Cystatin B Promotes the Proliferation, Migration, and Invasion of Intrahepatic Cholangiocarcinoma

Abstract

Background and Aims: Cystatin B (CSTB) has been demonstrated to play a significant role in the pathogenesis of a number of diseases, including the evolution and progression of multiple cancers. Nevertheless, the function of CSTB in intrahepatic cholangiocarcinoma (iCCA) is yet to be fully elucidated. Methods: By analyzing transcriptome sequencing data from the FU-iCCA cohort, the iCCA-27 cohort, and three public databases, we identified genes associated with iCCA prognosis and selected CSTB as the subject of our study. The expression of CSTB was examined between tumor tissues and adjacent normal tissues obtained from iCCA patients via Western blot analysis. The clinical significance of CSTB was analyzed through immunohistochemical staining of a tissue microarray. Subsequently, the biological effects of CSTB overexpression or knockdown on iCCA cells were evaluated in vitro and in vivo. Results: CSTB expression was markedly elevated in the CCA pathological tissues in comparison to the corresponding adjacent normal tissues. A correlation was identified between higher CSTB expression and poorer patient prognosis in the analysis of 176 iCCA patients. It is noteworthy that overexpression or knockdown experiments demonstrated that CSTB plays a role in the proliferation, migration, and invasion of cells. In subcutaneous tumor models in nude mice, the knockdown of CSTB resulted in smaller tumors in terms of size and weight, and a slower growth rate. Conclusions: CSTB plays a significant function in the regulation of iCCA progression and may serve as a promising biomarker for iCCA.

Keywords: CSTB; intrahepatic cholangiocarcinoma; invasion; migration; proliferation.

Figure 1

Gene screening process and elevated CSTB expression of malignant cells. (A) Gene screening process. (B) The expression of CSTB in pan-cancer. (C) Comparison of CSTB mRNA expression in cancer tissues and normal tissues in TCGA, GSE107943 (D), GSE76297 (E), sequencing data of 27 pairs of cancer and adjacent tissues (F), 24 pairs of cancer and adjacent tissues (G). (H) Comparison of CSTB protein expression in iCCA and adjacent tissues. (I) Representative images of immunohistochemical expression of CSTB in tumor and adjacent tissue microarrays (J) Comparison of expression of CSTB in immunohistochemistry of cancer and adjacent tissue microarrays from 176 patients. * p < 0.1; ** p < 0.01; *** p < 0.001; **** p < 0.0001; TCGA, The Cancer Genome Atlas; iCCA, intrahepatic cholangiocarcinoma. The original Western blot figures can be found in Supplementary File S1.

Figure 2

Expression pattern and prognostic value of CSTB in iCCA. Kaplan–Meier survival curves for OS (A) and RFS (B) of 176 iCCA patients after curative resection according to CSTB expression level in the ZSH cohort. Kaplan–Meier survival curves for OS of cholangiocarcinoma patients after curative resection according to CSTB expression level in the GSE89749 (C) High-CSTB mRNA (D) or CSTB protein (E) expression was significantly associated with poor OS in the FU-iCCA cohort. Comparison of CSTB mRNA expression (F) and CSTB protein expression (G) across four molecular subtypes identified in the FU-iCCA cohort. (H) CSTB mRNA expression in patients with different TNM stages in TCGA * p < 0.1; ** p < 0.01; *** p < 0.001; **** p < 0.0001; OS, overall survival; iCCA, intrahepatic cholangiocarcinoma. WB, Western blot; * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

CSTB promotes the multiplication of intrahepatic cholangiocarcinoma (iCCA) cells. (A) The expression of CSTB in four wild-type cells. The efficiency of knockdown CSTB (B) and overexpression CSTB (C) was verified at the protein and mRNA levels. According to the CCK8 detection (D,E) and colony formation assay (F,G), it was found that the knockdown of CSTB inhibited the proliferation of iCCA cells, and the overexpression of CSTB promoted the proliferation of iCCA cells. * p < 0.05, ** p < 0.01, **** p < 0.0001. The original Western blot figures can be found in Supplementary File S1.

Figure 4

CSTB promotes intrahepatic cholangiocarcinoma (iCCA) cell migration and invasion while decreasing intrahepatic cholangiocarcinoma (iCCA) cell apoptosis. According to the scratch healing assays (A,B) and migration assay (C,D), it was found that knockdown of CSTB inhibited the migration ability of iCCA cells, and the overexpression of CSTB promoted the migration ability of iCCA cells. (C,D) Cell migration and invasion abilities analyzed by the transwell migration assay (20×). (E) Flow cytometry analysis performed to assess the apoptosis in different groups of iCCA cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: Not significant.

Figure 5

Silencing CSTB inhibited tumor growth in vivo. (A) Establishment of the subcutaneous xenograft model with nude mice using an empty vector or CSTB knockdown CCLP1 cells. (B) Tumor growth curve representing each group of mice. (C) Tumors captured from distinct groups of mice. (D,E) Analysis of tumor volumes and weight among mice conducted through statistical means. **** p < 0.0001.

Figure 6

Transcriptome sequencing analysis results of the CCLP1 control group and knockdown group. (A) Differentially expressed genes in CCLP1-shCSTB compared to CCLP1-EV. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the differentially expressed genes.