Two pairs of shRNA were designed according to the bcl-2 sequence in GenBank (NM_000633), mainly following Tuschl’s rules [¹⁹]. The sequence was transcribed with the DNA polymerase III U6 promoter in plasmid pGCsi purchased from GeneChem Co. Ltd. (Shanghai, China). The plasmid pGCsi carries the GFP (green fluorescent protein) gene. In our previous study, we confirmed that both of them could successfully inhibit the expression of bcl-2 in HLECs [¹⁴]. In the present study, we use one of these shRNAs, called bcl-2 shRNA. Its forward sequence is: 5′-GAT CCC AAG TGA AGT CAA CAT GCC TGC TTC AAG AGA GCA GGC ATG TTG ACT TCA CTT TTT TT-3′.

SRA01/04 cells (a HLEC line) were cultured in DMEM medium supplemented with 10% fetal bovine serum, 2.05 mmol/l L-glutamine, 100 U/ml of penicillin and 100 μg/ml streptomycin at 37 °C, and 5% CO₂. For transfection, the cells were seeded in six-well plates at 1×10⁶ cells/well and allowed to grow overnight to 90%–95% confluency. In the PAMAM group, they were transfected with the mixture of 2 μg plasmid DNA and 12 μg PAMAM G5 (Sigma-Aldrich, St. Louis, MO) in 2 ml serum-free medium. In the liposome group, they were transfected with a mixture of 2 μg plasmid DNA and 4 μl Lipofectamine^TM 2000 (Invitrogen, Carlsbad, CA) in 2 ml serum-free medium. At 6 h after transfection, the medium was replaced by normal medium containing 10% fetal bovine serum and antibiotics up to 48 h after transfection. In addition to the medium control, cells were transfected with negative control shRNA.

At 24, 48, and 72 h after transfection, the samples were digested and centrifuged at 20,000× g for 10 min. After removing the supernatant, 200 μl of buffer was added and analyzed by flow cytometry. The difference in transfection efficiency between the PAMAM group and the liposome group at different times was compared. Transfected cells are green under a fluorescent microscope.

Total RNA from HLECs was prepared using Trizol® Reagent (Invitrogen, Carlsbad, CA). cDNA synthesis was conducted according to the RNA PCR kit protocol (Takara, Dalian, China). The GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene was used as a normalizing control. The designed paired primers were as follows: bcl-2, 5′-CAT CTC TGT TTT CTG GCC ACA-3′ (forward), 5′-ATC CTC CTC AGA AAC AGC AGC-3′ (reverse); GAPDH, 5′-ACC ACA GTC CAT GCC ATC AC-3′ (forward), 5′-TCC ACC ACC CTG TTG CTG TA-3′ (reverse). The PCR reaction was performed in a volume of 20 µl using SYBR green mix (Takara, Dalian, China) on an MXP3000 instrument (Stratagene Laboratories, La Jolla, CA). PCR results were analyzed using Opticon Monitor Analysis 2.0 software (Bio-Rad Laboratories, Inc., Hercules, CA). Each sample was tested at least three times.

Cells were lysed in a buffer containing 50 mmol/l Tris-HCl, pH 8.0, 150 mmol/l NaCl, 100 μg/ml phenylmethylsulfonyl fluoride, and 1% TritonX-100. Total protein in cell lysates was measured with the Bio-Rad colorimetric kit. Here, 50 μg protein lysate was separated by 12% SDS–PAGE and transferred onto polyvinylidene fluoride. After blocking in a 10% nonfat dry milk solution in washing buffer containing 10 mmol/l Tris (pH 7.6), 150 mmol/l NaCl, and 0.05% Tween-20, membranes were incubated for 1 h at room temperature with mouse monoclonal IgG1 of anti-bcl-2 (sc-7382; Santa Cruz Biotechnology, Santa Cruz, CA). Then they were washed four times and further incubated for 1 h with goat antimouse horseradish peroxidase–coupled secondary antibodies (Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature. Signals were detected with the ECL kit (Amersham Pharmacia Biotech UK Ltd., Little Chalfont, UK). β-Actin staining served as the internal standard for all membranes.

Apoptotic cells were measured with an Annexin V/FITC kit (Bipec Biopharma, Cambridge, MA) according to the manufacturer's instructions and analyzed by flow cytometry at 48 h after transfection. Experiments were conducted in triplicate. Furthermore, morphological alteration of cell apoptosis in SRA01/04 was observed under a confocal microscope using Hoechst 33258 (Sigma-Aldrich) staining approach. The expression of cytochrome c and the activity of cleaved caspase-3 were investigated by western blot as mentioned above. Anti-cytochrome c (sc-13561; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-cleaved caspase-3 (sc-22171; Santa Cruz Biotechnology) were used in western blot.

All experiments were performed in triplicate. The one-way ANOVA was used to determine the statistical significance of the data obtained. The software used was SPSS 11.0 (SPSS for Windows, version 11.0, SPSS Inc., Chicago, IL). A value of p<0.05 was taken to represent a statistically significant difference between group means.

The transfection rates of the PAMAM group and liposome group at 24, 48, and 72 h after transfection were 42.3±2.0%, 47.7±1.8%, 48.5±1.5%, and 35.1±1.8%, 41.1±1.7%, 42.0±2.3%, respectively. The difference was significant (p<0.05). We also observed the cells with a fluorescent microscope and found many green cells, which showed that they had been transfected, in the PAMAM group, liposome group, and negative control shRNA group; there were almost no green cells in the empty control group (Figure 1).

Real-time PCR showed that the bcl-2 mRNA expression of the PAMAM group and liposome group were greatly reduced compared with the negative control shRNA group and the empty control group. The expression in the PAMAM group was significantly lower than that in the liposome group (p<0.05; Figure 2).

Western blot showed that the bcl-2 protein expression of the PAMAM group and liposome group were greatly reduced compared with the negative control shRNA group (NC group) and the empty control group (EC group; Figure 3).

The percentage of HLECs undergoing apoptosis, measured by annexin V-FITC/PI staining, was greatly improved in the PAMAM group and liposome group at about 45.7±1.2% and 40.9±0.8%, respectively (Figure 4). Hoechst staining showed that bcl-2 shRNA transfected cells had a lower growth status, with nuclear fragmentation and condensation (Figure 5). The expression of cytochrome c and the activity of cleaved caspase-3 were greatly increased (p<0.05) in these two groups (Figure 6).