ADAMDEC1 promotes the malignant progression of cholangiocarcinoma by regulating NF-κB signaling pathway

Abstract

Cholangiocarcinoma (CCA), a highly aggressive form of cancer, is known for its high mortality rate. A Disintegrin and Metalloprotease Domain-like Protein Decysin-1 (ADAMDEC1) can promote the development and metastasis in various tumors by degrading the extracellular matrix. However, its regulatory mechanism in CCA remains unclear. Public databases and clinical tissue samples were used to evaluate whether ADAMDEC1 expression was correlated with the prognosis of CCA. We investigated the expression of ADAMDEC1-related regulatory genes and proteins in CCA and assessed the biological behaviors of CCA cells in vitro through functional experiments. Meanwhile, the interacting proteins of ADAMDEC1 and its involvement in the nuclear factor-kappa B (NF-κB) signaling pathway were screened and verified through bioinformatics analysis. The tumorigenicity of CCA was also assessed in a xenograft nude mouse model. Our results showed that ADAMDEC1 was highly expressed in tumor tissues from CCA patients and was positively correlated with poor prognosis. Interference cell lines targeting ADAMDEC1 in CCA cells were successfully constructed. Knockdown of ADAMDEC1 or MMP12 both affected the biological behaviors of CCA cells, and ADAMDEC1 silencing inhibited tumorigenicity and tumor growth of CCA in vivo. Moreover, ADAMDEC1 interacted with MMP12, modulating its expression and promoting the activation of the NF-κB signaling pathway. Our study uncovered the expression patterns and functional roles of ADAMDEC1 in CCA cells and tissues, highlighting its connection to the NF-κB pathway and MMP12 in the development of CCA. Therefore, ADAMDEC1 may serve as a potential therapeutic target for CCA.

Keywords: ADAMDEC1; CCA; MMP12; NF-κB; Tumorigenesis.

Fig. 1

The expression levels of the ADAMDEC1 gene in CCA tissues and cells. A ADAMDEC1 expression in 53 pairs of CCA tissues and paratumor tissues was assessed by IHC staining (×200). B The Kaplan-Meier curve showed overall survival data of patients with low ADAMDEC1 expression(n = 20) and high expression(n = 33). CThe expression of the ADAMDEC1 gene was analyzed using GEO datasets and the GEPIA 2.0 database in CCA tissues. D The mRNA and protein expression levels of the ADAMDEC1 gene in HIBEPIC, HCCC-9810, RBE, and QBC939 cell lines were detected by qRT-PCR and Western blot analysis, respectively. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2

The interference efficiency of ADAMDEC1 and the biological behavior of CCA cells in vitro. A Interference sequences were transfected into QBC939 and RBE cell lines. qRT-PCR and Western blot assays were conducted detect the mRNA and protein expression levels of the ADAMDEC1 gene in the interference target groups (siADAMDEC1-1, -2, -3). B The CCK-8 assay was used to analyze the proliferation properties of the cell lines. Cell proliferation was assessed at five time points to obtain relative growth data, with measurements repeated three times (**P < 0.01). C Flow cytometry analysis was performed to assess apoptosis. The proportion of apoptotic cells in the siADAMDEC1 group was compared with the siCtrl group in QBC939 and RBE cell lines, as shown in the histogram (***P < 0.001). D The wound healing assay was used to analyze migration ability. Migration was observed in the siADAMDEC1 group at 24 and 48 h in QBC939 and RBE cell lines(*P < 0.05, **P < 0.01). E Transwell chamber assays (+ ECM) were employed to determine invasive properties. The number of invading cells was counted from five different view fields. The transwell assay demonstrated that invasion ability was significantly affected in the siADAMDEC1 group in contrast with the siCtrl group. The count was repeated three times (***P < 0.001).

Fig. 3

The expression correlation between ADAMDEC1 and MMP12 in CCA. A According to the String and TIMER2.0 databases, the protein-protein interaction network indicates that the ADAMDEC1 gene has a direct interaction with MMP12, with findings consistent across both databases. B CO-IP experiment tested the co-expression of ADAMDEC1 and MMP12 in cell lines. C IF assay detected the co-localization of MMP12 and ADAMDEC1 protein expression in each group (×400). D IHC assay assessed MMP12 protein expression in five pairs of human CCA and paratumor tissues (×400). IHC assay measured the MMP12 protein expression level in a nude mouse xenograft model of CCA between two groups (×200).

Fig. 4

The interference efficiency of MMP12 and the biological behavior of CCA cells in vitro. A Interference sequences were transfected into QBC939 and RBE cell lines. qRT-PCR and Western blot assays were conducted to detect the mRNA and protein expression levels of MMP12 in the interference target groups (siMMP12-1, -2, -3). B The CCK-8 assay was used to analyze the proliferation properties of the cell lines. Cell proliferation was assessed at five time points to obtain relative growth data (**P < 0.01). C Flow cytometry analysis was performed to assess apoptosis properties. The proportion of apoptotic cells in the siMMP12 group was compared with the siCtrl group in QBC939 and RBE cell lines, as shown in the histogram(***P < 0.001). D The wound healing assay was used to analyze migration ability. The migration ability was observed in the siMMP12 group at 24 h and 48 h in the QBC939 and RBE cell lines(*P < 0.05, **P < 0.01). E Transwell chamber technology (+ ECM) was employed to determine invasive properties. A total of five different view fields were used to count the number of invading cells. The transwell assay demonstrated that invasive ability was affected in both the siMMP12 and siCtrl groups. The count was repeated thrice (**P < 0.01, ***P < 0.001).

Fig. 5

The subcutaneous tumor formation in nude mice and the expression levels of signature proteins in the NF-κΒ signaling pathway. A KEGG signaling pathway and GO analysis (BP, CC, MF) were performed based on ADAMDEC1-binding and related genes. B Western blot assay assessed the expression levels of TNFR2, p65, p-p65, IKB-α, and p-IKB-α proteins in the NF-κB signaling pathway in cell lines transfected with NF-κB activator 1 and /or siADAMDEC1. C Representative images of BALB/c nude mice and tumor masses were taken on day 38 after inoculation with ADAMDEC1 cells, with or without shRNA-mediated silencing of ADAMDEC1. D The weight and volume curves of subcutaneous tumors in nude mice (*P < 0.05). E HE staining showed changes in cancer cells and intracellular structures. Ki67 expression in the siADAMDEC1 group was detected by IHC staining (**P < 0.01).