双荧光素酶报告基因系统验证hsa-miR-326对BCL2L1、BAK1的靶向调控

The targeted-regulating role of hsa-miR-326 on gene BCL2L1 and BAK1 by dual luciferase reporter system

目的构建psiCHECK2荧光素酶报告基因载体,验证hsa-miR-326对BCL2L1和BAK1表达的影响。方法设计合成包含与hsa-miR-326结合序列及其突变型序列的BCL2L1和BAK1基因片段,构建荧光素酶报告基因载体,转染293T细胞,通过荧光素酶活性变化观察hsa-miR-326对BCL2L1和BAK1表达的影响。结果 hsa-miR-326对BCL2L1组的荧光表达有非常显著的抑制作用(P<0.01);而对BCL2L1-mut、BAK1和BAK1-mut组的荧光表达没有显著的抑制作用(P>0.05)。结论 hsa-miR-326可以调控BCL2L1的表达,但对BAK1的表达未见影响,推测hsa-miR-326可能通过下调BCL2L1参与血小板的凋亡。

Objective To identify the influence of hsa-miR-326 on the expression of BCL2L1 and BAK1,based on construction of psiCHECK2 luciferase reporter gene vector. Methods The potential fragments of hsa-miR-326 target genes BCL2L1 and BAK1 were predicted by the bioinformatics analyzing tools online. The target fragment of the BCL2L1 and BAK1 and their mutation sequence were connected to psiCHECK-2 vector. The constructs were cotransfected into 293T cells with hsa-miR-326 mimics or negative control（ mir-NC） by lipofectamineTM 2000（ lipo2000）,respectively. Dual luciferase reporter system was used to determine the luciferase activity. Results Luciferase assays revealed that hsa-miR-326 could significantly diminish luciferase activity from the reporter vector containing the 3＇ UTR of BCL2L1（ P〈0. 01）,While the suppression of luciferase activity was not found in BCL2L1-mut,BAK-1 and BAK1-mut（ P〉 0. 05）. Conclusion The results strongly suggest that hsa-miR-326 binds to the 3＇ UTR of BCL2L1,but not to the 3＇ UTR of BAK1. hsa-miR-326 might be related to the occurrence and development of storage platelet apoptosis by regulating the expression of the apoptosis related gene BCL2L1.