Effect of FABP4 DNA methylation on abnormal lipid metabolism in trophocyte treated with L-NAME / 实用医学杂志

Objective To investigate the effect of fatty acid binding protein 4(FABP4)DNA methylation on abnormal lipid metabolism in placental trophoblastic dyslipidemia. Methods Human placental trophoblast cell line(HTR-8)was treated with L-NAME of 100 μmol/L for 48 h. The lipid content in placental trophoblasts was detected by chemical enzyme-colorimetry. The FABP4 DNA methylation level in placenta trophoblasts was detected by nested-touch down methylation specific PCR (NT-MSP). the mRNA and protein expression of DNMT1 and FABP4 were detected by qRT-PCR and Western Blot,respectively,in trophoblast cells. Results The lipid content in trophoblasts significantly increased as compared with the control(P < 0.05). Expression of FABP4 mRNA and protein increased(P < 0.05),while FABP4 methylation level and expression of DNMT1 significantly decreased (P<0.05)after treatment with L-NAME. Conclusions FABP4 DNA methylation is involved in the regulation of lipid metabolism in placental trophoblastic cells of hypertensive disorder complicating pregnancy.

Role of homocysteine to promote the vascular smooth muscle cell proliferation by MiR-125 b methylation / 中国药理学通报

Aim To investigate the role of miR-125 b and its DNA methylation in homocysteine ( Hcy )-in-duced vascular smooth muscle cells( VSMCs) prolifera-tion. Methods VSMCs were stimulated with 0,50, 100, 200, 500 μmol · L-1 Hcy respectively. Then qRT-PCR was used to detect the mRNA levels of miR-125b,and nested-touchdown methylation-specific PCR ( ntMS-PCR) was used to detect the methylation levels of miR-125b. VSMCs were transfected with miR-125b precursor or the inhibitor of miR-125b ,then 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide ( MTT ) assay was used to reflect the proliferation of VSMCs. The distribution of CpG islands of miR-125b promoter region was analyzed by bioinformatics meth-ods. VSMCs were stimulated with 100 μmol·L-1 Hcy and transfected with or without DNA methylation inhib-itors 5-nitrogen impurity cytidine ( AZC) , then the ex-pression of miR-125b was detected by qRT-PCR. Re-sults The mRNA levels of miR-125 b were decreased in 100,200,500 μmol·L-1 Hcy group compared with 0 μmol·L-1 Hcy group. The precursor of miR-125b could inhibit the proliferation activity and the inhibitor of miR-125 b could increase the proliferation activity of VSMCs cells. Bioinformatics analysis indicated that MiR-125 b promoter region had a CpG island whose length was 792 bp ( 1881-2672 ) . The miR-125 b pro-moter region methylation levels increased after Hcy in-tervention ( P <0. 01 ) . The expression level of miR-125 b increased after AZC intervention ( P <0. 05 ) . Conclusions ① Hcy promotes vascular smooth mus-cle cell proliferation maybe by down-regulating the ex-pression of miR-125b. ② Hcy down-regulates the ex-pression of miR-125 maybe by up-regulating the methy-lation levels of miR-125b promoter region.

Effects of silencing LSD1 gene on Molt-4 cells apoptosis / 中国药理学通报

Aim To observe the effect of the LSD1 gene on the proliferation and apoptosis of Molt-4 cells, a kind of human acute T-lymphoblastic leukemia cells. Methods siRNA fragment based on LSD1 gene was designed, filtered out and then transfected into Molt-4 cells. The effects of LSD 1 siRNA on Molt-4 cell prolif-eration were observed by the method of MTS. The cell apoptosis was analyzed by flow cytometry. The states of histone H3K4, H3K9 methylation, histone H3 acetyla-tion, p15, DNA methyltransferase 1 (DNMT1), and apoptosis-related proteins like Bcl-2 , procaspase-3 were evaluated by Western blot. Results Silencing LSD1 gene inhibited cell proliferation. Molt-4 cell pro-liferation rate was ( 99. 65 ± 1. 21 )%, ( 83. 02 ± 1. 69)%, (65. 72 ± 2. 16)%,and (41. 15 ± 2. 23)%respectively after the treatment of Molt-4 cells with 0 , 30, 60, 120 nmol·L-1 of LSD1 siRNA after 48 hours ( P < 0. 05 ) . Cell proliferation rate was ( 99. 86 ± 1. 35)%,(65. 72 ± 2. 16)%,(48. 26 ± 1. 92)%,and ( 37. 86 ± 1. 66 )% respectively after the transfection of Molt-4 cells with 60 nmol · L-1 of LSD1 siRNA after 0 , 24 , 48 , 72 hours ( P<0. 05 ) . Cell apoptosis rate was ( 3. 35 ± 1. 26 )%, ( 12. 16 ± 1. 74 )%, ( 32. 74 ± 2. 47 )%, ( 54. 64 ± 2. 58 )% respectively after transfection of LSD1 siRNA in indicated concentrations for 48 hours ( P <0. 05 ) . At the same time, the ex-pression levels of apoptosis-related proteins like Bcl-2 , procaspase-3 decreased. LSD1 siRNA inhibited LSD1 and LSD1 mRNA, and accumulated histone mono-, and di-methylation H3K4 and histone H3 acetylation. However, alteration of H3K4 trimethylation, H3K9 methylation was not detected. LSD1 siRNA downregu-lated DNA demethylase DNMT1 and upregulated p15 . Conclusions LSD1 siRNA can inhibit Molt-4 cell proliferation and induce apoptosis. Its mechanism may be associated with epigenetic regulation. In addition, it is expected to become a new target for leukemia treat-ment.