CCND1基因3'-UTR区miR-15a-5p和miR-16-5p结合位点的荧光素酶报告载体构建及分析

摘要

Objective:To analysis the complementary sequences and the relationship between miR-15a-5p/ 16- 5p and CCND1 mRNA.Methods:To find the nucleotides of CCND13'- untranslated region (3'- UTR)and miR- 15a-5p/ 16-5p from pubmed and miRBase.After finding the binding area,luciferase assay was used to analyse the relationship between miR-15a-5p/ 16-5p and CCND1.Results:Target segments and mutant inserts sequen-cing confirmed that CCND1a/ Ma and CCND1b/ Mb luciferase report vector were successfully constructed.The results of the luciferase assays revealed that over expression of miR-15a-5p/ 16-5p could reduce the luciferase activity from the segment containing CCND1-b (P< 0 .05 ),whereas CCND1-a had no effect on luciferase activity (P > 0.05).Conclusion:The experimental data indicated that CCND13'- UTR region may have binding sites of miR- 15a-5p/ 16-5p.What's more,miR-15a-5p/ 16-5p could regulate the expression of CCND1 by targeting the putative binding site CCND1-b.%目的:构建miR-15a-5p和miR-16-5p的荧光素酶报告载体,利用此载体分析并验证miR-15a-5p和miR-16-5p与CCND1基因3'-UTR区域的确切结合位点,探讨miR-15a-5p和miR-16-5p与CCND1基因的相互作用关系.方法:通过Pubmed和miRBase数据库分别寻找到CCND1基因3'-UTR区域碱基序列和miR-15a-5p、miR-16-5p的碱基序列.找出理论结合位点后,构建荧光素酶报告载体,并用荧光素酶报告基因方法验证miR-15a-5p和miR-16-5p与CCND1基因之间的作用关系.结果:通过基因测序表明,本实验成功构建了CCND1a/Ma和CCND1b/Mb荧光素酶报告基因表达载体.将上述载体应用于荧光素酶报告基因实验,结果发现CCND1 b组的荧光素酶表达强度显著降低,差异有统计学意义(P<0.05).结论:miR-15a-5p和miR-16-5p与CCND1基因的3'-UTR区域存在结合位点,其具体结合位置在CCND1b区.这在理论上意味着miR-15a-5p和miR-16-5p可以抑制CCND1基因的表达.