RETRACTED ARTICLE: Slug down-regulation by RNA interference inhibits invasion growth in human esophageal squamous cell carcinoma

EC109 were kindly provided by Dr. Li (The First Affiliated Hospital, Zhangzhou University, Henan Key Laboratory of Tumor Pathology, Zhangzhou, China). TE13 was obtained from the Japanese Cell Resource Center for Biomedical Research (Sdai, Japan);EC109 cell lines showed the highest level of Slug expression and TE13 cell lines showed less Slug expression [17].

Three different double strand siRNA oligonucleotides were successfully made. The sequence was submitted to a Blast search against the human genome sequence to ensure that only the Slug gene was targeted. The following sequences were as followed Sense:5-gatccgcgattgacaccagattcaagagatctgggtactgcattggtcttttttc-3;antisense:5-agctttccaaaaaagaccaatgcagtacccagatctcttgaatctgggtactgcattggtca-3;Sense:5-tgccctcctcacaatagtc tttcaagagaagactattgtgaggagggcttttttc-3;Antisense:5-gatcgaaaaaagccctcctcacaatagtcttctcttgaaagactattgtgaggagggca-3;Sense:5-tgcaaatgtacccaatgatattcaagagatatcattgggtacatttgcttttttc-3;Antisense:5-gatcgaaaaaagcaaatgtacccaatgatatctcttgaatatcattgggtacatttgca-3. A validated medium GC scramble (SCR) double strand siRNA oligonucleotide was used as control for transfection(data not shown). It does not match any mammalian sequences currently available on online databases. The double strand siRNA oligonucleotides were cloned into the pGCL-GFP vector. The pGCL-GFP vectors with double strand siRNA oligonucleotides inserts were used as entry clone vectors and transferred into the vector pDC316-EGFP-U6 [Invitrogen, Carlsbad, CA, USA] using the Gateway BamHI and HindIII enzyme mix according to the manufacturer's directions [Invitrogen] to generate pEGFP-siRNA-Slug (siRNA-Slug)or pEGFP-siRNA-mock(mock siRNA).

The EC109 (TE13) cells were cultured in media for 7 days, plated in a six-well plate with 1 × 10⁶ cells per well using 2 ml culture medium without antibiotics, and grown to 60-80% confluence for the time of transfection. The Lipofectamine 2000 reagent (Invitrogen; Carlsbad, CA) was used following the protocol set by the manufacturer to transfect the cells with siRNA(cDNA). The following groups were set: [1] RNAi (cDNA) group: the cells were transfected with vector siRNA-Slug(Slug cDNA; [2] Negative transfection group: the cells were transfected with vector siRNA-mock(pEGFP); [3] Control group: the cells were transfected with only liposome and no plasmid. The transfected cells were cultured for 24-72 h, then medium was removed. Stably expressed clones, used for in vivo study and invasion study, were selected by using medium containing G418(250-300 ug/ml) for 28 days. Cells were routinely maintained in selection media containing 200-250 ug/ml of G418-sulfate to avoid overgrowth of nontransfected cells.

The nucleosomal DNA degradation was quantified by Cell Death Detection ELISA kit using antihistone antibody (Roche, Mannheim, Germany) as described previously [18]. Briefly, 1 × 10⁵ EC109 or TE13 cells were seeded in 5-cm culture dishes and allowed to adhere overnight. After treatment with siRNA-Slug (Slug cDNA) under the same schedule as described above for 72 h, following which both adherent and floating (apoptotic) populations were harvested. They were lysed in NP-40 lysis buffer and the nucleosomes in the supernatant were detected photometrically using an ELISA plate Reader (SpectraMax 190, Molecular Devices). The readings were expressed as degree of apoptosis considering the untreated control as 1.

The in vitro growth effects of siRNA-Slug or Slug cDNA on EC109 or TE13 cells were assessed using MTT (Sigma Chemical Co.) Briefly, 1 × 10⁴ cells were seeded in each well of 96-well microtiter plates and allowed to attach overnight. After treatment with siRNA-Slug (Slug cDNA) under the same schedule as described above for 24-72 h, 20 ml of 5 mg/ml MTT in PBS was added to each well, followed by incubation for 4 hours at 37°C. The formazan crystals were dissolved in DMSO. The optical density was determined with a microculture plate reader (Becton Dickinson Labware, Lincoln Park, NJ) at 540 nm. Absorbance values were normalized to the values obtained for the vehicle treated cells to determine the percentage of surviving cells. Each assay was performed in triplicate.

