Nested-MSP detection of arsenic trioxide-induced human multiple myeloma U266 cell line p16 gene demethylation and transcription.doc

 PAGE \* MERGEFORMAT 2 Nested-MSP detection of arsenic trioxide-induced human multiple myeloma U266 cell line p16 gene demethylation and transcription [Abstract] This study investigated the high-sensitivity DNA methylation detection method and DNA cloning and sequencing analysis to detect arsenic trioxide (As2O3) in to the role of methylation and demethylation of its possible mechanism of action analysis. Nested methylation-specific PCR method (nested  methylation specific PCR, n  MSP), DNA cloning and sequencing assay before and after the U266 cell line As2O3, the methylation status of p16 gene applied RT  PCR detection of p16, DNA methylation transferase 1 (DNMT1), DNMT3A, DNMT3B gene mRNA expression in the growth curve, MTT method, colony formation assay detection of As2O3 on the growth and proliferation of myeloma cells in vivo. DNA content by flow cytometry analysis of As2O3 on the multiple myeloma cell line U266 cycle. The results showed that: ① untreated group of U266 cell genomic DNA cytosine remained unchanged, while the role of U266 cells by As2O3 genomic DNA cytosine into thymine were, indicating existence of U266 cells, p16 gene methylation, As2O3 role abnormal methylation of p16 gene after the phenomenon was reversed; ② untreated group of cells did not express p16 gene, As2O3 for 72 hours increased expression of p16 gene, 0.5 μ mol / L group, 1.0 μ mol / group and 2.0 μ mol / group of p16 gene expression in positive bands gray value and β  -actin ratios were (0.22 ± 0.10), (0.59 ± 0.11), (0.68 ± 0.09), positive control gray-scale ratio (0.77 ± 0.13), the difference was very significant statistically significant (P