Human thyroid carcinoma TPC-1 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). TPC-1 cells were maintained in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Grand Island, NY, USA), 100 U/ml penicillin and 50 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and incubated at 37°C in a humidified atmosphere of 5% CO₂.

dTAT NP-NIS and the negative control (NC) plasmids were purchased from Shanghai GenePharma, Co., Ltd. (Shanghai, China). TPC-1 cells were incubated in six-well plates at a density of 1–2×10⁶ cells/well. Subsequently, 100 nmol/l plasmids were transfected Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and transfected into TPC-1 cells.

Following transfection, TPC-1 cells were seeded six-well plates at a density of 1–2×10⁶ cells/well for 24 h. Total RNA from the TPC-1 cells was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following treatment with DNase (Takara Biotechnology Co., Ltd., Dalian, China) a TaqMan miRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to synthesize cDNA using 1 µg RNA. Subsequently, a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to conduct RT-qPCR using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.). The sequences were as follows: NIS forward, 5′-ACSCACTGGAAGCACGGCGG-3′ and reverse, 5′-GTGGMRCCGTGCAKRTTGG-3′; β-actin forward, 5′-AGGCACCAGGGCGTGAT-3′ and reverse, 5′-TGCTCCCAGTTGGTGACGAT-3′. The cycling conditions used were 95°C for 4 min, 95°C for 15 sec and 60°C for 30 sec for 40 cycles. The RT-qPCR experiment was conducted 6 times. Relative expression of mRNA was quantified using the 2^−∆∆CT method (17).

TPC-1 cell total protein was extracted using a radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA). Protein concentrations were determined using a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal quantities of total protein (50 µg) were resolved using 10–12% SDS-PAGE (Bio-Rad Laboratories, Inc.) and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked in 5% non-fat milk for 2 h at room temperature and then incubated with rabbit anti-human NIS (1:500; sc-134515), caspase-3 (1:500; sc-98785), Akt (1:200; sc-8312) and phosphorylated-Akt (1:500; sc-33437) and PTEN primary antibodies (1:500; sc-9145; all Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. The membranes were washed with Tris-buffered saline containing Tween 20 (Sigma-Aldrich; Merck KGaA) and then incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit immunoglobulin G antibodies (1:1,000; sc-2922; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at room temperature. The membranes were visualized using an Immobilon Western chemiluminescent HRP substrate (EMD Millipore, Billerica, CA, USA), and the blots were densitometrically analyzed using Image Lab 3.0 software (Bio-Rad Laboratories, Inc.).

TPC-1 cells were seeded onto 96-well plates at a density of 1–2×10³ cells/well for 12, 24 and 48 h, and subsequently incubated with 20 µl MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA) for 4 h. Then, 150 µl DMSO (Invitrogen; Thermo Fisher Scientific, Inc.) was added to each well and shaken for 15 min after the medium was removed. The optical density (OD) of each well was detected at 490 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

TPC-1 cells were seeded onto six-well plates at a density of 1–2×10⁶ cells/well for 24 h and washed twice using phosphate-buffered saline (PBS). TPC-1 cells were resuspended using buffer solution (from a FITC Annexin V Apoptosis Detection kit; BD Biosciences, Franklin Lakes, NJ, USA), and incubated with 5 µl Annexin V-fluorescein isothiocyanate and 10 µl propidium iodide for 30 min at 4°C in the dark. Flow cytometry (FACSCalibur; BD Biosciences) was used to measure cell apoptosis rates of the TPC-1 cells.

TPC-1 cells were seeded onto six-well plates at a density of 1–2×10⁶ cells/well for 24 h and washed twice using PBS. TPC-1 cell was fixed using 4% paraformaldehyde for 30 min at 4°C. Then, fixed TPC-1 cells were washed twice using PBS and incubated with sodium citrate (0.1%) containing 0.1% Triton X-100 (Beyotime Institute of Biotechnology, Haimen, China) for 5 min at 4°C. TPC-1 cells were dyed by DAPI staining and incubated for 10 min at 4°C in the dark, then activated using ultraviolet. DAPI staining was observed and images captured using a fluorescence microscope (CKX41; Olympus Corporation, Tokyo, Japan) at 340 nm.

All values were expressed as the mean ± standard deviation. Statistical analyses were conducted using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Data were analyzed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference between values.

To evaluate the gene expression of NIS in TPC-1 cells transfected with dTAT NP and negative control, RT-qPCR was performed. As shown in Fig. 1, there was a significant increase in the relative gene expression of NIS of dTAT NP group, compared with the NC group (P<0.05).

In order to characterize the dTAT NP-NIS and NC plasmid influence on the protein expression of NIS, western blot analysis was performed. The expression of NIS protein was significantly higher than that of the NC group (P<0.05; Fig. 2).

We clarified the overexpression of NIS influence on cell growth of TPC-1 cell. The results from MTT assay showed the cell growth of TPC-1 cell was significantly suppressed after dTAT NP-NIS transfection at 24 and 48 h, compared with the NC group (P<0.05; Fig. 3).

To determine the effect of the overexpression of NIS apoptosis rates in TPC-1 cells, cellular apoptosis was observed in TPC-1 cells using flow cytometry. As shown in Fig. 4, cellular apoptosis of dTAT NP-NIS transfection was significantly higher than that of the NC group, after dTAT NP-NIS transfection for 24 h (P<0.05).

To validate the overexpression of NIS influence on cell nucleus apoptosis of TPC-1 cell, TPC-1 cells were stained with DAPI. After dTAT NP-NIS transfection at 24 or 48 h, there was a visible increase in the nuclear apoptosis rate of TPC-1 cell-transfected with dTAT NP-NIS compared with the NC group (Fig. 5).

To further clarify whether the effect of NIS expression on caspase-3 protein expression of TPC-1 cell, caspase-3 protein expression was analyzed using western blot analysis in TPC-1 cell. Western blot analysis displayed that caspase-3 protein expression was significantly activated in the dTAT NP-NIS transfected cells at 24 h, compared with the NC group (Fig. 6).

To further validate the effect of NIS expression on Akt signaling in TPC-1 cells, Akt and p-Akt protein expression levels were detected. Compared with the NC group, the relative Akt/p-Akt rate was significantly reduced by overexpression of NIS (Fig. 7).

To further evaluate the effects of NIS expression on PTEN signaling in TPC-1 cells, PTEN protein expression was evaluated. As shown in Fig. 8, relative PTEN protein expression was significantly elevated compared with the NC group (Fig. 8).