Overexpression of Cullin7 is associated with hepatocellular carcinoma progression and pathogenesis

Ethics statement

All procedures performed in the studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Clinical Research Ethics Committee of the Third Affiliated Hospital, Sun Yat-sen University. Written informed consent was obtained from all the participants, and the ethical guidelines under the Declaration of Helsinki were followed.

Cell lines

The HCC cell lines HepG2, Hep-3B, HuH7, and SMMC-7721 and the normal hepatic cell line LO2 were obtained from the Institute of Cell Biology of the Chinese Academy of Science (Shanghai, China). All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, USA), 100 U/ml penicillin or 100 μg/ml streptomycin, and incubated at 37 °C in a humidified incubator with an atmosphere of 5% CO₂.

Patients and tissue specimens

Paraffin embedded tissue sections were obtained from archived liver samples of patients with HCC (n = 162) at the Third Affiliated Hospital, Sun Yat-sen University between January 1, 2008 and December 31, 2009. All patients had not been pre-treated with chemotherapy or radiotherapy. In total, 162 normal liver tissue specimens were obtained from the periphery of the cancer site and utilized as controls. Histopathological diagnosis of all specimens was confirmed by two trained pathologists. The patient cohort included 144 men and 18 women with a median age of 53 years (range 19–72 years); 162 patients showed 37 as well differentiated, 112 as moderately differentiated, and 13 as poorly differentiated. According to the TNM system from the International Association for the Study of Hepatic Cancer, 64 patients in stage A, 11 in stage B, 67 in stage C, and 20 in stage D. Five biopsies of HCC tissues and the matched adjacent non-cancerous hepatic tissues were frozen and stored in liquid nitrogen until further use.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen). As previously described [16], First-strand cDNA was synthesized with a Super ScriptIII First-Strand Synthesis System (Invitrogen). qRT-PCR for cullin7 mRNA was performed on Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems) using One Step SYBR PrimeScript Plus RT-PCR kit (Takara Bio, Inc., Shiga, Japan) according to the manufacturer’s instruction. The cycling conditions for real-time PCR were as follows: 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The Cullin7-specific primers were as follows: sense, 5′-CCATCTCAGAGTCCCAACAC-3′, antisense, 5′-TTCAGCACCAC GGCATAG-3′. β-actin served as an endogenous control using the following primers: sense, 5′-GTCGTCGACACGGCTCC-3′, and antisense, 5′-TCGTCGCCCACATAGGAATC-3′. The expression levels of Cullin7 were corrected by reference to β-actin, and the relative amount of mRNA was calculated by the comparative ∆Ct method.

Vectors and retroviral infection

Cullin7-specific small hairpin RNA (shRNA; the target sequence, 5′- TGAGATCCTAGCTGAACTG-3′) were designed and synthesized by GeneChem Co, Ltd. (Shanghai, China). The Cullin7 overexpression plasmid, Cullin7-pcDNA3.1(+), was synthesized by Life Technologies (Thermo Fisher Scientific, Inc.). HepG2 cells were transfected with 2 μg of Cullin7 shRNA or Cullin7-pcDNA3.1(+) vector using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Western blotting

Total protein from cells or tissues was lysed using RIPA buffer (ProMab Biotechnology, USA). The concentration of the total protein was quantified using a bicinchoninic acid (BCA) protein assay kit (Boster, China). Equal amounts of protein lysates (30 μg each lane) were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After being blocked with 4% dry milk, the membranes were incubated with primary antibodies against Cullin7 (Cell Signaling) and β-actin at 4 °C overnight. Then, the membrane was further incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. Protein bands were visualized with enhanced chemiluminescence (ECL) reagents (Pierce; Thermo Fisher Scientific Inc., Waltham, MA, USA). β-actin was used as an internal reference for relative quantification.

Immunohistochemistry

Tumor specimens were fixed in formalin, dehydrated in an ethanol series, treated with xylene, and mounted in paraffin. Four-micrometer-thick tissue sections were deparaffinized, rehydrated, and incubated with 3% H₂O₂ in methanol. Subsequently, the antigen was retrieved with PH 8.0 EDTA high temperature-pressure repairing, and sections were incubated with 1% BSA. Slides were incubated with rabbit anti-human monoclonal Cullin7 antibody (dilution 1:150, Abcam) at 4 °C overnight. A normal nonimmune rabbit serum was used as a negative control. After incubation with a secondary antibody at room temperature for 60 min, slides were incubated with a streptavidin-peroxidase complex. The peroxidase reaction was developed with 3,3′-diaminobenzidine, and the slides were counterstained with hematoxylin. Sections were dehydrated, rendered transparent, covered with coverslips and sealed with neutral gum.

Tumor and normal tissue specimens were assessed by two independent pathologists. Cullin7 expression was localized predominantly in the nuclei. Sections were considered positive if expression was detected in more than 10% of the cells in tumor or normal tissue.

MTT assay

Cells were seeded on 96-well plates at initial density of (0.2 × 10⁴ per well).At each time point, cells were stained with 100 μl sterile 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide dye (0.5 mg/ml, Sigma, St Louis, MO) for 4 h at 37 °C, followed by removal of the culture medium and addition of 150 μl of dimethyl sulphoxide (Sigma). The absorbance was measured at 570 nm, with 655 nm as the reference wavelength. All experiments were performed in triplicates.

Colony formation assay

Colony formation assay performed as previously described [17]. Briefly, Cells were seeded in triplicate (500cells per 60-mm culture dish in complete medium) and incubated for 3 weeks in a humidified incubator at 37 °C. After seeding for 3 weeks, colonies were stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The colony formation ability was assessed by counting the number of colonies from each of the triplicate samples by using a microscope.

