摘要
目的:确定miR-494与CLPTM1L的靶向关系。方法:采用生物信息学方法预测miR-494与CLPTM1L基因的结合位点。采用PCR技术扩增CLPTM1L基因3'UTR片段,并克隆至pmir GLO载体,构建野生型及突变型重组双荧光素酶报告质粒。将培养的293T细胞分为4组,分别共转染miR-494或阴性对照和pmir GLO-CLPTM1L-W 3'UTR或pmir GLOCLPTM1L-M 3'UTR,检测4组细胞中荧光素酶活性。结果:酶切和测序证实成功构建了野生型及突变型重组双荧光素酶报告质粒pmir GLO-CLPTM1L-W 3'UTR和pmir GLO-CLPTM1L-M 3'UTR;与共转染miR-494 mimics和突变型CLPTM1L3'UTR质粒的293T细胞中荧光素酶活性(1.056 4±0.163 4)、共转染阴性对照序列和突变质粒的293T细胞中荧光素酶活性(0.961 3±0.177 9)或野生型质粒的293T细胞中荧光素酶活性(0.983 4±0.001 2)相比,共转染miR-494 mimics和野生型CLPTM1L 3'UTR质粒的293T细胞中荧光素酶活性(0.651 6±0.136 4)明显降低(F=4.476,P=0.040),其他3组间比较差异无统计学意义(P>0.05)。结论:CLPTM1L与miR-494存在靶向关系。
Aim： Analysis and identification of miR-494 potential target gene CLPTM1 L. Methods： Bioinformatics analysis suggested CLPTM1 L was a target of miR-494. PCR was performed to obtain wild-type and mutant CLPTM1 L 3＇UTR fragments,which were cloned into pmir GLO and constructed the recombinant plasmids pmir GLO-CLPTM1L-W and pmirGLO-CLPTM1L-M,respectively. 293 T cells were cultured. Two recombinant plasmids were co-transfected into 293 T cells with the miR-494 mimics or scrambled oligonucleotide（ negative control）,and the relative luciferase activity was determined by luciferase reporter assay. Results： With identification of restriction enzyme digestion and sequencing,the sequences of CLPTM1L-W 3＇UTR and CLPTM1L-M 3＇UTR were cloned into pmir GLO successfully. Compared with the group of miR-494 mimics co-transfected with pmir GLO-CLPTM1L-M（ 1. 056 4 ± 0. 163 4） and the groups of miR-NC co-transfected with pmir GLO-CLPTM1L-W（ 0. 983 4 ± 0. 001 2） or pmir GLO-CLPTM1L-M（ 0. 961 3 ± 0. 177 9）,the relative luciferase activity in group of miR-494 mimics co-transfected with pmir GLO-CLPTM1L-W showed a significant decrease（ 0. 651 6 ± 0. 136 4; F = 4. 476,P = 0. 040）. No difference was found among the aforementioned three groups（ P〈0. 05）.Conclusion： CLPTM1 L is a target gene of miR-494.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2015年第3期430-433,共4页
Journal of Zhengzhou University(Medical Sciences)
