The human HCC HepG2, HuH-7 and HEK293 (human embryonic kidney cells) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). SMMC7721 was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. The cells were maintained in a humidified condition that contained 5% CO₂ at 37°C and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine, followed by the addition of 100 U/ml penicillin and 100 μg/ml streptomycin.

The most effective lentiviral vectors carrying shRNA were previously screened in our laboratory (20). Adenoviral vectors harboring TRAIL and lentiviral vectors carrying shBcl-xL, shcIAP2, shSurvivin and shRaf-1 were previously constructed by our laboratory (20,25). Construction and purification of the vectors were performed as per the standard protocol. The titration of recombinant lentiviruses and adenoviruses was performed using a TCID50 assay on HEK293 cells. Recombinant lentiviral and adenoviral vectors were short for LV and Ad5, respectively. The cells were subsequently divided into the Mock, LV-shSNAIL, Ad5.TRAIL and Ad5. TRAIL + LV-shSNAIL groups.

Infected cells were collected and washed with phosphate-buffered saline (PBS). Total RNA was extracted from the cells of the target cells, and cDNA was generated with a PrimeScript^® RT reagent kit (Takara, Chiga, Japan) at a total volume of 20 μl according to the manufacturer’s instructions. cDNA was then used in each amplification reaction. Reactions were performed using SYBR^® Premix Ex Taq™ (Takara), under the following PCR conditions: denaturation at 95°C for 30 sec, followed by 40 cycles of annealing at 95°C for 5 sec and extension at 60°C for 30 sec. Each sample was also subjected to melting curve analysis to confirm amplification specificity. GAPDH was used as a control housekeeping gene. The expression of SNAIL was assessed by normalization of the cycle threshold (Ct) of these genes to that of GAPDH. A Ct value was obtained from each amplification curve by using the software provided by the manufacturer (Roche, Mannheim, Germany).

The analysis of HCC cell viability was determined by an MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. HCC cells were plated in 96-well microtiter plates equally according to the density standards of 5×10³ cells/well. Each well was provided suitable culture medium with the condition in 5% CO₂ at 37°C until grown to a high density. Each group of cells was incubated for 24–96 h following treatment with various viruses. Subsequently, 5 mg/ml MTT was added to each well and incubated for 4 h under the same condition. The original culture of each well was abandoned. The crystal substances produced from the cells were dissolved in 150 μl dimethyl sulfoxide (DMSO) and agitated for 10 min. Cell viability was assessed as optical density (OD) at a wavelength of 490 nm for immune monitoring by the enzyme-linked immunosorbent spot assay (Bio-Rad, Hercules, CA, USA).

Whole-cell lysates were obtained following centrifugation at 120,000 × g for 10 min of the target cells of all the groups. Total proteins were separated by electrophoresis on 12% polyacrylamide and transferred to 0.45 μm nitrocellulose (NC) membrane. The samples were washed three times using PBS after Ponceau S Staining kit dyeing. Bands were incubated with primary antibodies, rabbit polyclonal anti-SNAIL Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and anti-TRAIL (Cell Signaling Technology, Danvers, MA, USA), respectively. Conjugates were combined with secondary goat anti-rabbit alkaline phosphatase antibody (Abcam, Cambridge, MA, USA), which was marked with HRP (horseradish peroxidase). GAPDH detection was performed using rabbit polyclonal anti-GAPDH and mouse monoclonal anti-GAPDH (Abcam). The fluorescent compounds were detected by ECL (Pierce, Rockford, IL, USA). At the same time, primary antibodies, rabbit polyclonal anti-p53 (Cell Signaling Technology), anti-Bcl-xL (Cell Signaling Technology), anti-cIAP2 (Abcam, Cambridge, UK), anti-survivin (Cell Signaling Technology), anti-Raf-1 (Cell Signaling Technology), respectively, were applied to the membranes and the process was repeated as above.

HCC cell lines were placed into 6-well plates and incubated with recombinant lentiviruses or adenoviruses, respectively. After 48 h, when the cells were infected, the medium was washed with PBS twice. The cells were then stained with Hoechst 33258 (25 μg/ml). Hoechst 33258 was excited by 405-nm violet diode. The percentage of apoptotic cells was analyzed using a fluorescence microscope.

The statistical analyses were performed using the Student’s t test and one-way ANOVA to determine the significance. Results were presented as mean ± standard deviation (SD). P<0.05 was considered statistically different.

