CLIC1 Drives Angiogenesis in Hepatocellular Carcinoma by Modulating VEGFA

Clinical Samples

A tissue microarray was constructed by Shanghai Zhuoli Biotechnology Co., Ltd. Sixty-seven pairs of HCC tissues and paracarcinoma tissues were fixed in formaldehyde and embedded in paraffin for further pathological analysis.

IHC Analysis

IHC was performed in 67 pairs of HCC tissues and paracarcinoma tissues. Formalin-fixed paraffin-embedded tumor tissues of 4 μm thickness were dewaxed in xylene and rehydrated using decreasing concentrations of ethanol. Endogenous peroxidase activity was blocked with 3% H₂O₂ methanol solution for 10 min at room temperature. The slides were further placed in EDTA buffer for antigen retrieval and blocked with 200 μL of 5% fetal bovine serum (FBS) for 5 min followed by overnight incubation of primary antibodies at 4°C. The primary antibodies used were against CLIC1 (1:100, 14545-1-AP, Proteintech), CD34 (1:1000, GB13013, Servicebio), and VEGFA (1:300, GB11034, Servicebio). After incubation, the slides were washed in phosphate-buffered saline and treated with the corresponding biotinylated secondary antibody for 60 min at room temperature. 3′-Diaminobenzidine was used as the chromogenic substrate. After counterstaining with Mayer's hematoxylin, the slides were dehydrated, cleared, and mounted. The IHC score of CLIC1 was calculated as P Score × I Score (P Score: 0: positive cells <10%; 1: positive cells <25%; 2: positive cells <50%; 3: positive cells <75%; 4: positive cells >75%; and I Score: 1: weak; 2: moderate; 3: strong).^22,23 Patients with IHC scores ≥4 were defined as having high CLIC1 expression, and patients with IHC scores <4 were defined as having low CLIC1 expression.

Cell Culture

HCC cell lines including BEL7402 (Accession number: CBP60192) and Huh7 (Accession number: CBP60202) were obtained from Cobioer Biosciences. And the vascular endothelial cell line EA.hy926 (Accession number: GNHu39) was obtained from the bank of the Chinese Academy of Sciences. The BEL7402 cell line was cultured in Roswell Park Memorial Institute medium 1640 supplemented with 10% FBS. The Huh7 and EA.hy926 cell lines were cultured in Dulbecco's modified Eagle medium supplemented with 10% FBS. All cells were cultured in an incubator at 37°C with 5% CO₂.

Cell Transfection

The BEL7402 cell line and Huh7 cell line used for coculture with EA.hy926 and preparation of conditioned media (CMs) were infected with control/CLIC1 overexpression plasmids and control/CLIC1 siRNA (Genechem Company). For xenograft assay in nude mice, the BEL7402 cell line was infected with negative control (NC)/CLIC1 overexpression lentivirus (GV492, Genechem Company). And the Huh7 cell line for xenograft assay was infected with NC/CLIC1 RNA interface (RNAi) lentivirus (GV248, Genechem Company). The RNAi sequence targeting CLIC1 was 5′-TCGGTACTTGAGCAATGCCTA-3′. Infected cells were incubated with media with puromycin (6 μg/mL) for 1 week, and then the expression of CLIC1 was verified by western blot.

EA.hy926 Migration Assay

To examine the role of vascular endothelial growth factor (VEGF) in angiogenic activity in CMs of BEL7402/Huh7 cells, we collected the CMs from different groups as follows: (1) Huh7 NC/CLIC1-knockdown, (2) Huh7 NC/CLIC1 overexpression, (3) Huh7 CLIC1 overexpression + AntiVEGF (Bevacizumab) (A2006, Selleck Chemicals), (4) BEL7402 NC/CLIC1-knockdown, (5) BEL7402 NC/CLIC1 overexpression, and (6) BEL7402 CLIC1 overexpression + AntiVEGF. The levels of VEGFA were measured using an ELISA kit (ELH-VEGF-CL, Raybiotech) according to the manufacturer's instructions.

For the EA.hy926 migration assay, 4 × 10⁴ EA.hy926 cells were seeded in the upper chamber with FBS-free media, and 4 × 10⁴ HCC cells (Huh7/BEL7402) or CMs of HCC cells were placed in the lower chamber of a 24-well transwell plate (8 μm, Corning). After 24 h of incubation at 37°C and 5% CO₂, the cells were fixed with methanol and stained with 0.5% crystal violet. Cells on the upper surface were removed with cotton swabs, and cells that migrated to the lower surface were counted under an optical microscope. The experiment was repeated 3 times.

Animal Models

Twenty 4- to 6-week-old male nude mice were randomly divided into 4 groups and maintained at the Zhejiang Academy of Medical Sciences. A total of 5 × 10⁶ HCC cells (Huh7/BEL7402) infected with lentivirus were subcutaneously injected into the right side of the mouse dorsal flanks. ²⁴ The xenograft tumors were measured by a Vernier caliper once a week. Tumor volume was estimated by the following formula: V = 1/2 × length × width × width. The mice were sacrificed when the tumor volumes reached 1500 mm³ or in the fourth week. The reporting of this study conforms to ARRIVE 2.0 guidelines. ²⁵ In animal experiments, the authors fully follow the requirements of animal ethics and comply with the “Guide for the Care and Use of Laboratory Animals, Eighth Edition.” ²⁶ The authors have made their efforts to minimize the number of animals utilized and to decrease their suffering.

