SHARPIN promotes cell proliferation of cholangiocarcinoma and inhibits ferroptosis via p53/SLC7A11/GPX4 signaling

Abstract

SHARPIN is a tumor-associated gene involved in the growth and proliferation of many tumor types. A function of SHARPIN in cholangiocarcinoma (CCA) is so far unclear. Here, we studied the role and function of SHARPIN in CCA and revealed its relevant molecular mechanism. The expression of SHARPIN was analyzed in cholangiocarcinoma tissues from patients using immunohistochemistry, quantitative PCR, and western blot analysis. Expression of SHARPIN was suppressed/overexpressed by siRNA silencing or lentiviral overexpression vector, and the effect on cell proliferation was determined by the CCK-8 assay and flow cytometry. Accumulation of reactive oxygen species was measured with MitoTracker, and JC-1 staining showed mitochondrial fission/fusion and mitochondrial membrane potential changes as a result of the silencing or overexpression. The ferroptosis marker solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and the antioxidant enzymes superoxide dismutase 1 (SOD-1) and SOD-2 were analyzed by western blot. The results showed that SHARPIN expression was increased in CCA tissue, and this was involved in cell proliferation. SHARPIN silencing resulted in accumulated reactive oxygen species, reduced mitochondrial fission, and a reduced mitochondrial membrane potential. Silencing of SHARPIN inhibited the ubiquitination and degradation of p53, and downregulated levels of SLC7A11, GPX4, SOD-1, and SOD-2, all of which contributed to excessive oxidative stress that leads to ferroptosis. Overexpression of SHARPIN would reverse the above process. The collected data suggest that in CCA, SHARPIN-mediated cell ferroptosis via the p53/SLC7A11/GPX4 signaling pathway is inhibited. Targeting SHARPIN might be a promising approach for the treatment of CCA.

Keywords: SHARPIIN; cholangiocarcinoma; ferroptosis; mitochondria; proliferation.

FIGURE 1

SHARPIN expression is increased in CCA tissue. (A) SHARPIN mRNA levels were analyzed for 36 tumor cases and 9 controls using the web‐based tool Gene Expression Profiling Interactive Analysis (GEPIA). (B) SHARPIN protein levels in tumor cells and adjacent tissue from seven CCA patients were detected with immunohistochemistry. (C, D) Immunofluorescence was performed to show the expression of SHARPIN in tumors and adjacent tissue, and expression was quantified. SHARPIN is stained red, and nuclei are stained blue. (E) SHARPIN mRNA levels in CCA tissue were higher than in normal tissue. (F, G) SHARPIN expression was higher in tumor tissue compared to adjacent normal tissues, as determined by western blot analysis. Values (mean ± SD) from quintuplicate experiments are shown. ***p < 0.001 vs adjacent group.

FIGURE 2

SHARPIN promotes the growth of CCA cell lines. (A, B) Hccc9810 and HuCCT1 cell proliferation was analyzed by CCK8 assay following siRNA transfection to silence SHARPIN and lentiviral vector to overexpression SHARPIN. (C, E) Fluorescence microscopy of the EdU assay and quantitative analyses, with EdU stained red and nuclei stained blue. (D, F) Flow cytometry demonstrates cell proliferation after EdU staining with quantitative analysis. Values (mean ± SD) from quintuplicate experiments are shown. **p < 0.01, ***p < 0.001 vs Scr siRNA group, ^# p < 0.05, ^## p < 0.01 vs SHARPIN NC group.

FIGURE 3

The SHARPIN expression influenced the content of intracellular reactive oxygen species (ROS). (A, B) Fluorescence images of intracellular ROS production loaded with the ROS probe DCF‐DA. ROS signal is shown in green and nuclei in blue. (C, D) Flow cytometry analysis of intracellular ROS generation showed changes after SHARPIN silencing. Values (mean ± SD) from quintuplicate experiments are shown. **p < 0.01 vs Scr siRNA group, ^# p < 0.05 vs SHARPIN NC group.

FIGURE 4

SHARPIN could alter the mitochondrial membrane potential and mediate fusion or fission in CCA cell lines. (A, B) After cells were silenced with siSHARPIN and overexpression with lentiviral vector, mitochondria were stained with Mito Tracker (red), and the mean fluorescence intensity was quantified. (C, D) Mitochondrial membrane potential (Δψm) was measured using the JC‐1 probe. The distribution of JC‐1 aggregates (PE channel) and monomers (FITC channel) was determined by flow cytometry. Values (mean ± SD) from quintuplicate experiments are shown. ***p < 0.001 vs Scr siRNA group, ^# p < 0.05 vs SHARPIN NC group.

FIGURE 5

Changing the expression of SHARPIN would determine cell fate with ferroptosis. (A) Western blots showing expression of p53, SHARPIN, FSP1, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), superoxide dismutase 1 (SOD‐1), and superoxide dismutase 2 (SOD‐2). β‐actin served as an internal reference. (B–H) Quantitative analysis of p53, SHARPIN, FSP1, GPX4, SLC7A11, SOD‐1, and SOD‐2 based on band density (I) Co‐immunoprecipitation assay of ubiquitin bound to p53 protein in the Hucc9810 CCA cell line following SHARPIN silencing. Values (mean ± SD) from quintuplicate experiments are shown. *p < 0.05, **p < 0.01 vs Scr siRNA group, ^# p < 0.05 vs SHARPIN NC group.

FIGURE 6

Mechanism of SHARPIN regulated cell ferroptosis via ubiquitin‐mediated p53 degradation and the SLC7A11/GPX4 signaling cascade.