In our previous study (20), we detected the expression of SNAIL in HepG2. Based on the results, LV-shSNAIL was found to suppress the expression of SNAIL at the mRNA and protein level in HepG2 cells (20). The mRNA level was measured by employing quantitative PCR, while western blot analysis was used to determine the protein expression level. The results verified that the level of SNAIL was obviously reduced following LV-shSNAIL infection in other HCC cells (Fig. 1), which was consistent with the anterior study (20). The findings showed that the construction of LV-shSNAIL was successful in silencing SNAIL expression in various types of HCC cells.

Recent findings have shown that, suppressing SNAIL may inhibit invasion and metastasis in human HCC (26). Moreover, as described above, silencing SNAIL reduced viability in HCC cells. It is well known that the main function of TRAIL is the induction of tumor apoptosis, as identified by findings from our laboratory (25,27). Therefore, we first evaluated the status of TRAIL expression in different groups of three types of HCC cells, and the level of TRAIL was detected among the cells using western blot analysis. Analysis of the results showed that TRAIL expression was markedly increased following the addition of Ad5.TRAIL + LV-shSNAIL (Fig. 2). These data showed that TRAIL was effectively expressed following Ad5.TRAIL vector infection of HCC cells, and that knockdown of SNAIL increased TRAIL expression.

We also examined the effect of SNAIL and TRAIL on HCC by assessing the state of cell proliferation. In order to study the change in HCC cell viability for response to the SNAIL silencing and TRAIL, respectively, we detected cell viability in the four groups, from the point when reagents were added until 96 h, once every 24 h by MTT assay (Fig. 3). The extent of inducing apoptosis was also investigated. The samples were stained with Hoechst 33258 for 48 h and then analyzed by fluorescence microscopy (Fig. 4). The results revealed that silencing SNAIL or upregulated TRAIL decreased proliferation and increased apoptosis in HCC cells.

Since TRAIL-induced apoptosis was weakened in HCC cells as compared to other TRAIL-sensitive cells, previous studies focused on apoptosis induction by acting on TRAIL for HCC cells (28). Therefore, we examined the change of TRAIL-induced apoptosis in different groups. As shown in Fig. 4, HCC cell apoptosis was highest in the group infected with LV-shSNAIL and Ad5.TRAIL compared to the other groups. This result suggested that silencing SNAIL enhanced sensitivity that TRAIL induced HCC cell apoptosis.

Previous authors reported that p53 sensitized TRAIL-induced apoptosis in various types of cancer (29,30) and blocking SNAIL activated p53, which regulated apoptosis (31). To determine whether p53 was associated with silencing of SNAIL to increase TRAIL-induced apoptosis in HCC, we detected the expression level of p53 in different groups in HuH-7 cells. The results revealed that p53 expression was highest in groups to which LV-shSNAIL was added than those where LV-shSNAIL was not added (Fig. 5).

Resistance of HCC cells to TRAIL-induced apoptosis has been shown to be associated with Bcl-xL, cIAP2 and survivin factors, which are located in the downstream gene of the NF-κB pathway (14,15,32). Moreover, Hall et al suggested that blocking the activation of Raf kinase inhibited NF-κB binding to the Mcl-1 promoter thereby enhancing the sensitization of TRAIL-mediated apoptosis in cancer cells (13). Among them, Raf-1 kinase, a member of the Raf kinase family, is an important signaling molecule acting on the downstream kinases MEK and ERK, which pathway converges on NF-κB activation (13,33). Accordingly, we investigated the expression levels of these proteins in HCC cells to analyze the mechanism associated with the enhancement of TRAIL-induced apoptosis by SNAIL silencing. The results showed that Bcl-xL, cIAP2, survivin and Raf-1 expression were reduced following the addition of LV-shSNAIL than without its addition to HuH-7 cells, and particularly low in LV-shSNAIL + Ad5.TRAIL (Fig. 5).

In order to determine whether Bcl-xL, cIAP2, survivin and Raf-1 participated in the suppression of SNAIL to mediate HCC cell sensitivity to TRAIL-induced apoptosis, we used lentiviral vectors carrying shRNA to infect HuH-7 for the knockdown of Bcl-xL, cIAP2, survivin and Raf-1. The effect of TRAIL-induced apoptosis of HuH-7 cells was analyzed by fluorescence microscopy following the knockdown of each gene. Infection of LV-shBcl-xL, LV-shSurvivin or LV-shRaf-1 HuH-7 cells showed obvious sensitivity to TRAIL-induced apoptosis, whereas the sensitivity of shcIAP2 was not affected (Fig. 6).

In conclusion, Bcl-xL, survivin and Raf-1 downregulation was conducive to enhancement of HCC cell sensitivity to TRAIL-induced apoptosis by silencing SNAIL. In other words, silencing of SNAIL enhanced TRAIL-induced apoptosis by affecting the NF-κB pathway.