Polypeptides From Chlamy Farreri (PCF)’s Chemoprevention Against Chronic UVB Exposure Induced HaCaT Cell Malignant Transformation By Targeting P16and RASSF1A Methylation

Objective To establish chronic UVB-induced malignant transformation model of HaCaT cells and investigate the mechanism of Polypeptide from Chlamys farreri(PCF) protecting HaCaT cells from UVB-induced malignant transformation through methylation pattern of P16, RASSF1A and expression of GADD45a, DNMTS. Methods The optimal doses of UVB radiation for induction of malignant transformation of HaCaT cells were determined by CCK-8assay. The degree of malligant transformation was detected by cell morphology, soft agar colony formation, zymographic analysis of MMP-9activity. Cells were divided into four groups:control group, UVB model group, UVB+2.84mmol/L PCF group, UVB+2.84mmol/L vitamin C. Effect of PCF on UVB-induced malignant transformation of HaCaT cells were detected by Plating efficiency assay, soft agar colony formation, and cell cycle assay. Time course changes of DNMTs in HaCaT cells subjected to chronic UVB exposure were assayed by RT-PCR. Protein expression levels of DNMT3b, GADD45a, RASSF1A and P16in HaCaT cells subjected to chronic UVB exposure were determined by Western blot. Methylation specific-high resolution melting (MS-HRM) analyses were carried out to quantify the extent of methylation of the P16and RASSF1A. Results The results of experiment suggest that10mJ/cm2UVB irradiation for20times make up the cell malignant transformation. Pretreatment PCF or VC(2.84mmol/L) against chronically UVB irradiation-damaged malignant transformation and the results showed that PCF is significantly superior than VC for cellular malignant transformation (P<0.05). The mRNA expression of DNMT1and DNMT3a had no statistical significance VS the control group. The mRNA and protein expression of DNMT1started to rasise at4times and reached a peak at20times(P<0.05). The protein expression of GADD45a reached a peak at4times and decreased after4times until reached to the bottom at20times(P<0.05). The protein expression of P16and RASSF1A decreased after4times until reached to the bottom at20times(P<0.05). Chronic UVB exposure induced RASSF1A and P16hyperethylation and down-regulation expression of protein, which was reversed by pretreatment PCF or VC, and PCF is significantly superior than VC (P<0.05). Pretreatment PCF or VC attenuated expression of DNMT3b and increased protein expression of GADD45a(P<0.05). Conclusion PCF’s anti-malignant transformation effects in HaCaT cells were mediated by inhibiting the hypermethylation of RASSF1A and P16induced by chronic UVB exposure. This effect is likely to be contributed by DNMT3b and GADD45a.