人Bcl-6 3'UTR区报告质粒及其表达载体的构建和功能检测  

Construction of human Bcl-6 3'UTR reporter vector and expression vector and their functional assessment

作　　者：韩白玉[1,2] 崔瀚之[3] 燕翔[4] 黄鹏 黄华龙 范忠义[4] 窦京涛[1] 

机构地区：[1]解放军总医院内分泌科 [2]北京军区第264医院内分泌科 [3]解放军309医院肿瘤科 [4]解放军总医院肿瘤科 [5]解放军61213部队

出　　处：《南方医科大学学报》2015年第10期1451-1456,共6页Journal of Southern Medical University

基　　金：中国人民解放军第三〇九医院课题(2015MS-010)

摘　　要：目的通过构建bcl-6基因野生型、突变体3'UTR区及其编码序列(CDS),观察miR-127对bcl-6的直接靶向调控作用及bcl-6表达载体回复miR-127抑制细胞周期和细胞生长的功能。方法利用PCR方法扩增bcl-6基因3'UTR区序列及其CDS,分别构建在pc DNA3.0-Luc和pc DNA3.0-Flag载体上,在bcl-6基因3'UTR质粒基础上应用重组PCR方法构建miR-127结合位点突变的突变体报告基因质粒,应用荧光素酶报告基因系统检测miR-127对bcl-6的直接靶向调控作用,在肝癌细胞Hep G2中检测过表达及敲低miR-127引起bcl-6基因表达抑制后细胞周期和细胞生长的改变,同时应用表达载体回复bcl-6蛋白水平,检测bcl-6在miR-127调控细胞周期和细胞生长中的必要性。结果构建的重组质粒经酶切鉴定和测序证实构建正确,bcl-6 3'UTR野生型和突变体报告质粒与miR-127共转293T细胞和Hep G2细胞后荧光素酶报告基因检测显示miR-127明显降低野生型报告质粒的活性,但对突变体活性没有影响,miR-127可引起Hep G2细胞G2/M期阻滞并抑制细胞生长,bcl-6可以逆转miR-127对细胞周期和细胞生长的影响。结论成功构建bcl-6基因3'UTR区野生型、突变体报告质粒和bcl-6基因的表达载体,荧光素酶报告基因和回复实验证实均具有生物学功能。Objective To observe the direct regulation of miR-127 on Bcl-6 and the effect of Bcl-6 in rescuing miR-127-induced cell cycle and cell growth inhibition. Methods The 3＇UTR and coding region of human bcl-6 gene were amplified by PCR and cloned into pc DNA3.0-Luc and pc DNA3.0-Flag vectors, respectively. Mutations were introduced into the seed sequences of the predicted miR-127 target sites within the Bcl-6 3＇UTR using recombinant PCR. Luciferase assay was used to verify the direct targeted regulation of miR-127 on Bcl-6. In Hep G2 cell models with overexpression or knockdown of miR-12, the changes of cell cycle and cell growth were investigated after transfection with the constructed vectors. Results The recombinant plasmids were successfully obtained as confirmed by double digestion and sequence identification. Luciferase assay showed that in293 T and Hep G2 cells, miR-127 inhibited the activation of wild-type Bcl-6 3＇UTR reporter vector but not mutated Bcl-6 3＇UTR vector. Overexpression of miR-127 induced cell cycle arrest at G2/M phase and suppressed the growth of Hep G2 cells, and these effects were reversed by Bcl-6 overexpression. Conclusion We successfully cloned wild-type and mutated 3＇UTR reporter vectors and expression vector of bcl-6 gene and confirmed their biological functions.

关 键 词：BCL-6基因 3'UTR区 荧光素酶报告基因 细胞周期 

分 类 号：Q78[生物学—分子生物学]