Western Blot

Western blotting was performed according to the directions described in our previous research. ²⁷ The primary antibodies used were as follows: CLIC1 (1:1000, 14545-1-AP, Proteintech) and β-actin (1:1000, A5441, Sigma).

Statistical Analysis

Unpaired Student's t-test was used to assess the differences between 2 groups, and a multivariate linear regression analysis was performed to determine the correlation between CLIC1 expression and clinicopathological inference.

High Expression of CLIC1 is Correlated with Higher MVD

IHC was performed on 67 pairs of HCC and paracarcinoma tissues to evaluate the expression levels of CLIC1 and CD34. We analyzed the MVD of slices stained with CD34. The results indicated that CLIC1 expression was higher in HCC tumor tissues than in paracarcinoma tissues (Figure 1A). Patients with increased CLIC1 expression showed a higher MVD (Figure 1B) (P = .013).

High Expression of CLIC1 is Associated with Poor Survival

Sixty-seven HCC patients were divided into 2 groups according to CLIC1 expression. The number of CLIC1 low-expression patients was 35, and the number of CLIC1 high-expression patients was 32. The results showed that the expression of CLIC1 was correlated with TNM staging, pathological differentiation, and vascular invasion (Table 1). Kaplan-Meier curves indicated that patients with lower expression of CLIC1 had better overall survival (P < .001) and recurrence-free survival (P = .046) (Figure 1C and D). Then, univariate and multivariate Cox regression analyses were performed to analyze the risk factors for HCC, and the extent of the expression of CLIC1 was verified as an independent prognostic factor for HCC patients (Table 2).

CLIC1 Induces VEGFA Secretion in HCC Cell Lines

CMs were collected from CLIC1 low-expression and CLIC1 overexpression HCC cell lines. The concentration of VEGFA was measured in the CM of the different groups. VEGFA in CM from CLIC1-knockdown Huh7 cells was approximately 2652 pg/mL, compared with 2497 pg/mL of the control group (Figure 2C, P = .619). On the contrary, VEGFA in CM from CLIC1 overexpression Huh7 cells was approximately 2386 pg/mL, compared with 707.9 of the control group (Figure 2C, P < .001). VEGFA in CM from CLIC1-knockdown BEL7402 cells was approximately 1471pg/mL, compared with 1839 pg/mL of the control group (Figure 2D, P = .017). On the contrary, VEGFA in CM from CLIC1 overexpression BEL7402 cells was approximately 2696 pg/mL, compared with 1013 pg/mL of the control group (Figure 2D, P < .001).

Coculture with CLIC1 Overexpression Cell Lines Facilitates the in Vitro Migration of EA.hy926 Cells

To investigate the migration of endothelial cells under different culture conditions with HCC cells, we cocultured EA.hy926 cells with CLIC1 low-expression and CLIC1 overexpression HCC cell lines in Transwells (8 μm). In a coculture system with CLIC1-knockdown Huh7 cells and BEL7402 cells, the migration ability of EA.hy926 cells was significantly repressed (Figure 3A, P = .552, P = .036), and in contrast, it was promoted in a coculture system with CLIC1 overexpression Huh7 cells and BEL7402 cells compared with the control group (Figure 3A, P = .037, P = .004). Subsequently, we added AntiVEGF Bevacizuma to the CLIC1-overexpressing Huh7 cells and BEL7402 cells, which significantly inhibited the migration of EA.hy926 cells (Figure 3A, P = .052, P < .001).

CM from CLIC1 Overexpression Cell Lines Promotes the in Vitro Migration of EA.hy926 Cells

We then analyzed the impact of the CM derived from CLIC1 low-expression and CLIC1 overexpression HCC cells on the migration ability of endothelial cells. The results were consistent with those achieved in the HCC cell-endothelial cell coculture system. CM from CLIC1 overexpression Huh7 cells and BEL7402 cells promoted the migration of EA.hy926 cells (Figure 3B, P < .001, P = .056), and CM from CLIC1-knockdown Huh7 cells and BEL7402 cells repressed the migration ability of endothelial cells (Figure 3B, P = .056, P = .193). Meanwhile, consistent with the above results, adding Bevacizuma to CM from CLIC1 overexpression Huh7 cells and BEL7402 cells significantly inhibited this migration (Figure 3B, P < .001, P = .065).

CLIC1 Promotes Angiogenesis in Vivo

To determine whether CLIC1 could regulate angiogenesis in vivo, we developed xenograft tumors using CLIC1-knockdown Huh7 cells, CLIC1-overexpressing BEL7402 cells, and HCC cells transfected with control lentivirus. The results showed that the growth of xenograft tumors derived from CLIC1-knockdown Huh7 cells was restrained compared to that of the control group. The growth of CLIC1-overexpressing BEL7402 cells showed was slightly different from the control group, but the difference was not statistically significant. IHC staining of the xenograft showed results similar to those obtained in human HCC tissues. MVD was higher in tumors with CLIC1 overexpression. In the tumor tissues derived from CLIC1-knockdown cells, microvessels could rarely be seen (Figure 4).