Human follicular thyroid carcinoma cell lines FTC-133 and FTC-238 were purchased from Guangzhou Jennio Biotech Co. (China). The cell lines were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium containing 2 mM glutamine, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 Ag/ml streptomycin in a humidified CO₂ incubator at 37°C.

All specimens were obtained from the General Surgery Department of The Second Affiliated Hospital of Dalian Medical University (Liaoning, China) and included 101 samples of follicular thyroid cancer and the corresponding peritumoral tissues (3 cm from the tumor edge). For the use of these clinical materials for research purposes, prior consents from the patients and approval from the Ethics Committees of The Second Affiliated Hospital of Dalian Medical University were obtained, and all the procedures have been performed in compliance with the Helsinki Declaration. All specimens had confirmed pathological diagnosis and were staged according to the 2013 thyroid carcinomas staging system of the International Union against Cancer (UICC). These tissues were snap-frozen in liquid nitrogen and stored at −80°C until used.

Real-time PCR was used to analyze gene expression. Total RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized using the QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer's protocol. Real-time PCR was carried out on an ABI Prism 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using QuantiTect SYBR Green PCR kit (Qiagen). The primer sequences used for amplification were as follows: forward, 5′-CTTTGCCCTGTACTTCTCG-3′ and reverse, 5′-CAGCACTGGAATGGAGAGA-3′ for ST6GalNAcII; and forward, 5′-CTCCTCCACCTTTGACGCTG-3′ and reverse, 5′-TCCTCTTGTGCTCTTGCTGG-3′ for GAPDH. The relative expression level of target gene was normalized to that of the respective GAPDH.

Extracted proteins were electrophoresed under reducing conditions in 10% sodium dodecylsulfate-polyacrylamide gels, and then blotted onto a polyvinylidene difluoride membrane. After blocking with 5% skimmed milk in PBS containing 0.1% Tween-20 (PBST), the membrane was incubated with antibody (1/1,000 diluted; Abcam) and then with peroxidase-conjugated anti-rabbit IgG (1/10,000 diluted; GE Healthcare UK Ltd., Little Chalfont, UK). A GAPDH antibody (1/200 diluted; Santa Cruz Biotechnology) was used as a control. All bands were detected using ECL Western Blot kit (Amersham Biosciences, UK), and the bands were analyzed with LabWorks™ (ver 4.6, UVP; Bio-Imaging Systems).

FTC-238 cells were incubated in appropriate antibiotic-free medium with 10% fetal bovine serum (Gibco), transferred to a 6-well tissue culture, and incubated at 37°C in a CO₂ incubator to obtain 60–80% confluence. The cell cultures were stably co-transfected with a plasmid vector containing the puromycin-resistance marker and the specific short hairpin RNA (shRNA) (ST6GalNAcII), respectively, which was prepared according to the protocol. Scrambled shRNA was used as the negative control. The transfection efficiency calculated by the percentage of fluorescent cells was about 82%, and cell viability was 89% by trypan blue dye exclusion assay. Four weeks later, we used puromycin to screen the cells stably expressing shRNA. Several colonies were picked and expanded for further study. The knockdown had no effects on the cell morphology.

The human ST6GalNAcII coding sequences were obtained from Takara Company (Dalian, China) and were inserted into the pEGFP-N2 vector (Invitrogen, Carlsbad, CA, USA) at the sites of EcoRI, XhoI. Cells were transfected with 5 µg of target gene expression vector or empty vector (EV) in 100-mm dishes using PolyFect transfection reagent (Qiagen) according to the manufacturer's instruction. After 4 weeks of screening, the cell lines stably expressing ST6GalNAcII (FTC-133/ST6GalNAcII) and empty vector (FTC-133/mock) were established. Then cells were collected for gene expression assay and for further explorations. The cell transfection efficiency was 79% and the survival rate was 87%.

Invasiveness of tumor cells was examined using 24-well Transwell units (Corning, Corning, NY, USA) with 8-mm pore size poly-carbonate filter coated with Matrigel (BD Biosciences) to form a continuous thin layer. Cells (3×10⁵) were harvested in serum-free medium containing 0.1% BSA and added to the upper chamber. The lower chamber contained 500 ml 90% RPMI-1640 and 10% FBS. At the end of incubation, the cells on the upper surface of the filter were completely removed by wiping with a cotton swab. The filters were fixed in methanol and stained with Wright-Giemsa. Cells invading the Matrigel that reached the lower surface of the filter were counted with light microscope at a magnification of ×400. In migration assay, the upper chamber was not coated with Matrigel. Samples were acquired in triplicate and data expressed as the average cell number in five fields.

