Lipofectamine transfection and TRIzol reagents were purchased from Invitrogen (Grand Island, NY, USA). MG132 was purchased from Selleckchem (Houston, TX, USA). Antibodies against TRIM16, E-cadherin, N-cadherin, vimentin, HA, Myc, snail, slug, ZEB1, ZEB2 and β-actin antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-α-catenin antibody and Matrigel were from BD (Franklin Lakes, NJ, USA). Unless otherwise noted, all other chemicals were from Sigma (St. Louis, MO, USA).

Liver cancer cell lines HepG2, SMMC-7721, HCCLM3, MHCC97H, and HEK 293 Phoenix ampho-packaging cells were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Chinese Academy of Sciences; liver cancer cell lines were routinely cultured as previously described (9). Cell lines were maintained at 37°C in an atmosphere containing 5% CO₂ in Dulbecco's modified Eagle's medium or RPMI-1640 supplemented with 10% fetal bovine serum.

Sixty-one tumor and para-cancerous tissues which, were used for qRT-PCR and western blot analysis, were randomly collected from HCC patients who underwent curative resection with informed consent between 2011 and 2014 at the Department of Laparoscopic Surgery, First Affiliated Hospital of Dalian Medical University. Study protocols were approved by the Hospital Ethics Committee of Dalian Medical University, and written informed consent was obtained from patients based on the Declaration of Helsinki.

pBabe retroviral construct containing human TRIM16 cDNA and pSuper with shRNA against human TRIM16 were prepared as described previously (10). The generation of retrovirus supernatants and transfection of cancer cells were conducted as described previously. Infected cells were selected by adding 2 μg/ml puromycin to the culture medium for 48 h and then maintained in complete medium with 0.5 μg/ml puromycin. Empty retroviral-infected stable cell lines were also produced by the above protocols. siRNA against ZEB2 expressed in pSuper vector were prepared as described previously (11). The generation of retrovirus supernatants and transfection of cancer cells were conducted as described above except infected cells were selected by adding 400 μg/ml of G418. The expression of TRIM16 and ZEB2 was confirmed by qRT-PCR and western blot analysis.

Invasion assay was performed using Matrigel (BD)-coated Transwell inserts (Costar, Manassas, VA, USA) containing polycarbonate filters with 8-μm pores as detailed previously (12). According to the manufacturer's instructions, the inserts were coated with 50 μl of 1 mg/ml Matrigel matrix. 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 were added to lower chamber. After 24-h incubation, cells that migrated to the lower surface of the membrane were fixed and stained. For each membrane, five random fields were counted at ×10 magnification. Motility assays were similar to Matrigel invasion assay except that the Transwell insert was not coated with Matrigel.

Cell lines were plated on culture slides for 24 h. In addition, the cells were rinsed with phosphate-buffered saline fixed with 4% paraformaldehyde in PBS. Next, cell membrane was permeabilized using 0.5% Triton X-100. These cells were then blocked for 30 min in 10% BSA in PBS and incubated with primary antibodies in 10% BSA overnight at 4°C. After three washes in PBS, the slides were incubated for 1 h in the dark with FITC-conjugated secondary antibodies. Slides were stained with DAPI for 5 min to visualize the nuclei, and examined using a confocal imaging system (LSM 780) (Carl Zeiss, Jena, Germany).

Standard methods were used for western blot analysis. Cell lysates were prepared by extraction with lysis buffer. Proteins (10 μg) were separated by SDS-PAGE under reducing conditions and blotted onto a polyvinylidene difluoride membrane. Membranes were probed with specific antibodies. Blots were washed and probed with respective secondary peroxidase-conjugated antibodies, and the bands visualized by chemiluminescence.

Total RNA was extracted using TRIzol reagent and cDNA was synthesized using SuperScript II reverse transcriptase. qRT-PCR and data collection were performed with an ABI PRISM 7900HT sequence detection system. The primers used in this study are listed in Table I.

Different cells were resuspended in PBS at a concentration of 1×10⁷ cells/ml in metastasis assays. Cell suspension (0.1 ml) was injected into tail veins of nude mice. The mice were sacrificed by CO₂ 60 days after inoculation.

Data are presented as means ± standard deviation (SD). Data were analyzed by SPSS/Win11.0 software (SPSS, Inc., Chicago, IL, USA) in t-test (unpaired, two-tailed), and results were considered significant at P<0.05.

To investigate whether TRIM16 might be involved in HCC, the mRNA expression level of TRIM16 in HCC tissues and their matched normal adjacent tissues was determined by qRT-PCR in 61 samples (Fig. 1A). As compared with normal tissues, HCC specimens showed lower expression of TRIM16 (Fig. 1B). We then analyzed TRIM16 expression in HCCs without or with metastasis property; we found that TRIM16 mRNA lower expression was significantly correlated with metastasis property in HCC tissues (Fig. 1C). We examined TRIM16 protein expression in the same HCC samples by western blot analysis (Fig. 1D). We observed that the level of TRIM16-positive cells was markedly lower in HCC tissues than the level in the normal liver tissues (Fig. 1E). Most importantly, TRIM16 lower expression was consistently significantly correlated to distant metastasis in these HCC samples (Fig. 1F). As showed in Fig. 2, expression level of TRIM16 protein (Fig. 2A) and mRNA (Fig. 2B) in invasive HCC cell lines was lower than that in the no-invasive HCC cell lines. These data demonstrated that the downregulation of TRIM16 might be relevant to invasive property of HCC.