In vitro tumorigenecity and anchorage-independent growth were assayed using standard methods. Stably transfected cells were harvested and suspended in culture medium. One percent agarose was coated onto 35-mm plates, and further overlaid with cell suspension (5,000 cells in 0.5% agarose). Plates were incubated in humidified tissue culture incubator at 37°C, 5% CO₂, for 2 weeks. Visible colonies were counted under a phase-contrast microscope.

Cellular proteins were extracted and separated on SDS-PAGE gels, and Western blotting analyses were carried out as described previously(19). Antibodies used were anti-Slug(G-18:1:200), anti-Bcl-2 (N-19: 1:250) and anti-E-cadherin(G-10: 1:200). All were from Santa Cruz Biotechnology; anti-β-actin (AC-15:1:500) from Sigma-Aldrich.

Tumor samples(metastasis node) fixed in 10% neutral buffered formalin were embedded in paraffin using automatic embedding equipment, after which 5-μm sections were prepared. Immunohistochemical analysis for Slug (dilution at 1:100; Santa Cruz Biotechnology)was done on paraffin-embedded sections of mice according to the manufacturer's instruction. After washing with phosphate-buffered saline, the sections were incubated with biotinylated secondary antibody. Positive staining of Slug was seen in the cytoplasm of tumor cells. The results of the immunohistochemical stainings were evaluated by the percentage of positively stained carcinoma cells. TUNEL staining for tumor tissue was based on the protocol of the Dead End Colorimetric TUNEL System (Promega). The tissue sections were viewed at ×100 magnification and images were captured with a digital camera. Percentage of apoptotic cells was defined as TUNEL-positive cells among 1000 tumor cells. Percentage of TUNEL-positive cells in each group were calculated from three tumor

specimens.

The BD BioCoat Matrigel™ invasion chambers with polyethylene terephthalate-filters coated with matrigel basement membrane matrix (6 wells, 8 μm pore size; BD Biosciences, Franklin Lake, NJ) were re-hydrated just before the assay using FBS-free DMEM according to the manufacturer's instructions. The chambers were assembled using freshly prepared matrigel-coated filters and DMEM containing 0.8 mL NIH-3T3 as a chemoattractant in the lower compartment. Stably siRNA Slug transfected or Slug cDNA-transfected cells were harvested by trypsinization, and suspended in DMEM containing 10% FCS. The cells (at a concentration of 1.25 × 10⁵ cells/2 ml) were added to the invasion chamber containing a matrigel-coated filter. The assembled chambers were incubated for 24 h at 37°C. At the end of the incubation, nonmigrating cells, which remained on the upper surface of the filter, were completely removed by wiping with a cotton swab. The cells on the bottom surface of the filter were fixed with 100% ethanol for 30 sec and stained with toluidine blue for 10 min. Cells migrated in the lower chamber were counted. Experiments were repeated thrice.

Immunodeficient male mice, 4 to 6 weeks old, were purchased from Shang Hai Animal Center. All animals were maintained in a sterile environment and cared for within the laboratory animal regulations of the Ministry of Science and Technology of the People's Republic of China (http://​www.​most.​gov.​cn/​kytj/​kytjzcwj/​200411/​ t20041108). Autoclaved cages containing food and water were changed once a week. Mouse body weight was measured every 3 to 4 days. On the day of tumor cell inoculation, tumor cells at 60% to 80% confluence were trypsinized and resuspended in fetal bovine serum-free culture medium. Animals were injected in the abdominal cavity with 5 × 10⁶ cells(siRNA Slug transfected cells, siRNA-mock transfected cells or control cells). The experiments were terminated 21 days. Animals were sacrificed by CO₂ inhalation, autopsy was carried out for assessment of metastases macroscopically. The number of the seeded tumor in the abdominal cavity is used for assessment of metastases.

Recent data indicate that Slug expression is relevant for melanoma metastasis [20]. We undertook experiments to assess whether Slug is involved in ESCC invasion and metastasis. We tested whether Slug knockdown affected the invasion capabilities of ESCC cells by using an in vitro invasion assay. In Slug-silenced EC109 cells, invasion was significantly reduced (Figure 1 A). Western blot analysis resulted in Slug protein at significantly lower levels in siRNA Slug-transfected EC109 cells (Figure 1 C, P < 0.05). The results indicate that Slug-silence affected the invasion capabilities of ESCC cells. We also tested the effects of Slug cDNA transfection on the invasion capability of ESCC cell line TE13. Expression of Slug was significantly increased in Slug cDNA-transfected cells(Figure 1 D, P < 0.05) and the invasion capability was significantly elevated(Figure 1 B, P < 0.05). Compared to untreated cells, no further decrease or increase in invasion capability was observed in mock transfected cells. Together, these data show that Slug modulates invasion of ESCC cells in vitro.