Cell invasion and motility assay

Cell invasion and motility assay was performed as described by Guo et al. [13]. Briefly, cell invasion was measured in Matrigel-coated Transwell inserts containing polycarbonate filters with 8-μm pores. The inserts were coated with 50 μL of 1 mg/mL Matrigel matrix according to the manufacturer’s recommendations. Cells (2 × 10⁵) in 200 μL of serum-free medium were plated in the upper chamber, whereas 600 μL of medium with 10% fetal bovine serum was added to the lower well. After 24 h of incubation, the top cells were removed, and the bottom cells were counted. The cells that migrated to the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. For each membrane, five random fields were counted at 10 × magnification. Motility assays were similar to the Matrigel invasion assays except that the Transwell insert was not coated with Matrigel.

Wound healing assay

Wound healing assay was performed as described by Guo et al. [13]. Briefly, the different cells were respectively seeded in six-well plates at a density of 1 × 10⁶ per well with complete culture medium. When cell confluency reaches 90%, then a single wound was created using a sterile plastic pipette tip. After scratching, the cells were gently washed twice with PBS and the complete culture medium was replaced. Cell migration was photographed and the width of the wound areas were measured using an inverted microscope at different time points.

Statistical analysis

Data were analyzed using SPSS version 13.0. (Chicago, IL, USA). The relationship between the expression of Cullin7 and the clinico-pathological parameters was evaluated by χ2 analyses. A value of P < 0.05 was considered statistically significant.

Overexpression of Cullin7 in HCC cell lines

A western blot analysis revealed that Cullin7 protein was highly expressed in the HepG2, Hep-3B, HuH7 and SMMC-7721 HCC cell lines; however, it was weakly expressed in LO2 cells (Fig. 1a). Consistent with the upregulation of Cullin7 protein, the real-time PCR results showed that all HCC cell lines displayed significantly higher, up to 14-fold, expression of Cullin7 mRNA compared with the LO2 cell line (Fig. 1b).

Overexpression of Cullin7 in HCC tissue

The expression of Cullin7 protein was also discovered to be increased in all five HCC tissues compared with adjacent normal hepatic tissues using western blotting (Fig. 1c). A real-time PCR analysis revealed that the tumor/normal ratio of Cullin7 mRNA expression was as high as 22-fold in one case of five paired primary HCC tissues (Fig. 1d). Furthermore, protein quantification demonstrated that four HCC tissues displayed much more than a 4-fold increase of Cullin7 protein compared with adjacent normal hepatic tissues (Fig. 1e).

Overexpression of Cullin7 in archived HCC tissues

In this study, 162 archived HCC tissues were examined using IHC staining. Immunohistochemical analysis revealed overexpression of Cullin7 predominantly in the nuclei (Fig. 2a, b). The expression of Cullin7 protein was detected in 69.1% (112/162) of HCC specimens. In contrast, Cullin7 was barely detectable and only present in 29.0% (47/162) of the normal hepatic tissues. Cullin7 was discovered to be upregulated in HCC compared with normal hepatic tissues. Statistical analyses showed that there was a striking difference in overexpression of Cullin7 between HCC and adjacent non-cancerous hepatic tissues (P < 0.01) (Table 1).

Groups	n	Cullin7
		negative	positive (%)
HCC	162	50	112(69.1)
Normal	162	115	47 (29.0)

There was a significant difference in the expression of Cullin7 between HCC and adjacent normal hepatic tissue (P<0.01)

Upregulation of Cullin7 promotes the proliferation, migration, and invasion capacities of HCC cells

To further investigate the biological role of Cullin7 expression in HCC progression, a HepG2 cell line of HCC that stably overexpressed Cullin7 was established (Fig. 3a, b). As shown in Fig. 3a and b, the mRNA and protein expression levels of Cullin7 in HepG2 cells were significantly increased compared with the vector cell lines. Furthermore, the results of MTT assays demonstrated that Cullin7-infected HepG2 cells grew faster than the control HepG2 cells by day 5 after plating (Fig. 3c). The effect of Cullin7 was also verified by the colony formation assay (Fig. 3d). Meanwhile, the influence of Cullin7 on cell migration was evaluated by a wound healing assay. The assay revealed that Cullin7-infected HepG2 cells had evidently faster closure of the wound area compared with the control HepG2 cells (Fig. 3e). HepG2 Cullin7 cells showed over two-fold more cell migration through Transwell membranes than the control cells after 24 h of incubation (Fig. 3f). HepG2 Cullin7 cells also exhibited a higher ability to invade Matrigel (Fig. 3f).

Deregulation of Cullin7 inhibits the proliferation, migration, and invasion capacities of HCC cells

The impact of Cullin7 expression on proliferation, migration and invasion was evaluated in Cullin7-knockdown cells. As shown in Fig. 4a and b, the HepG2 cells displayed significantly decreased Cullin7 expression at both the mRNA and protein levels compared with the control cells. The depletion of Cullin7 expression caused significantly compromised viability in HepG2 cells (Fig. 4c). The colony formation assay revealed that silencing endogenous Cullin7 in HepG2 cells could lead to decreases in both colony number and colony size (Fig. 4d). The effect of Cullin7 knockdown on cell migration was first assessed with a wound healing assay. Cullin7-knockdown HepG2 cells had significantly reduced closure of the wound area compared with the respective control cells (Fig. 4e). This result was confirmed using a Boyden chamber assay. HepG2 Cullin7-RNAi cells showed a lower degree of cell migration than the control cells in the Transwell migration assays (Fig. 4f). Furthermore, Cullin7-RNAi cells also exhibited a lower ability to invade Matrigel (Fig. 4f).