Effect of YTH Domain Family Protein 2 on the Sodium Arsenite-Induced Malignant Transformation of Skin Cells.

Objective To investigate the effect of liquid-liquid phase separation(LLPS)of YTH domain family protein 2(YTHDF2)on the sodium arsenite-induced malignant transformation of skin cells,providing a new intervention target for the prevention and control of sodium arsenite-induced carcinogenesis.Methods The HaCaT cell model of malignant transformation was constructed by continuous treatment with 1 μmol/L sodium arsenite for 22 weeks,including cells with normal YTHDF2 LLPS(YTHDF2-wt)and cells with inhibited YTHDF2 LLPS(YTHDF2-mut).Confocal microscopy was employed to observe and characterize the LLPS droplets formed by YTHDF2 during sodium arsenite-induced malignant transformation of skin cells.Cell proliferation,scratch healing,and colony formation assays were performed to detect malignant phenotypes.Western blotting,quantitative reverse transcription PCR,and immunofluorescence experiments were conducted to examine the effects of YTHDF2 LLPS on the mRNA and protein levels of phosphatase and tensin homolog deleted on chromosome ten(PTEN)during sodium arsenite-induced malignant transformation of skin cells.Results After 4 weeks of sodium arsenite treatment,LLPS droplets of YTHDF2 appeared in YTHDF2-wt cells,and the number of droplets gradually increased as the treatment time was prolonged(<i>F</i>=35.252,<i>P</i><0.001),while no phase-separated droplets were observed in YTHDF2-mut cells.Compared with YTHDF2-mut cells,YTHDF2-wt cells showed enhanced proliferation at the time points of 48 h(<i>t</i>=3.654,<i>P</i>=0.006)and 72 h(<i>t</i>=5.458,<i>P</i><0.001)after 22 weeks of sodium arsenite treatment.The scratch healing rate of YTHDF2-wt cells was increased at the 8th(<i>t</i>=12.137,<i>P</i><0.001)and 22th(<i>t</i>=4.484,<i>P</i>=0.011)weeks of sodium arsenite treatment.The number of colonies formed by YTHDF2-wt cells was higher at the 4th(<i>t</i>=3.365,<i>P</i>=0.027),8th(<i>t</i>=5.580,<i>P</i>=0.005),and 22th(<i>t</i>=3.328,<i>P</i>=0.029)weeks of sodium arsenite treatment.Compared with YTHDF2-mut cells,YTHDF2-wt cells showed down-regulated protein(<i>t</i>=-3.119,<i>P</i>=0.036)and mRNA(<i>t</i>=4.051,<i>P</i>=0.015) levels of PTEN after 22 weeks of sodium arsenite treatment.Immunofluorescence results showed that after 4 weeks of sodium arsenite treatment,YTHDF2 LLPS droplets in YTHDF2-wt cells were localized to stress granules,translation-related membrane-less organelles.Conclusions During sodium arsenite-induced malignant transformation of skin cells,YTHDF2 undergoes LLPS and localizes to stress granules,translation-related membrane-less organelles.YTHDF2 LLPS participates in sodium arsenite-induced malignant transformation of skin cells by down-regulating the mRNA level of the key tumor suppressor PTEN.