This study analyzed the effect of Slug on the cell growth of EC109 and TE13 cell lines. EC109 cell lines were transfected with siRNA Slug, TE13 cell lines were transfected with pSlug cDNA, and their viability was followed for 72 hours using a MTT cell proliferation assay. Cell growth over the 72-hour period for the EC109 and TE13 cell lines transfected with siRNA Slug and Slug cDNA is shown in Figure 2A and 2B, respectively. Each value is measured in triplicate. Significant growth inhibition was found in siRNA Slug-transfected EC109 cell lines compated with mock siRNA control (P < 0.05)(Figure 2A). On the contrary, Slug cDNA promotes growth in TE13 cell lines compated with mock cDNA transfected TE13, although the difference was not significant (P = 0.085, Figure 2B). Figure 2A and 2B showed that the growth curves for Slug siRNA-transfected EC109 cells were significantly lower than those for control cells, and the growth curves for Slug cDNA-transfected TE-13 cells were significantly higher than those for control cells. Furthermore, colony formation assay in monolayer culture showed that the number of surviving colonies of Slug siRNA-transfected cells was markedly decreased compared to those of control cells, and the number of surviving colonies of Slug cDNA-transfected cells was increased compared to those of control cells, although the difference was not significant (P = 0.072, Figure 2C), suggesting that knockdown of Slug expression inhibits colony formation, and slug overexpression seems to enhance colony formation.

We evaluated the effects of Slug siRNA transfection on apoptosis induction in EC109 cells using an ELISA kit to quantitatively measure fragmented DNA. After the same treatment schedule described above, a significantly higher level of DNA fragmentation was detected after Slug siRNA transfection compared to those of control cells (Figure 3A, P < 0.05). We further evaluated the effects of Slug siRNA transfection on apoptosis induction using an TUNEL kit. After the same treatment schedule described above, a significantly higher level of cell apoptosis index was detected after Slug siRNA transfection compared to those of control cells (Figure 3.B, P < 0.05). Western blot shown a significantly higher level of Slug protein in Slug-transfected EC109 cells for 72 h compared with controls (Figure 3 C, *P < 0.05, *P < 0.01).

It has been demonstrated that Slug is involved in the control of apoptosis [14] and also involved in the EMT, linked to the acquisition of the invasive phenotype [19], we analyzed the expression of several Slug targets in stable siRNA Slug-transfected EC109 cells and Slug cDNA-transfected TE13 cells by western blotting and Q-PCR. In siRNA Slug-transfected EC109 cells, anti-apoptotic Bcl-2 was down-regulated (Figure 4 A, P < 0.05). By contrast, in Slug cDNA-transfected TE13 cells, Bcl-2 was up-regulated (Figure 4A, P < 0.05). E-cadherin, a well-known target of Slug previoussly discribed [20], was up-regulated in siRNA Slug-transfected cells(P < 0.05), but in Slug cDNA-transfected cells, E-cadherin was down-regulated (Figure 4 B, P < 0.05).

We first tested the effect of siRNA Slug-transfected cells in EC109 pseudometastatic models. The mice were injected with stable siRNA Slug-transfected (18 animals) or siRNA mock-transfected (18 animals) EC109 cells(5×10⁶ )at the site of the abdominal cavity on day 0. After 21 days, the animals were sacrificed and autopsy was carried out to remove organs. Liver metastasis occurred in only four mice in parental groups, and three in mock-transfected mice, no liver metastasis occurred in siRNA Slug-transfected groups. There were many off-white nodus in peritoneum, retina, mesentery, intestinal and gastric wall(Figure 5 A-I, magnification ×200). The node that less than 2 mm is round, and irregular when the node is more than 3 mm. Autopsy showed a reduction of seeded tumor nodes in abdominal cavity organs in pEGFP-siRNA Slug-transfected groups. The number of seeded tumor was 91.16 + 8.7, 90.4 + 9.04 and 33.17 + 6.49 in control groups, siRNA mock groups and Slug siRNA groups respectively. Differences of seeded tumor reached statistical significance (Figure 5 J, P = 0.037).

Furthermore, we analyzed the expression of Slug in the tumor sections from control, mock and Slug siRNA transfected groups using immunohistochemistry (Figure 6 a and 6b). Positive staining of Slug was seen in the cytoplasm of tumor cells. We observed significant Slug expression in control and mock-transfected tumor sections. However, expression levels were drastically reduced in siRNA Slug-transfected sections of mice. TUNEL staining in the same samples was increased from 1.6% positive area in the mock siRNA transfected group to 14.2% positive areas in the siRNA Slug transfected groups(Figure 6 c and 6d).