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

Construction and analysis of luciferase reporter vector of miR-15a-5p and miR-16-5p binding sites in the 3'-UTR region of CCND1

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。

Objective：To analysis the complementary sequences and the relationship between miR-15a-5p/16-5p and CCND1 mRNA.Methods：To find the nucleotides of CCND1 3＇-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 sequencing 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 CCND1 3＇-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.