Transfection of the Human Sodium/Iodide Symporter (NIS) Gene with Liposomes and the Expression of the NIS Protein in Human Lung A549 Cancer Cells

Human lung A549cancer cell thawing and culture conditions

Human lung A549 adenocarcinoma cells (1×10⁶/ml) were thawed and grown as monolayers at 37°C under an atmosphere of 5% CO₂ under air in 25-cm tissue culture flasks containing 10% FBS-DMEM culture medium. When the cells grew to 90% confluence, they were transfected with the hNIS vectors or vacancy vectors based on the manufacturer’s instructions.

Recombinant pcDNA3-hNIS plasmid amplification, purification and identification

The recombinant pcDNA3-hNIS plasmid was amplified, purified and identified as described previously^[6]. Briefly, the DH5α bacillus coli was thawed, cultured in a LB plate with ampicillin, X-gal (5-bromine, 4-chlorine, 3-indole, ß-galactan), and IPTG (iso-propyl-ß-D-thiogalactoside). Then the recombinant pcDNA3-hNIS plasmid was transfected into DH5α bacteria for propagation at 37°C for 16 h. The propagated recombinant pcDNA-hNIS plasmid was extracted from the culture and purified by the Plasmid Mini Kit, and then identified by a restriction EcoRI endonuclease digestion (the endonuclease point is G↓AATTC).

Human NIS cDNA sequencing assay

Based on established Genebank sequences: four primers were designed for the sequence detection of the amplified hNIS pcDNA3. P1: 5’-CTG CCG AGC ATC CTC CCA C-3’; P2: 5’-CAC TGT AAG CAC AGG CCA GG-3’; P3: 5’-CAT GCC TCT GCT GGT GCT G-3’; P4: 5’-TGG CCC ATC CTG AGG TTC C-3’. The amplified human NIS cDNA gene was detected bilaterally from both ends. Combing these 4 detection results resulted in the whole sequence of the inserted gene. The sequencing was accomplished by the Shanghai United Gene Ltd Co.

Transfection of lung A549 cancer cells by the recombinant pcDNA3-hNIS plasmid

In this study, Lipofectamine-2000 was employed to mediate hNIS gene transfection.

• ①Grouping: Cultured A549 cells were divided into an experimental group transfected with the recombinant pcDNA3-hNIS plasmid and a control group transfected only with the plasmid pcDNA3.

• ②One day before transfection: The human lung A549 cancer cells were digested with 0.05% steapsin and 0.53 mmol/L EDTA, and then cultured in the medium with 10% FBS-DMEM.

• ③The day of transfection: (a) The recombinant pcDAN3-hNIS plasmid (24 μg) and 1.5 ml of Opti-MEM I were mixed in an Eppendorf tube at room temperature for 5 min. (b) At the same time, 60 μl of Lipofectamin 2000 and 1.5 ml of Opti-MEM I were mixed in another Eppendorf tube at room temperature for 5 min. (c) The recombinant pcDAN3-hNIS plasmid, Lipofectamin 2000 and Opti-MEMI were mixed completely and held at room temperature for 20 min. (d) A 3 ml mixture of the recombinant pcDAN3-hNIS plasmid, Lipofectamin 2000 and Opti-MEMI was slowly lysed in 75-cm² flasks containing human lung A549 cancer cells, and incubated for transfection at 37°C under an atmosphere of 5% CO₂ in air.

• ④The 2nd day of transfection: The suspend liquid was discarded and replaced with 10% FBS-DMEM for continuing culture.

• ⑤The 3rd day of transfection: Lung cancer A549 cells were digested with 0.05% steapsin containing 0.53 mmol/L EDTA, and then placed into 6-well plates; 2 ml of 10% FBS-DMEM medium was added to each well for continuing culture.

Detection of the hNIS protein transfected by the recombinant pcDAN3 plasmid in human lung A549 cancer cells

The NIS gene expression in the lung cancer cells was detected and assayed by Western blot immunoblotting and immoncytohistochemistry.

• ①Western blots: (a) Human lung A549 cancer cells were cultured in 10-cm plates. (b) When the cultured cells grew to 70% confluence, the recombinant pcDNA3-hNIS plasmid and vacancy pcDNA3 vector were transfected to the A549 cancer cells and 24 h later, the culture fluid was replaced with 10 ml of 10% FBS DMEM followed by culturing at 37°C under an atmosphere of 5% CO₂ in air. (c) After 3 days of culture, the culture fluid was discarded and the plate placed on ice. The cells were washed with 0°C PBS twice and followed by the addition of 1 ml of fission buffer fluid and a 20-min incubation. (d) The cells on the plate were collected and put into iced-centrifuge tubes. (e) The cells were centrifuged at 12,000 rpm (4°C) for 2 min, the supernatant collected, subjected to electrophoresis, membrane transfer and development of an image.

• ②Immunohistochemical staining: (a) Preparation of the specimen: The A549 cells were incubated in 4 portions of 6-well plates. Two of the 6-well plates were the experimental group transfected with the pcDNA3-hNIS gene and other two of the 6-well plates were the control group transfect with only the empty pcDNA3 vector. The A549 cells were plated in 6-well plates at a density of 5×10⁵ cells/ml in 10% FBS-DMEM medium and incubated for 96 h at 37°C in 5% CO₂. Then the culture medium was discarded and the cells were dried under normal atmosphere for the immunochemical staining. (b) Immunohistochemistry staining procedure: The lung cancer cells transfected with the recombinant pcDNA3-hNIS plasmid were incubated with monoclonal rat-anti human primary antibody followed by sheep anti-rat as the second antibody. The cells were then incubated with the horseradish peroxidase (HRP) for 30 min at 37°C. The antigen-antibody complex was visualized using diaminobenzidine. (c) Judgment of the staining results: Expression of the NIS protein on the cell membrane surface was judged by the yellow brown staining: when the stained cells were assessed as 0~1, 2~25, 26~50 and 51~100 %, they were judged as negative (-), positive (+), distinctly positive (++) and strongly positive (+++).