吕小云

    (青海大学基础部，青海 西宁 810016)

    吕小云(1970～)，男，土族，博士在读，副教授

    微星RNA对p16基因表达的调控

    吕小云

    (青海大学基础部，青海 西宁 810016)

    摘要目的了解mir346和mir639对周期蛋白依赖激酶抑制剂2A(p16)基因表达的调控。方法以定点突变法为基础，设计获得c.-21 C >T和c.-34 G>T p16INK4a5′-UTR突变型。用选定的mirRNA和包含野生型和突变型p16INK4a5′-UTR的pGL3质粒共转染。24小时后进行双萤光素酶试剂(Promega)检测分析和实时PCR定量分析。结果Mir346和mir636 相对mir639 和mir935在MDF7和Mel28细胞有较高表达。随着mir346的导入，MCF7细胞野生型荧光酶活性显著降低(57%)；突变型荧光酶活性也明显降低(39%)。并且在mir346存在时，野生型和突变型都显示出相同程度的表达量。mir639对WM266细胞荧光酶活动没有表现出显著影响。结论mir346在MCF7细胞可显著下调p16的表达，而mir639在WM266细胞对p16的表达没有显著影响。

    关键词mirRNAmir63p16INK4aUTR

    中图分类号R555

    文献标识码识码A

    DOI：10.13452/j.cnki.jqmc.2015.02.010

    AbstractObjectiveTo investigate the impact of mir346 and mir639 on the regulation of p16INK4a5′-UTR expression.Method A site-directed mutagenesis approach was developed to obtain the c.-21 C>T and the c.-34 G>T p16INK4a 5′-UTR mutations.With the chosen mirRNA and pGL3-based luciferase vectors containing the wild type or the mutated p16INK4a5′-UTR transfected the cancer cell line.The cells were harvest in 24 h and then luciferase assays and real time PCR analysis were carried out using the dual-luciferase reagent.Result Mir346 and mir636 showed a higher expression in MDF7 and Mel28 cell lines compared to mir639 and mir935.The lusiferase activity of wild type p16 in MCF7 cell line decreases notably(57%)and variant type decreases also down to 39%.Both the wild and variant p16 expressed the same level when mir346 existing.mir639 have not show a notable impact on WM266 cell line.Conclusion mir346 can download the expression of p16INK4a5′-UTR in MCF7 cell and mir639 has no direct impact on in WM266 cell line. ......