ZHX1 Promotes the Proliferation, Migration and Invasion of Cholangiocarcinoma Cells

Abstract

Zinc-fingers and homeoboxes 1 (ZHX1) is a transcription repressor that has been associated with the progressions of hepatocellular carcinoma, gastric cancer, and breast cancer. However, the functional roles of ZHX1 in cholangiocarcinoma (CCA) have not been determined. We investigated the expression and roles of ZHX1 during the proliferation, migration, and invasion of CCA cells. In silico analysis and immunohistochemical studies showed amplification and overexpression of ZHX1 in CCA tissues. Furthermore, ZHX1 knockdown using specific siRNAs decreased CCA cell proliferation, migration, and invasion, whereas ZHX1 overexpression promoted all three characteristics. In addition, results suggested EGR1 might partially mediate the effect of ZHX1 on the proliferation of CCA cells. Taken together, these results show ZHX1 promotes CCA cell proliferation, migration, and invasion, and present ZHX1 as a potential target for the treatment of CCA.

Fig 1. ZHX1 was overexpressed in cholangiocarcinoma (CCA).

(A) ZHX1 amplification frequency was analyzed in various cancers using TCGA data. ZHX1 gene was found to be amplified in various cancers including CCA. Cholangio (Cholangiocarcinoma), Colorectal (Colorectal adenocarcinoma), Lung Squ (Lung squamous cell carcinoma), ccRCC (kidney renal clear cell carcinoma), AML (acute myeloid leukemia) (B) Using cBioPortal online platform, the correlation between ZHX1 gene amplification and expression of ZHX1 mRNA was plotted. (C) Using NCBI Gene Expression Omnibus database (GSE32225), the ZHX1 expression in normal bile duct and cholangiocarcinoma tissues was analyzed. (D) Immunohistochemistry showed ZHX1 was overexpressed in some CCA tissues (N = 10), and notably, ZHX1 overexpression was observed in CCA cells that had invaded lymph nodes.

Fig 2. ZHX1 regulated the proliferation of CCA cells.

(A) The effect of knockdown or overexpression of ZHX1 on the level of mRNA was examined by real-time PCR. For ZHX1 knockdown, CCA cells were treated with 100 nM of scrambled siRNA (SCR) or ZHX1 siRNA. SCR siRNA-treated samples were used as controls. For the gain-of-function study, ZHX1-overexpressing CCA cells (ZHX1-over) were generated from HuCCT1 cells, and empty control vector-expressing (Mock) cells were used as a control. GAPDH was used as an internal control. (B) Knockdown and overexpression efficiencies were determined by western blotting. (C) ZHX1 protein levels were quantified using image J software, and β-actin was used as an internal control. SCR siRNA-treated controls in the knockdown study, and Mock cells in the gain-of-function study were used as controls. (D) Cell proliferation was measured 3 to 5 days after SCR or ZHX1 siRNA transfection. SCR siRNA-treated controls in the knockdown study and Mock cells in the gain-of-function study were used as controls to calculate relative cell proliferation. Bar graphs show the means ± SEs of three independent experiments. *, P < 0.05, **, P<0.01, versus SCR or Mock.

Fig 3. ZHX1 promoted the migration of cholangiocarcinoma cells.

Migration was examined using a Boyden chamber assay and a wound healing assay. (A) ZHX1 siRNA significantly inhibited the FBS-induced migrations of SNU478 and SNU1196 cells compared with SCR siRNA. Scale bar represents 50 μm. (B) The number of migrated cells were counted and SCR siRNA-treated controls were used as controls to calculate the percentage of cell migration in ZHX1-knockdown cells. The values are shown as the bar graph. Results are the means ± SEs of three independent experiments. (C) Overexpression of ZHX1 significantly increased the migration of HuCCT1 cells versus Mock cells. Scale bar represents 50 μm. (D) The number of migrated cells were counted and Mock cells were used as controls to calculate the percentage of cell migration in ZHX1-overexpressing cells. Results are the means ± SEs of three independent experiments. (E) Migration of cholangiocarcinoma cells was examined using a wound healing assay. Cells were scratched one day after seeding, washed twice with 1X PBS, and then the fresh media containing 100 ng/ml of mitomycin C and 0.1% FBS was added. Pictures were taken at 0 hour and 21 hours after scratching. Five separate experiments were performed. (F) Migration was quantified by measuring the scratch widths. Mock cells were used as controls. The bar graphs show the means ± SEs of five independent experiments. *, P < 0.05, **, P<0.01, versus SCR or Mock.

Fig 4. ZHX1 accelerated the invasion of cholangiocarcinoma cells.

A matrigel invasion assay was used to examine the invasive ability of CCA cells. (A) ZHX1 siRNA decreased the FBS-induced invasion of SNU478 and SNU1196 cells compared with SCR siRNA. (B) The numbers of invaded cells were counted and SCR siRNA-treated cells were used as controls to calculate the percentage of cell invasion. Results are the means ± SEs of three independent experiments. (C) Overexpression of ZHX1 significantly increased the invasion of HuCCT1 cells versus Mock cells. Scale bar represents 50 μm. (D) The numbers of invaded cells were counted and Mock cells were used as controls to calculate the percentage of cell invasion. Results are the means ± SEs of three independent experiments. *, P < 0.05, **, P<0.01, versus SCR or Mock.

Fig 5. ZHX1 regulated EGR1 expression in cholangiocarcinoma cells.

(a) Real-time PCR was used to assess EGR1 mRNA level changes. EGR1 mRNA levels were measured two days after SCR or ZHX1 siRNA transfection in SNU478 or SNU1196 cells and after plating Mock and ZHX1-overexpressing HuCCT1 cells. (b) Cell proliferation was observed 3 to 4 days after SCR or EGR1 siRNA transfection in SNU478, SNU1196 and HuCCT1 cells. SCR values were used as controls to calculate relative cell proliferations. Results are presented in the bar graphs as the means ± SEs of three independent experiments. *, P < 0.05, **, P<0.01, versus SCR or Mock.