Construction of arsenic-resistant A216T missense mutant PML-RARα plasmid and the effect of realgar on its protein products and its mechanism#br#
PAN Yiming1 LI Ruibai1 HUANG Ziming1 LIU Yu1 FEI Mingming2 CHEN Lingyan2 HOU Li1
1.Department of Hematology and Oncology, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100700, China;
2.School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Zhejiang Province, Wenzhou 325035, China
Abstract：Objective To construct PMLA216T-RARα overexpression plasmid and to investigate the effect of realgar on its protein product and its mechanism. Methods The mRNA sequences of PML and RARα, and the fusion sites of PML-RARα were obtained from the database and literature. After designing primers, the target fragments were obtained by polymerase chain reaction, and the two fragments were joined by seamless cloning technique, inserted with mutations, and ligated with vector plasmids, and verified by sequencing. The plasmid was transfected into HL-60 cells, and the changes of PML-RARα, p-mTOR, p62, and LC3B levels were observed by Western blot after 24 h treatment with serum starvation method; and the changes of PML-RARα, p-mTOR, p62, and LC3B levels were observed by Western blot after 24 h treatment with different concentrations of realgar. Results The sequencing results showed that the plasmid was successfully constructed and the sequence conformed to the expected design. The levels of PMLWT-RARα and PMLA216T-RARα after 1, 2, 4, and 8 μmol/L realgar treatment were lower than those without realgar treatment (0 μmol/L), and the differences were statistically significant (P < 0.05); the level of PMLWT-RARα after 0.5 μmol/L realgar treatment was lower than that without realgar treatment, and the difference was statistically significant (P < 0.05). The level of PMLA216T-RARα was higher than that of PMLWT-RARα after the same realgar concentration treatment, and the difference was statistically significant (P < 0.05). The levels of PMLA216T-RAR α and p-mTOR in 5% FBS and untreated FBS were lower than those in 10% FBS, and the levels of p62 and LC3B were higher than those in 10% FBS, and the differences were statistically significant (P < 0.05). The levels of PMLA216T-RAR α after 4, 8, 16, 32, and 64 μmol/L realgar treatment were lower than those without realgar treatment, and the differences were statistically significant (P < 0.05); after 8, 16, 32, and 64 μmol/L realgar treatment, the levels of p-mTOR and p62 were lower than those without realgar treatment, and the LC3B level was higher than that without realgar treatment, and the differences were statistically significant (P < 0.05); the levels of p-mTOR and p62 after 16 μmol/L realgar treatment were lower than those after 8 μmol/L realgar treatment, and the LC3B level was higher than that after 8 μmol/L realgar treatment, and the differences were statistically significant (P < 0.05). Conclusion The missense mutant PMLA216T-RARα overexpression plasmid is successfully constructed, and high concentration of realgar may downregulate PMLA216T-RARα through selective autophagy.
潘一鸣1 李蕊白1 黄子明1 刘宇1 费明明2 陈凌燕2 侯丽1. 耐砷A216T错义突变型PML-RARα质粒的构建及雄黄对其蛋白产物影响及机制[J]. 中国医药导报, 2021, 18(33): 16-20.
PAN Yiming1 LI Ruibai1 HUANG Ziming1 LIU Yu1 FEI Mingming2 CHEN Lingyan2 HOU Li1. Construction of arsenic-resistant A216T missense mutant PML-RARα plasmid and the effect of realgar on its protein products and its mechanism#br#. 中国医药导报, 2021, 18(33): 16-20.
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