摘要
目的分析并鉴定miR-4483对TIMP-1表达水平的靶向调控作用，并探讨miR-4483的抗肝纤维化机制。方法应用生物信息学方法预测miR-4483靶基因为TIMP-1。采用重叠PCR技术将靶基因3’UTR片段克隆至pGL3-M载体构建野生型及突变型重组质粒，和pRL-TK、miRNA前体共转染入大鼠肝星状细胞系HSC-T6，检测荧光素酶的表达。将miRNA前体转染HSC-T6后检测TIMP-1的mRNA和蛋白质表达水平。结果酶切和测序证实重组质粒构建成功，共转染miR-4483前体组可下调野生型质粒的荧光素酶活性（P〈0．05），不影响突变型质粒的荧光素酶活性，而共转染miR-NC组，突变型质粒和野生型质粒荧光素酶活性比较差异无统计学意义（P〉0．05）；上调miR-4483水平后，TIMP-1mRNA和蛋白的表达水平与转染miR-NC和空白组比较差异有统计学意义（P〈0．05）。结论TIMP-1是miR-4483的靶基因，miR-4483通过负向调控TIMP-1表达而起到抗肝纤维化作用。
Objective To analyze and identify the target regulation effect of miR4483 on TIMP- 1 expression levels, and study the anti liver fibrosis mechanism of miR-4483. Methods The target gene of miR-4483 was TIMP-1, predicted by bioinformatics method. The wild-type and mutant recombinant plasmids（ pGL3-TIMP-1-wt and pGL3-TIMP-1-mut） were constructed by overlap extension PCR of target gene 3 ＇ UTR fragment cloning into the vector pGL3-M, which were co-transfected into HSC-T6 cells with the miRNA-4483 mimics and pRL-TK for detection of luciferase expession. Real-time RT-PCR and West- ern blot were performed to examine the mRNA and protein expression levels of TIMP-1 after miRNA-4483 mimics transfected into HSC-T6 cells. Results The recombinant plasmid was confirmed by restriction enzyme digestion and sequencing. The co-transfected miRNA-4483 mimics can down-regulate the lucifer- ase activity of the wild-type plasmids （ P 〈 0.05 ）, but can not influence the ]uciferase activity of the mu- tant-type plasmids. There was mo significant difference in the luciferase activity between the wild-type plasmids and mutant plasmids co-transfected with miR-NC （ P 〉 0.05 ）. Up-regulation of miR4483 levels can dramatically suppress the TIMP-1 mRNA and protein expression levels in HSC-T6 cells（P 〈0.05）. Conclusions TIMP-1 is the target gene of miR-4483, amd miR4483 has anti liver fibrosis effect by down-regulation of TIMP-1 expession levels.
出处
《中国实用医刊》
2014年第7期41-44,共4页
Chinese Journal of Practical Medicine
