Effects of expression of growth arrest and DNA damage inducible gene 45α interfered by small interference RNA on the invasion and migration of a cutaneous squamous cell carcinoma cell line A431

Abstract

Objective To evaluate effects of targeted silencing of growth arrest and DNA damage inducible gene 45α (Gadd45α) by small interference RNA (siRNA) on the invasion and migration of a cutaneous squamous cell carcinoma cell line A431. Methods Real-time fluorescence-based quantitative PCR and Western blot analysis were performed to detect the mRNA and protein expression of A431 and Colon16 cells respectively, and then A431 cells with highly expressed Gadd45α served as a research object. Cultured A431 cells were divided into 3 groups: experimental group transfected with Gadd45α-siRNA-1, negative control group transfected with negative control siRNA, and blank control group receiving no treatment. After the RNA interference, the mRNA and protein expression of Gadd45α in the above 3 groups were measured by real-time fluorescence-based quantitative PCR and Western blot analysis, respectively, and the effect of the RNA interference on the invasion and migration abilities of A431 cells was evaluated by Transwell and wound scratch assays. Results Gadd45α mRNA and protein were highly expressed in the A431 cells. After the RNA interference, the experimental group showed markedly lower mRNA and protein expressions of Gadd45 in the A431 cells (0.286 ± 0.013, 0.33 ± 0.007, respectively) compared with the negative control group (1.028 ± 0.183, 0.87 ± 0.002, respectively) and blank control group (1.001 ± 0.057, 0.86 ± 0.004, respectively) , and there were significant differences in the mRNA and protein expressions of Gadd45 among the 3 groups (F= 5 893.857, 2 763.000, both P < 0.001) . The number of A431 cells crossing the polycarbonate membrane was higher in the experimental group (66.33 ± 3.79) than in the negative control group (26.00 ± 4.36) and the blank control group (28.33 ± 4.16) , and there was a significant difference among the 3 groups (F= 20.084, P= 0.002) . Moreover, the wound scratch assay showed that the number of migrating A431 cells per high-power field was significantly higher in the experimental group than in the negative control group and the blank control group (315.33 ± 6.66, 154.67 ± 2.08, 130.67 ± 3.51 respectively; F= 1 676.255, P < 0.001) . Conclusion Gadd45α is highly expressed in the cutaneous squamous cell carcinoma cell line A431, and targeted silencing of Gadd45α gene can enhance the in vitro invasion and migration abilities of A431 cells. Key words: Neoplasms, squamous cell; RNA interference; Neoplasm invasiveness; Cell migration assays; Growth arrest and DNA damage-inducible 45α