Inhibitory effect of survivin-shRNA on A431 cutaneous squamous cell carcinoma

Abstract

Objective: To observe the effect of survivin-shRNA on A431 cutaneous squamous cell carcinoma (SCC) and explore the molecular biological mechanism of nuclear factor κB (NF-κB) in the inhibition of survivin-shRNA on cutaneous SCC.

Methods: Survivin-shRNA adenovirus vector was constructed to screen out the best interference sequence. Nude mice were subcutaneously inoculated with the cultured A431 cell suspension to duplicate A431 transplanted tumor models. These mice were randomly divided into the blank control group, the rAd-EGFP negative control group, the rAd-survivin-shRNA transfection group and the Res positive control group, with 5 mice in each group. The corresponding reagents were injected into the tumors. All the mice were sacrificed on the 20^th day. The tumor tissues were isolated, with tumor volume and tumor weight measured, the tumor growth curves were accordingly plotted and the tumor inhibition rate was consequently calculated. Hematoxylin-eosin (HE) staining was used to observe the cellular morphology of the tumors. TUNEL was applied to the detection of cell apoptosis. Western blot was applied to the detection of the expression of Survivin, inhibitor of NF-κB (IκB), P65, P53 and Caspase-3.

Results: As to the transplanted tumors, tumor volume and tumor weight were decreased in nude mice in the rAd-survivinshRNA transfection group and the Res positive control group, in comparison with the blank control group and the rAd-EGFP negative control group, the differences were of statistical significance (p < .05); The results of TUNEL showed that the apoptosis rates were significantly increased in the rAd-survivin-shRNA transfection group and the Res positive control group, in comparison with the blank control group and the rAd-EGFP negative control group, the difference was statistically significant (p < .05); In the rAd-survivin-shRNA transfection group and the Res positive control group, the microscopic observation showed a small amount of sparsely-distributed tumor cells, degenerative liquefactive necrosis, cancer cell shrinkage and round-shape, and karyopycnosis; The expressions of Survivin and P65 proteins were decreased in the rAd-survivin-shRNA transfection group and the Res positive control group, in comparison with the blank control group and the rAd-EGFP negative control group, the differences were statistically significant (p < .05), while the expressions of IκB, P53 and Caspase-3 proteins were significantly increased, in comparison with the blank control group and the rAd-EGFP negative control group, the differences were statistically significant (p < .05).

Conclusions: Survivin-shRNA can inhibit the growth of the transplanted tumors in cutaneous SCC nude mice, one of the mechanisms is to promote the apoptosis of tumor cells and inhibit the growth of SCC transplanted tumors by inhibiting the NF-κB signaling pathway and then activating the tumor suppressor gene P53.