Effects of silencing Gpr48 gene expression on PKC signaling pathway activation and cell invasion in skin squamous cell carcinoma cell line A431

Abstract

Objective To analyze the effects of silencing G-protein-coupled receptor 48 (Gpr48) gene expression on protein kinase C (PKC) signaling pathway and cell invasion in skin squamous cell carcinoma (SCC) . Methods Gpr48 interfering shRNA (shRNA-Gpr48 group) and negative control shRNA were infected into skin SCC cell line A431. And the relative expression of Gpr48 was detected by real-time quantitative reverse transcription polymerase chain reaction (RT- PCR) . Expressions of PKC-related antibody proteins were determined by Western Blot. Transwell chamber assay was used for cell invasion. Statistical analysis was performed with SPSS 20.0-software. The independent sample t test was used to compare the differences of the above indexes in shRNA-Gpr48 group and shRNA-NC group. The effects of Gpr48 deletion on activation of A431 cell line PKC and and cell invasion stimulated by tripropylamine (TPA) were determined. Results qRT-PCR results showed the relative expression of Gpr48 in the shRNA-Gpr48 group was lower than that in the shRNA-NC group (0.27±0.06) vs (1.01±0.12) (t = 17.441, P < 0.001) . The mRNA expression of p-PKCδ (0.463±0.035) vs (0.721±0.074) , and NF-κB p65 was lower in the shRNA-Gpr48 group compared to the shRNA-NC group (0.631±0.079) vs (0.834±0.102) (t = 9.966, 4.975, all P < 0.01) while the mRNA expression of Notch1 was higher (0.981±0.037) vs (0.562±0.041) (t = 23.991, P < 0.001) in the shRNA-Gpr48 group. Western Blot results showed that the protein expression of p-PKCδ (0.221±0.034) vs (0.292±0.046) , and NF-κB p65 (0.512±0.047) vs (0.636±0.051) (t =3.925, 5.653, all P < 0.01) was lower in the shRNA-Gpr48 group compared to the shRNA-NC group while the expression of Notch1 was higher in the shRNA-Gpr48 group (0.524±0.063) vs (0.421±0.076) (t = 3.299, P < 0.01). The cell invasion rate in the shRNA-Gpr48 group was lower than that in the shRNA-NC group (35.03±1.54) ﹪ vs (51.83±2.76) ﹪ (t = 16.809, P < 0.01) . Conclusion Silencing Gpr 48 expression may inhibit cancer cell proliferation and invasion by inhibiting PKC activation, decreasing expressions of p-PKCδ and NF-κB p65 and up-regulating the expression of tumor suppressor gene Notch1. Key words: Skin; Squamous cell carcinoma; G-protein-coupled receptor 48; Signal pathway