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

Construction of arsenic-resistant A216T missense mutant PML-RARαplasmid and the effect of realgar on its protein products and its mechanism

目的构建PML^(A216T)-RARα过表达质粒,并研究雄黄对其蛋白产物的影响及机制。方法通过资料库及文献获得PML、RARα的mRNA序列,以及PML-RARα的融合位点。设计引物后,通过聚合酶链反应获得目的片段,通过无缝克隆技术连接两个片段,插入突变,并与载体质粒相连接,测序验证。转染质粒至HL-60细胞,血清饥饿法处理24 h后用Western blot观察PML-RARα、p-mTOR、p62、LC3B水平变化;不同浓度的雄黄处理24 h后,用Western blot观察PML-RARα、p-mTOR、p62、LC3B水平变化。结果测序结果显示质粒构建成功,序列符合预期设计。1、2、4、8μmol/L雄黄处理后PML^(WT)-RARα和PML^(A216T)-RARα水平低于未经雄黄处理(0μmol/L)的PML^(WT)-RARα和PML^(A216T)-RARα水平,差异有统计学意义(P<0.05);0.5μmol/L雄黄处理后PML^(WT)-RARα水平低于未经雄黄处理的PML^(WT)-RARα水平,差异有统计学意义(P<0.05)。相同浓度雄黄处理后PML^(A216T)-RARα水平高于PML^(WT)-RARα水平,差异有统计学意义(P<0.05)。5%FBS和未经FBS处理的PML^(A216T)-RARα、p-mTOR水平低于10%FBS处理的PML^(A216T)-RARα、p-mTOR水平,p62、LC3B水平高于10%FBS处理的PML^(A216T)-RARα水平,差异有统计学意义(P<0.05)。4、8、16、32、64μmol/L雄黄处理后PML^(A216T)-RARα水平低于未经雄黄处理的PML^(A216T)-RARα水平,差异有统计学意义(P<0.05);8、16、32、64μmol/L雄黄处理后p-mTOR、p62水平低于未经雄黄处理的p-mTOR、p62水平,LC3B水平高于未经雄黄处理的LC3B水平,差异有统计学意义(P<0.05);16μmol/L雄黄处理后p-mTOR、p62水平低于8μmol/L雄黄处理后的p-mTOR、p62水平,LC3B水平高于8μmol/L雄黄处理后的LC3B水平,差异有统计学意义(P<0.05)。结论错义突变型PML^(A216T)-RARα过表达质粒构建成功,高浓度雄黄可能通过选择性自噬下调PML^(A216T)-RARα。

Objective To construct PML^(A216T)-RARαoverexpression 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-RARαwere 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 PML^(WT)-RARαand PML^(A216T)-RARαafter 1,2,4,and 8μmol/L realgar treatment were lower than those without realgar treatment(0μmol/L),and the differences were statistically significant(P<0.05);the level of PML^(WT)-RARαafter 0.5μmol/L realgar treatment was lower than that without realgar treatment,and the difference was statistically significant(P<0.05).The level of PMLA216T-RARαwas higher than that of PML^(WT)-RARαafter the same realgar concentration treatment,and the difference was statistically significant(P<0.05).The levels of PML^(A216T)-RARαand 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 PML^(A216T)-RARαafter 4,8,16,32,and 64μmol/L realgar treatment were lower than those without realgar treatment,and the differences were statistically significant(P<0.05);after 8,16,32,and 64μmol/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 16μmol/L realgar treatment were lower than those after 8μmol/L realgar treatment,and the LC3B level was higher than that after 8μmol/L realgar treatment,and the differences were statistically significant(P<0.05).Conclusion The missense mutant PML^(A216T)-RARαoverexpression plasmid is successfully constructed,and high concentration of realgar may downregulate PML^(A216T)-RARαthrough selective autophagy.