The SGC-7901 gastric cancer cell line was a gift from Dr Yang Zhang (Department of Biochemistry and Molecular Biology, Zhongshan Medical College, Sun Yat-Sen University, China) (20). The cell line was cultured in high-glucose DMEM (Gibco, BRL, Guangzhou, China) supplemented with 10% fetal bovine serum at 37˚C with 5% CO₂.

Id1-specific siRNA used for Id1 knockdown and the control siRNA were synthesized by GenePharma (Shanghai GenePharma Co., Ltd.). The sequences of siRNA targeting the Id1 coding region were as follows: sense, 5′-CUCGGAAUCCGAAGUUGGADTDT-3′ and antisense, 5′-UCCAACUUCGGAUUCCGAGDTDT-3′ (21). The siRNAs were then transfected into the PC3 cells by Lipofectine 2000 (Invitrogen, USA), according to the manufacturer's instructions.

The full-length Id1 cDNA was amplified from total cDNA of SGC-7901 cells by PCR, and was then subcloned between the KpnI and EcoRI sites of pcDNA3.1(+) vector. Purified plasmids were sequence-verified by Invitrogen (Shanghai, China). The plasmid was transfected into SGC-7901 cells by Lipofectine 2000. The primers used for PCR were as follows: forward, 5′-GATGGTACCATCATGAAAGTCGCCAGTG-3′ and reverse, 5′-GATGAATTCTCAGCGACACAAGATGCGA-3′.

SGC-7901 cells were seeded in 96-well plates at a concentration of 5,000 cells/well in a volume of 150 μl of cell culture medium. After 24 h, transfection was performed. The plates were incubated at 37˚C with 5% CO₂ for 48 and 72 h. MTT solution (20 μl) (5 g/l, dissolved in PBS) was added to each well and the plates were incubated at 37˚C for another 4 h. Subsequently, the supernatant was discarded and l50 μl dimethylsulfoxide was added to dissolve the insoluble MTT formazan. The absorbance values at 570 nm were detected by a multi-well plate reader (Tecan).

SGC-7901 cells were transfected with the above-mentioned siRNA or plasmid for 72 h and were then presented for flow cytometry assay. For cell cycle analysis, DNA labeling was performed using the Cycletest Plus DNA Reagent kit (BD Biosciences Pharmingen, USA), and the samples were analyzed using a flow cytometer (Beckman Counter, USA). For the detection of apoptotic cells, labeling tests involving both propidium iodide (PI) and annexin-V were performed using an Annexin-V staining kit (Invitrogen, USA), according to the manufacturer's instructions. Briefly, at least 1×10⁶ cells were harvested by trypsinization, incubated with FITC-labeled annexin-V and PI stock solutions for 10 min at room temperature and analyzed using a flow cytometer (Beckman Counter).

SGC-7901 cells were transfected for 72 h, harvested and lysed for total protein extraction. Protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, China). Equal amounts of protein were separated by 15% SDS-PAGE and transferred onto PVDF membranes. The membranes were rinsed with TBST and incubated in blocking buffer (5% dried milk in PBS) for 1 h at 37˚C, followed by incubation with primary antibodies at 4˚C overnight. The antibody against cyclin D1 used in western blotting was purchased from Beyotime (China), antibody against β-actin was purchased from PTG (USA), and other antibodies were purchased from Santa Cruz (USA). After washing with TBST three times, the membranes were incubated with their corresponding secondary antibodies for 1 h. The blots were visualized by an enhanced chemiluminescence detection system (Amersham). The expression of β-actin was used as a normalization control for protein loading.

Data were expressed as the means ± SD. Statistical analyses were performed using Student's t-test. P<0.05 indicated statistical significance.

The effect of siRNA or pcDNA3.1-Id1 on the levels of Id1 was evaluated using both reverse transcriptase-PCR and western blotting. As shown in Fig. 1, significantly decreased Id1 mRNA and protein levels were detected in siRNA transfected cells compared to the control group (siControl), indicating that Id1 siRNA successfully downregulated the Id1 gene in SGC-7901 cells. Furthermore, pcDNA3.1-Id1 upregulated the levels of Id1 in SGC-7901 cells.

To evaluate the effect of Id1 on the proliferation of SGC-7901 cells, MTT assay was performed. As shown in Fig. 2, Id1 was involved in the proliferation of SGC-7901 cells; the inhibitory effect was evident after 72-h transfection with Id1-siRNA. However, the upregulation of Id1 in SGC-7901 cells could not promote cell proliferation.

To examine the effect of altered Id1 expression on cell apoptosis, transfected cells were analyzed by Annexin V-FITC/PI double staining. As shown in Fig. 3A, compared to the control group, no significant change was found in the annexin V-positive or annexin V and PI-double positive cell fractions in the Id1 siRNA-transfected group. Moreover, Id1 overexpression in SGC-7901 cells transfected with pcDNA3.1-Id1 showed no impact on cell apoptosis; it suggested that Id1 is not involved in the apoptosis of SGC-7901 cells. Bax and Bad are important in the apoptotic pathway. In this study, although the changes of Id1 levels did not affect the apoptosis of SGC-7901 cells, we detected the protein levels of Bax and Bad in SGC-7901 cells. As shown in Fig. 4A, the expression of Bax and Bad increased when cells were transfected with Id1 siRNA, as expected, whereas their expression decreased when cells were transfected with pcDNA3.1-Id1.

Cell cycle distribution was analyzed by flow cytometry after the cells were transfected for 72 h. As shown in Fig. 3B, the proportion of the G2/M phase was decreased in Id1 siRNA-transfected cells compared to the control group. The cell cycle did not change when Id1 was overexpressed in SGC-7901 cells. We further analyzed the changes of certain cell cycle regulators. Cyclin D1 was decreased in the Id1 knockdown group, while p16 and p21 were increased; cyclin D1 was elevated in the Id1 overexpression group, while p16 and p21 were decreased (Fig. 4B).

The correlation between Id1 and p-Akt expression in SGC-7901 cells was examined by western blotting. As shown in Fig. 5, Akt phosphorylation at Thr308 and Ser473 was reduced in Id1 siRNA-treated SGC-7901 cells, but enhanced in pcDNA3.1-Id1-treated SGC-7901 cells. These results suggest that the expression of p-Akt is associated with Id1 expression in SGC-7901 cells.