To investigate the effect of TRIM16 knockdown on hepatoma carcinoma cell migration and invasion, HepG2 human hepatoma cell line, was first infected with shTRIM16 or the control. We used western blot and qRT-PCR analyses to detect transfection efficiency. Compared with the control cells, the HepG2 cells, transfected with the TRIM16 shRNA plasmid, displayed significantly decreased TRIM16 expression at the protein and mRNA levels (Fig. 3A and B). Boyden chamber assay was first used to assess the influence of TRIM16 on HCC cell migration to detect the effect of TRIM16 on HCC cells. As indicated by the Transwell assay and Matrigel assay, knockdown of TRIM16 expression significantly inhibited HCC cell migration and invasion (Fig. 3C and D). These results further demonstrated that silencing TRIM16 promotes HCC cell migration and invasion in vitro.

We also used MHCC97H cells to establish a stable cell line that constitutively overexpressed TRIM16. The transfection efficiency was confirmed using western blot and qRT-PCR analyses. As shown in Fig. 4A and B, the MHCC97H cells that had been transfected with the TRIM16 expression plasmid displayed significantly increased TRIM16 expression both at the protein and mRNA levels compared with the vector cells. In order to measure the effect of TRIM16 on HCC cell migration and invasion, the effect of TRIM16 on HCC cell migration was assessed by the Boyden chamber assay. The overexpression TRIM16 markedly reduced the migratory (Fig. 4C) and invasive (Fig. 4D) capacity of MHCC97H cells. These results indicate that ectopic TRIM16 expression inhibits HCC cell migration and invasion in vitro.

We then investigated the functional relevance of TRIM16 for metastasis in vivo. HepG-2-shTRIM16 #2 and its control cells were injected into nude mice through the tail vein. We observed that silencing TRIM16 not only significantly increased the number of mice with distant metastasis (Fig. 5A), but also markedly increased the number of metastatic tumors in the lungs of each mouse (Fig. 5B and C). Therefore, the in vivo results further demonstrate the critical role of TRIM16 in HCC metastasis.

During the establishment of these cell lines, we observed that HepG-2-shTRIM16 cells exhibited fibroblastic morphology, compared to their respective control cells (data not shown). This observation was further confirmed by western blot and immunofluorescence analyses of epithelial and mesenchymal molecular marker expression. We showed that TRIM16 knockdown decreased the levels of epithelial markers (E-cadherin and α-catenin) and increased the levels of mesenchymal markers (N-cadherin and vimentin) (Figs. 6A and 7A). Moreover, expression levels of mRNA correlated with the corresponding protein levels (Fig. 6B), suggesting that TRIM16 affected the expression of epithelial and mesenchymal markers at the transcript level. Conversely, MHCC97H-TRIM16 cells reverted to a mesenchymal phenotype as compared to their respective control cells (data not shown). Consistent with this, ectopic TRIM16 expression resulted in an increase in expression of epithelial markers, and a decrease in expression of mesenchymal markers (Figs. 6C and D and 7B).

To better understand the mechanisms by which TRIM16 engaged in the EMT program, we assessed the expression of the EMT regulating genes, such as Snail, Slug, ZEB1 and ZEB2, on HepG2-shTRIM16, MHCC97H-TRIM16 and their control cells. The results shown that HepG2-shTRIM16 cells exhibited greatly increased ZEB2 expression at protein level (Fig. 8A), whereas, ectopic TRIM16 in MHCC97H cells markedly decreased ZEB2 expression at protein levels (Fig. 8C). While ZEB2 mRNA level had no significant change in either TRIM16 knockdown or ectopic expression (Fig. 8B and D), indicating that the regulation function of TRIM16 on ZEB2 expression only took place on the post-transcriptional level.

To understand how TRIM16 exerts its effects on ZEB2, 293T cells were co-transfected with HA-tagged TRIM16 and Myc-tagged ZEB2 constructs. Immunoprecipitation and subsequent immunoblot analyses revealed that Myc-tagged ZEB2 co-immunoprecipitated with HA-tagged TRIM16 (Fig. 9A), confirming a physical interaction between the two proteins. The physical interaction between TRIM16 and ZEB2 suggested that TRIM16 maybe target ZEB2 for ubiquitination and subsequent degradation. Next, in order to investigate if the regulation of TRIM16 on ZEB2 protein is dependent on the ubiquin-proteasome pathway, we combined the treatment of proteasome inhibitor MG132 with ectopic TRIM16 treatment. We found that ZEB2 degradation caused by TRIM16 ectopic expression was completely abrogated by proteasome inhibition (Fig. 9B), demonstrating that the regulation of ZEB2 expression by TRIM16 is indeed mediated via proteasome-dependent pathway.

We transfected HepG2-shTRIM16 cells with ZEB2 siRNAs to silence ZEB2 gene expression to investigate whether TRIM16-induced metastatic capacity was mediated by ZEB2 expression (Fig. 10A and B). It was found that knockdown of ZEB2 in HepG2-shTRIM16 cells were accompanied by the reduction of migratory and invasive capacities (Fig. 10C). Collectively, these results show that ZEB2 mediates shTRIM16-induced migration and invasion in HCC cells.