The tumorigenicity of ST6GalNAcII in vivo was investigated using a xenograft tumor model in the nude mice. Forty-eight 5-week-old male athymic nude mice were provided with sterilized food and water and equally divided into three groups. Approximately, 1×10⁷ cells (with or without ST6GalNAcII shRNA interference and control shRNA) were subcutaneously inoculated into the right flank of each nude mouse. Once bearing palpable tumors (about 4 weeks after tumor cell inoculation), mice were sacrificed and their tumors were isolated, weighed, and photographed. Experiments were repeated three times.

LY294002 (Sigma) was used to suppress the activity of the PI3K/Akt signaling in FTC-238 cells. Briefly, the tumor cells (1×10⁴ cells/well) were incubated with dimethyl sulfoxide (DMSO) or the PI3K inhibitor LY294002 (20 mmol/l) dissolved in DMSO, and collected after 24 h. Tumorigenicity was analyzed when PI3K/Akt signaling was blocked in xenograft tumor model. Sixty female athymic nude mice (5-week-old) were divided into 4 groups and 1×10⁷ FTC-238 cells (with DMSO, LY294002, control shRNA, Akt shRNA, respectively) were injected subcutaneously into the axillary regions of each nude mouse, respectively. Once bearing palpable tumors (about 4 weeks after tumor cell inoculation), mice were sacrificed and their tumors were isolated and weighed. Changes in protein expression were measured by western blot analysis.

Tumors were removed from the mice and immunohistochemical (IHC) staining was conducted using formalin-fixed paraffin-embedded sections of tissues by the avidin-biotin-peroxidase complex (ABC) method. Four-micron sections of formalin-fixed paraffin-embedded tissues were cut with a microtome and dried overnight at 37°C on a silicanized slide (Dako, Carpinteria, CA, USA). Samples were deparaffinized in xylene at room temperature for 80 min and washed with a graded ethanol/water mixture and then with distilled water. The samples were soaked in a citrate buffer and then microwaved at 100°C for 10 min. The following steps were used. Before addition of the primary antibodies, endogenous peroxidase activity was blocked by incubation in methanol containing 1% H₂O₂ for 20 min, followed by 60 min incubation with normal donkey serum to reduce background staining. The primary antibodies, goat anti-human, ST6GalNAcII antibodies (Abcam, Cambridge, UK), were incubated at 4°C for 8 h, followed by incubation with the biotinylated secondary antibodies (donkey anti-goat IgG; Santa Cruz Biotechnology) for 30 min and ABC complex for 30 min. The primary and secondary antibodies were used at 1:80 and 1:100 dilutions, respectively. The peroxidase binding sites were demonstrated by the diaminobenzidine method. A phosphate-buffered solution instead of the primary antibody was used in the protocols for negative controls. The level of expression level is measured by Image-Pro Plus software 6.0.

Each experiment was performed at least in triplicate, and the measurements were performed in three independent experiments. The data were expressed as mean ± standard deviation (SD) from the triple tests of each group. SPSS 17.0 software was used for statistical analysis and Student's t-test was selected to determine the significance of differences among the examined groups. P<0.05 was considered to be statistically significant.

Real-time PCR and western blot analysis was used to evaluate the expression level of ST6GalNAcII in mRNA (Fig. 1a) and protein expression (Fig. 1b). These data indicated that ST6GalNAcII may be associated with metastasis of human follicular thyroid carcinoma cells (FTC-238).

Owing to the high expression of ST6GalNAcII in FTC-238 cells, we silenced ST6GalNAcII with shRNA, in order to elucidate the effect of ST6GalNAcII on the invasion and metastasis of thyroid cancer cells. As shown in Fig. 2a and b, the expression level of ST6GalNAcII was significantly reduced in FTC-238 transfectants compared to the control transfectants (P<0.05).

After ST6GalNAcII shRNA transfection, Transwell invasion assay was performed to further evaluate the invasion capability of cells with ST6GalNAcII knockdown on tumor cells in vitro. The invasion capability of FTC-238 cells transfected with ST6GalNAcII shRNA showed a significant reduction, as compared with control shRNA group, suggesting that cell invasion capability was inhibited by treatment with ST6GalNAcII shRNA (Fig. 2c).

