules to adhesion sites [23]. Specifically, TLN1 is located in focal adhesions (FAs), which modulate integrin signaling and promote metastasis in various types of cancers, including prostate cancer [24], colon cancer [25], triple-negative breast cancer (TNBC) [26], and acute myeloid leukemia [27]. However, the significance of TLN1 in CHOL and its underlying molecular mechanisms remain unclear. Based on predictions from the GEPIA database, we observed elevated TLN1 expression in CHOL. Our findings further revealed a positive correlation between TLN1 and LAYN expression. To validate this relationship, we conducted a Co-IP assay to confirm the interaction between LAYN and TLN1. Subsequently, we observed that the knockdown of TLN1 expression in CHOL cells reversed the promotional effects of LAYN overexpression on CHOL cell proliferation, migration, invasion, and EMT. Moreover, overexpression of LAYN promoted the phosphorylation of TLN1 as well as the expression of ITGB1, a pathway downstream of TLN1, and the phosphorylation of c-Jun, ERK, and p38. This suggests that the TLN1/ITGB1 axis mediates the stimulatory effects of LAYN on CHOL cell migration, invasion, and EMT. The role of the TLN1/ITGB1 axis in tumor metastasis has been previously reported. Zhang et al. showed that silencing TLN1 in TNBC cells significantly attenuated tumor cell migration by interfering with the dynamic formation of FA with ITGB1, thereby modulating EMT [28]. Our observational data are consistent with these results.

To gain a deeper understanding of the mechanisms underlying the LAYN regulatory pathway in CHOL, we explored the upstream regulators of LAYN in CHOL. We identified a potential interaction between the transcription factor CREB1 and the promoter region of LAYN according to hTFtarget database predictions. CREB1, an oncogene, encodes a versatile transcription factor that binds to promoter cis-regulatory elements containing TGANNTCA sequences [12,29]. This transcription factor is widely expressed in various tissues and is involved in the regulation of both coding and noncoding genes. CREB1 is frequently deregulated in human cancers and modulates the proliferative and/or migratory state of tumor cells, making it a promising target for oncology therapy [30,31]. For example, the CREB1/miR-433 reciprocal feedback loop regulates the proliferation and metastasis in CRC [32]. However, the specific mechanism of action of CREB1 in CHOL remains unclear. In our study, CREB1 was predicted to be highly expressed in CHOL by the GEPIA database, and the expression of CREB1 was positively correlated with LAYN and TLN1, and the binding of CREB1 to the LAYN promoter was further confirmed by ChIP experiments. Next, we found that the knockdown of CREB1 expression in CHOL cells reversed the promotional effects of LAYN overexpression on the proliferation, migration, invasion, and EMT of CHOL cells, as well as the regulation of the TLN1/ITGB1 axis. This suggests that CREB1 mediates the TLN1/ITGB1 axis to promote the migration, invasion, and EMT of CHOL cells through transcriptional regulation of LAYN. This is similar to the findings of Wang et al. who reported that CREB1 is overexpressed in nasopharyngeal carcinoma (NPC) and promotes SRGN expression in NPC by targeting the promoter region of SRGN, which promotes NPC growth and metastasis [33].

Collectively, our findings underscore the significant overexpression of LAYN in CHOL, which drives tumor metastasis via the activation of the TLN1/ITGB1 pathway. The function of LAYN in CHOL is intricately governed by the transcription factor CREB1. Our study sheds light on the intricate mechanisms underlying the CREB1/LAYN/TLN1/ITGB1 interaction network, which is crucial for the initiation and progression of CHOL, thereby broadening our understanding of the molecular mechanisms underlying CHOL metastasis. Consequently, the CREB1/LAYN/TLN1/ITGB1 axis has emerged as a promising prognostic biomarker and potential therapeutic target for CHOL, offering a novel perspective for targeted therapeutic interventions aimed at inhibiting CHOL cell growth and metastasis.

Ethic approval

This study was approved by Animal Ethics Committee of Xiangya Hospital, Central South University (No.202300195).

Data available

All data can be obtained from the corresponding author.

Funding

This study was supported by 10.13039/501100004735 Natural Science Foundation of Hunan Province (No. 2023JJ30910 ).

CRediT authorship contribution statement

Shuai Liang: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Shuhua Zhou: Writing – review & editing, Validation, Investigation, Formal analysis, Data curation. Yangshuo Tang: Writing – review & editing, Visualization, Software, Methodology, Data curation. Moyan Xiao: Writing – review & editing, Visualization, Methodology, Data curation, Conceptualization. Ke Ye: Writing – review & editing, Validation, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Multimedia component 1

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Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e36595.
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