AIM

To investigate the gene knock-down effect by the phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA)-targeted double-stranded RNA (dsRNA) and its effect on cell proliferation and cycle distribution in SW948.

METHODS

Two PIK3CA-targeted dsRNAs were constructed and transfected into SW948 cells. Transfections were performed using lipofectamine™ 2000. The transfection effectiveness was calculated basing on the rate of fluorescence cell of SW948 at 6 h after transfection. Total messenger RNA was extracted from these cells using the RNeasy kit, and semiquantitative reverse transcription polymerase chain reaction was performed to detect the down-regulation of PIK3CA, AKT1, MYC, and CCND1 gene expression. Cells were harvested, proteins were resolved, and western blot was employed to detect the expression levels of PIK3CA, AKT1, MYC, and CCND1 gene. Cell proliferation was assessed by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay and the inhibition rate was calculated. Soft agar colony formation assay was performed basing on colonies greater than 60 μm in diameter at ×100 magnification. The effect on cell cycle distribution and apoptosis was assessed by flow cytometry. All experiments were performed in triplicate.

RESULTS

Green fluorescence was observed in SW948 cell transfected with plasmid Pgenesil-1, and the transfection effectiveness was about 65%. Forty-eight hours post-transfection, mRNA expression of PIK3CA in SW948 cells was 0.51 ± 0.04 vs 0.49 ± 0.03 vs 0.92 ± 0.01 vs 0.93 ± 0.03 (P = 0.001 ) in Pgenesil-CA1, Pgenesil-CA2, negative and blank group respectively. mRNA expression of AKT1 was 0.50 ± 0.03 vs 0.48 ± 0.01 vs 0.93 ± 0.04 vs 0.92 ± 0.02 (P = 0.000) in Pgenesil-CA1, Pgenesil-CA2, negative and blank group respectively. mRNA expression of MYC was 0.49 ± 0.01 vs 0.50 ± 0.04 vs 0.90 ± 0.02 vs 0.91 ± 0.03 (P = 0.001) in the four groups respectively. mRNA expression of CCND1 was 0.45 ± 0.02 vs 0.51 ± 0.01 vs 0.96 ± 0.03 vs 0.98 ± 0.01 (P = 0.001) in the four groups respectively. The protein level of PIK3CA was 0.53 ± 0.01 vs 0.54 ± 0.02 vs 0.92 ± 0.03 vs 0.91 ± 0.02 (P = 0.001) in Pgenesil-CA1, Pgenesil-CA2, negative and blank group respectively. The protein level of AKT1 in the four groups was 0.49 ± 0.02 vs 0.55 ± 0.03 vs 0.94 ± 0.03 vs 0.95 ± 0.04, P = 0.000). The protein level of MYC in the four groups was 0.51 ± 0.03 vs 0.52 ± 0.04 vs 0.92 ± 0.02 vs 0.95 ± 0.01 (P = 0.000). The protein level of CCND1 in the four groups was 0.54 ± 0.04 vs 0.56 ± 0.03 vs 0.93 ± 0.01 vs 0.93 ± 0.03 (P = 0.000). Both Pgenesil-CA1 and Pgenesil-CA2 plasmids significantly suppressed the growth of SW948 cells when compared with the negative or blank group at 48 h after transfection (29% vs 25% vs 17% vs 14%, P = 0.001), 60 h after transfection (38% vs 34% vs 19% vs 16%, P = 0.001), and 72 h after transfection (53% vs 48% vs 20% vs 17%, P = 0.000). Numbers of colonies in negative, blank, CA1, and CA2 groups were 42 ± 4, 45 ± 5, 8 ± 2, and 10 ± 3, respectively (P = 0.000). There were more than 4.5 times colonies in the blank and negative control groups as there were in the CA1 and CA2 groups. In addition, the colonies in blank and negative control groups were also larger than those in the CA1 and CA2 groups. The percentage of cells in the CA1 and CA2 groups was significantly higher in G₀/G₁ phase, but lower in S and G₂/M phase when compared with the negative and control groups. Moreover, cell apoptosis rates in the CA1 and CA2 groups were 5.11 ± 0.32 and 4.73 ± 0.32, which were significantly higher than those in negative (0.95 ± 0.11, P = 0.000) and blank groups (0.86 ± 0.13, P = 0.001). No significant difference was found between CA1 and CA2 groups in cell cycle distribution and apoptosis.

CONCLUSION

PIK3CA-targeted short hairpin RNAs can block the phosphoinositide 3-kinase-Akt signaling pathway and inhibit cell growth, increase apoptosis, and induce cell cycle arrest in the PIK3CA-mutant colon cancer SW948 cells.