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目的 探讨miR-492是否参与红细胞OK血型抗原表达的转录后调控.方法 化学合成2个BSG基因3'UTR片段,分别包含BSG rs 8259 TT或BSG rs 8259 AA位点.合成时两端加上XhoⅠ和NotⅠ酶切位点,克隆至pUC57载体,随后构建至psiCHECK-2载体,测序验证.分别将不同浓度的miR-492 mimic与构建的psiCHECK2-BSG-T或psiCHECK2-BSG-A重组质粒共转染K562细胞,设置空白对照,各转染实验重复3次.利用双荧光素酶报告基因检测试剂盒测定海肾荧光素酶活性,并按萤火虫荧光素酶活性进行均一化处理,数据统计分析.结果 测序结果显示,成功构建了分别包含BSG rs 8259 TT或BSG rs 8259 AA位点的重组psiCHECK-2质粒.双荧光素酶报告基因检测结果表明,miR-492 mimic 浓度大于100nmol/L时,对psiCHECK2-BSG-T有显著抑制作用.而mir-492 mimic对psiCHECK-BSG-A无抑制作用.结论 miR-492可能参与了调控BSG rs 8259 TT基因型个体红细胞OK抗原的表达.

Objective To investigate whether miR-492 is involved in the post-transcriptional regulation of OK blood group antigen expression on red blood cells.Methods Two 3'-UTR fragments of the BSG gene were synthesized with a chemical method,which respectively encompassed the BSG rs8259 TT or BSG rs8259 AA sites.The fragments were added with Xho Ⅰ and Not Ⅰ restriction enzyme cutting sites at both ends and cloned into a pUC57 vector,which in turn was constructed into a psiCHECK-2 vector and verified by sequencing.K562 cells were transfected with various combinations of miR-492 mimic and constructed psiCHECK2-BSG-T or psiCHECK2-BSG-A recombinant plasmid.A blank control group was set up.Each transfection experiment was repeated three times.The activity of Renilla reniformis luciferase was determined and normalized with that of firefly luciferase,and detected with a dual-luciferase reporter assay system.The data were subjected to statistical analysis.Results The sequencing results confirmed that the recombinant psiCHECK2 plasmids containing the BSG rs8259 TT or rs8259 AA sites were constructed successfully.The results of dual-luciferase report gene detection showed that the miR-492 mimic could significantly inhibit psiCHECK2-BSG-T at a concentration over 100 nmol/L.However,it could not inhibit psiCHECK-BSG-A.Conclusion miR-492 may be involved in the regulation of OK antigen expression on red blood cells with the BSG rs8259 TT genotype.