Identification of chondroitin polymerizing factor (CHPF) as tumor promotor in cholangiocarcinoma through regulating cell proliferation, cell apoptosis and cell migration

Figures & data

Table 1. Expression patterns of CHPF in cholangiocarcinoma tissues and normal tissues revealed in immunohistochemistry analysis

CHPF expression	Tumor tissue		Normal tissue
	Cases	Percentage	Cases	Percentage
Low	38	51.4%	5	100%
High	36	48.6%	0	-

P < 0.001

Table 2. Relationship between CHPF expression and tumor characteristics in patients with cholangiocarcinoma

Features	No. of patients	CHPF expression		P value
		Low	High
All patients	74	38	36
Age (years)				0.492
<59	36	17	19
≥59	38	21	17
Gender				0.812
Male	38	19	19
Female	36	19	17
Grade				<0.001***
1	10	10	0
2	38	26	12
3	23	0	23
lymphatic metastasis (N)				0.214
N0	58	32	26
N1	16	6	10
T Infiltrate				0.146
T1	5	5	1
T2	34	17	17
T3	32	15	17
T4	3	1	2

Table 3. Relationship between CHPF expression and tumor characteristics in patients with cholangiocarcinoma analyzed by Spearman rank correlation analysis

Tumor characteristics	index
Grade	Pearson correlation	0.724
	Significance (two tailed)	P < 0.001***
	n	71

Figure 1. CHPF was upregulated in cholangiocarcinoma

The expression of CHPF in tumor tissues of cholangiocarcinoma was detected by IHC and compared with normal tissues, showing that CHPF was upregulated in cholangiocarcinoma and associated with tumor grade. The representative images were randomly selected from at least three independent experiments.

Figure 2. Construction of cholangiocarcinoma cell models with CHPF knockdown

(A) Fluorescence imaging was performed to evaluate the efficiency of lentivirus transfection. (B) qPCR was utilized to detect the knockdown efficiency of CHPF in HCCC-9810 and QBC939 cells. (C) The successful knockdown of CHPF in HCCC-9810 and QBC939 cells was further verified by western blotting. The representative images were randomly selected from at least three independent experiments. The data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 3. CHPF knockdown inhibited cell proliferation and promoted cell apoptosis and cell cycle arrest in cholangiocarcinoma cells

(A) MTT assay showed that knockdown of CHPF significantly inhibited cell proliferation of HCCC-9810 and QBC939 cells. (B) The results of flow cytometry demonstrated that knockdown of CHPF obviously promoted cell apoptosis of HCCC-9810 and QBC939 cells. (C) Human Apoptosis Antibody Array was performed to identify the differential expression of apoptosis- related proteins between shCtrl and shCHPF groups of HCCC-9810 cells. (D) The detection of cell cycle indicated that knockdown of CHPF significantly promoted the arrest of cell cycle in G2 phase. The data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 4. CHPF knockdown inhibited cell migration and expression of EMT-related proteins

(A) Wound-healing assay showed that cell migration of HCCC-9810 and QBC939 cells was significantly suppressed by CHPF knockdown. (B) The results of western blotting showed the downregulation of EMT-related proteins in shCHPF group of both HCCC-9810 and QBC939. The representative images were randomly selected from at least three independent experiments. The data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 5. CHPF overexpression promoted cholangiocarcinoma development in vitro.

(A) qPCR and western blotting were performed to verify the overexpression of CHPF in HCCC-9810 cells. (B) MTT assay was performed to show the effects of CHPF overexpression on HCCC-9810 cell proliferation. (C, D) Flow cytometry was applied to reveal the regulatory effects of CHPF overexpression on cell apoptosis (C) and cell cycle distribution (D). (E) A wound-healing assay was performed to demonstrate the regulation of cell migration by CHPF overexpression. The representative images were randomly selected from at least three independent experiments. The data were shown as mean ± SD. *P < 0.05, **P < 0.01

Figure 6. CHPF knockdown inhibited tumor growth of cholangiocarcinoma in vivo.

(A) Volume of tumors was measured and calculated throughout the culture of animal models and showed obviously showed down growth of tumor in shCHPF group. Inset showed the photo of the removed tumors. (B, C) The bioluminescence intensity obtained by in vivo imaging showed apparently smaller tumors in shCHPF group. (D) The weight of the tumors was measured, which showed that tumors in shCHPF group were lighter. (E) The IHC analysis of Ki-67 expression in tumors showed obvious higher levels in shCtrl group. The representative images were randomly selected from at least three independent experiments. The data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001