Effect of ten-eleven translocation protein on the expression of amyloid precursor protein and β-site APP cleaving enzyme-1 in SH-SY5Y and IMR-32 cells in folic acid deficiency and the underlying mechanisms

Abstract

Objective To investigate the effect of ten-eleven translocation protein on the proliferation of human neuroblastoma cell lines SH-SY5Y and IMR-32 and the expression of amyloid precursor protein, PS1, and β-site APP cleaving enzyme-1 in the absence of folic acid and possible mechanisms involved. Methods SH-SY5Y and IMR-32 cells were cultured in vitro and divided into the folic acid deficiency group (0 mg/L), the low folic acid group (1mg/L), and the normal control group (4mg/L). The MTT method was used to observe cell proliferation, and RT-PCR was adopted to detect the mRNA expression of APP, PS1, BACE1, DNMTs and TETs in the cells in real-time. Besides, we generated a stable low-level TET1 expression cell line, and compared the expression with that in a negative control group. Furthermore, the expression of fluorescent protein was observed by fluorescence inverted microscope, cell proliferation was measured by the MTT assay, and mRNA levels of TET1, APP, PS1, and BACE1 were detected by RT-PCR. Results (1) In the folic acid deficiency group and the low folic acid group, cell proliferation of SH-SY5Y after 120 h and of IMR-32 cell after 144 h significantly decreased (P<0.001) .The mRNA levels of APP, PS1, BACE1, DNMT1, DNMT3a, DNMT3b, TET1, TET2, and TET3 in SH-SY5Y cells increased (F=80.315, 35.386, 101.979, 786.407, 80.331, 131.545, 28.000, 9.165, and 102.167, all P<0.05); the mRNA levels of APP, PS1, BACE1, DNMT1, DNMT3b, TET1, TET2, and TET3 in IMR-32 cells also rose (F=12.283, 93.669, 40.815, 157.234, 24.835, 147.594, 54.794, and 73.068, all P<0.05). (2) Generation of a stable low-level TET1 expression cell line: The mRNA level of TET1 in the low expression group (SH-SY5Y-shTET1) was 0.25±0.02, which was significantly lower than that in the negative control group (1.00±0.09) (P=0.007); the mRNA level of TET1 in the low expression group (IMR-32-shTET1) was 0.28±0.07, significantly lower than that in the negative control group (1.00±0.01) (P=0.003). (3)The proliferative ability of the low expression groups (SH-SY5Y-shTET1 and IMR-32-shTET1) was significantly higher than that in the negative control group (P<0.01). The mRNA levels of APP and BACE1 decreased (P<0.01 or P<0.05). Conclusion In the human neuroblastoma cell lines SH-SY5Y and IMR-32, folic acid deficiency up-regulates the expression of TETs, increases the expression of APP and BACE1 in the cells by TET protein demethylation, and inhibits cell growth. Key words: Folic acid; DNA methyhransferases; Fen-eleven; Amyloid protein; β-site App-cleavingenzyme