Nude mice bearing FTC-238, FTC-238-control shRNA and FTC-238-ST6GalNAcII shRNA1 xenografts were used to analyze the differences by measuring tumor volumes. Fig. 2d showed a significant reduction of mean tumor volume in nude mice bearing FTC-238-ST6GalNAcII shRNA, as compared with control shRNA group.

IHC staining analysis of the tumor section revealed that ST6GalNAcII was reduced in tumors derived from FTC-238-ST6GalNAcII shRNA cells compared to control group (Fig. 2e).

The above results indicated that knockdown of ST6GalNAcII could inhibit the invasion ability of follicular thyroid carcinoma cells.

After elucidating whether the effect of ST6GalNAcII suppresses the invasion ability of FTC-238, we transfected FTC-133 cells with ST6GalNAcII expression vector to determine the effect of overexpression of ST6GalNAcII on tumor cell invasion ability of FTC-133. As shown in Fig. 3a and b, increased levels of mRNA and protein of were detected in FTC-133 transfectants.

Transwell invasion assay revealed that the invasion capability of FTC-133 cells transfected with ST6GalNAcII expression vector was obviously increased compared with the FTC-133/mock cells (Fig. 3c).

Nude mice were inoculated with tumor cells FTC-133, FTC-133/mock, and FTC-133/ST6GalNAcII. Tumor volumes were increased obviously in nude mice bearing FTC-133/ST6GalNAcII, as compared to the FTC-133/mock group (Fig. 3d).

High expression levels of ST6GalNAcII in tumor cells of FTC-133/ST6GalNAcII, was also detected using IHC staining, as shown in Fig. 3e.

Therefore, the upregulation of the ST6GalNAcII gene was able to increase the invasion ability of follicular thyroid carcinoma cells.

Having established the pivotal role of PI3K/Akt pathway in tumor cells, we investigated whether ST6GalNAcII activated the PI3K/Akt pathway and whether this pathway played a central role in ST6GalNAcII-mediated cell invasion. Fig. 4a shows that following the decreased expression level of ST6GalNAcII, the expression and activity of the PI3K/Akt pathway was inhibited. PI3K expression decreased the protein and phosphorylation levels of Akt. The degree of phosphorylation of Akt at Ser473 and Thr308 and its downstream effector NF-κB was also downregulated after ST6GalNAcII silencing. No variation could be detected in the total amount of Akt protein, indicating a true decrease in the phosphorylation status. In addition, as illustrated in Fig. 4b, overexpression of ST6GalNAcII in FTC-133 cells showed the reverse tendency. The above suggested that the variation of ST6GalNAcII expression levels alters the PI3K/Akt signaling pathway.

After inhibition of PI3K and Akt by LY294002 and Akt siRNA, the invasion ability was significantly inhibited in FTC-238 cells. By western blotting, FTC-238 cells treated with LY294002, and Akt siRNA treatment exhibited apparently decreased expression levels of the main signal molecules of PI3K/Akt pathway (Fig. 5a). As shown in Fig. 5b, the inhibition of PI3K/Akt pathway made the FTC-238 cells less invasive. In Transwell invasion assay, the invasive capability of FTC-238 cells transfected with Akt siRNA obviously decreased in the Transwell migration and invasion assay. Similar results were also observed in vivo analysis where reduced tumor weight was measured in the mouse group bearing FTC-238 tumors with impaired PI3K/Akt signaling (Fig. 5b). Altered expression levels of the main signal molecules of the PI3K/Akt pathway in the mouse group bearing FTC-238 tumors with LY294002 or Akt shRNA treatment were also validated using IHC staining. These data implicated a role of PI3K/Akt signaling in regulating the invasive properties of FTC-238 cells.

The ST6GalNAcII expression status was detected in thyroid cancer with the corresponding pericarcinomatous tissue samples by immunohistochemistry staining (Table I). The data as shown in Table I are number of cases, and the expression of ST6GalNAcII was classified as high if >30% of tumor cells are stained and as low if <30% of cancer cells were stained. It is shown that follicular thyroid cancer tissues had a higher expression level of ST6GalNAcII compared with transitional tissues (P=0.004). There was no significant association between ST6GalNAcII expression and age, or distant metastasis in follicular thyroid carcinoma patients (P>0.05). Interestingly, the expression of ST6GalNAcII was also closely correlated with histological grade, lymph node metastasis, and clinical stage (P=0.004, 0.002, 0.006, respectively).