Ficolin-3 Activates Complement and Triggers Necroptosis in Cholangiocarcinoma Cells via the RIPK1/RIPK3/MLKL Signaling Pathway

Abstract

Ficolin 3 (FCN3) is a pattern recognition molecule that activates the complement system via the lectin pathway. While its immunological roles are known, the specific mechanisms by which FCN3 affects cholangiocarcinoma (CCA) pathogenesis remain unclear. In this study, we investigated FCN3 expression in CCA and benign cells, as well as tumor versus non-tumor tissues, using RT-qPCR and Western blotting analyses. The effects of FCN3 on CCA cell proliferation, migration, and invasion were analyzed through CCK-8, EdU, transwell, and wound-healing assays, with in vivo studies supporting these findings. The complement-mediated cytotoxicity of CCA cells was assessed using human serum with or without heat inactivation and an anti-C6 blocking antibody. Immunocytochemical staining was used to examine membrane attack complex (MAC) deposition, and an immunoprecipitation assay was adopted to evaluate the interaction between FCN3 and MASP family members. The role of FCN3 in inducing necroptosis was explored through transmission electron microscopy (TEM) and Western blotting analysis, focusing on the RIPK1/RIPK3/MLKL pathway. The results of the study demonstrate that FCN3 expression was significantly lower in CCA cells and tissues. Overexpressing FCN3 suppressed cell proliferation and migration, enhanced complement-mediated cytotoxicity via MASP2 binding, and increased MAC deposition. FCN3 also induced necroptosis through activating the RIPK1/RIPK3/MLKL pathway. These results highlight FCN3 as a tumor suppressor in CCA and suggest its potential as a therapeutic target for this malignancy.

Keywords: cholangiocarcinoma; complement activation; ficolin‐3; membrane attack complex; necroptosis.

FIGURE 1

The expression of FCN3 in CCA cells and tissues. The differential expression of FCN3 at the mRNA and protein levels between benign and tumor cells (A, B), between adjacent non‐tumor and CCA tissues (C, D) by RT‐qPCR (10 paired) and Western blotting analysis (7 paired). (E) The differential expression of FCN3 at the mRNA level between 147 adjacent non‐tumor and 98 CCA tissues in the GEO dataset. *p < 0.05; ***p < 0.001.

FIGURE 2

FCN3 significantly inhibits proliferation, migration, and clonal formation of CCA cells in vitro. Overexpression of FCN3 inhibited the proliferation, migration, and clonal formation of TFK1 and QBC939 shown by CCK‐8 (A), EDU (B), Transwell (C), scratch wound‐healing (D), and clone formation assays (E). ***p < 0.001.

FIGURE 3

FCN3 significantly inhibits the tumor formation of CCA cells in vivo. Real‐time comparison images of tumors formed by TFK1 and QBC939 groups FCN3 overexpression (A), knockdown (C), and matched control cells in nude mice. Statistical maps of tumor volume and weight in nude mice (B, D). The expression differences of FCN3 at the mRNA and protein levels between the overexpression group, knockdown group, and their respective control groups in tumor tissues of nude mice were detected by RT‐qPCR (E) and Western blotting analysis (F). The impact of FCN3 overexpression and knockdown on the proliferation levels of tumor tissues in nude mice was analyzed by IHC (G). **p < 0.01; ***p < 0.001.

FIGURE 4

Enrichment pathway analyses. KEGG pathway analysis were performed using R code based on 208 differential genes obtained by transcriptome sequencing (A), GO (B) and KEGG databases (C).

FIGURE 5

FCN3 activates complement by binding MASP2 and promotes human serum complement‐mediated cytolysis on CCA cells. (A) The sensitivity of primary CCA cells to complement‐mediated killing was validated using a human serum complement‐mediated cytotoxicity assay. (B) Overexpression of FCN3 in CCA cells enhanced the complement‐mediated cytolysis effect. However, when heat‐inactivated human pooled AB serum (Hi‐HPS) (complement inactivated by heating the serum at 56°C for 30 min) or a C6 blocking antibody were added to inhibit the complement pathway, the cytolysis effect on CCA cells was significantly weakened. (C) Co‐IP experiments confirmed the interaction between FCN3 and MASP2. (D) Results from the STRING database further supported a strong interaction between FCN3 and MASP2. (E) In TFK1 cells, red fluorescence represents the localization and expression levels of C3b, while purple fluorescence indicates the position and expression levels of C5b‐9. Blue fluorescence marks the position of the nucleus. ***p < 0.001.

FIGURE 6

FCN3 mediates the necroptosis of CCA cells through the RIPK1/RIPK3/MLKL pathway. (A) Western blotting analysis showed that the phosphorylation levels of RIPK1, RIPK3, and MLKL were increased in the FCN3 overexpression (FCN3‐OE) group, while they were reduced in the FCN3 knockdown (FCN3‐KD) group. This suggested that FCN3 upregulation enhanced the activation of the necroptosis pathway. (B) TEM confirmed that CCA cells in the FCN3‐OE group exhibited prominent features of necroptosis, such as swelling of organelles and rupture of the plasma membrane. (C) The FCN3 overexpression group showed a higher number of apoptotic and necrotic cells compared to the FCN3 knockdown group, indicating that FCN3 promoted both apoptotic and necroptotic cell death in CCA cells. ***p < 0.001.