摘要
目的分析前列腺亮氨酸拉链(PrLZ)基因3′非编码区(3′-UTR)可能的微小RNA(miRNA)调控位点,构建PrLZ基因3′-UTR荧光素酶报告载体,为研究PrLZ基因转录后的miRNA调控提供有效的工具。方法使用PCR方法从PrLZ阳性的C4-2细胞中扩增含PrLZ基因3′-UTR区序列,插入到T-Vector载体上,提高PCR产物的连接、克隆效率,通过Xbal和BamHI限制性内切酶双酶切后将其连入经相同内切酶双酶切后的荧光素酶报告基因载体pLUC中,构建pLUC-PrLZ 3′-UTR载体,使用生物信息学方法预测PrLZ基因3′-UTR可能是miR-34a的作用靶点,使用慢病毒载体构建对照载体(质粒名称-NC)和过表达miR-34a(质粒名称-miR-34a),包装成病毒颗粒后分别感染293-T细胞,然后通过X-tremeGENE HP DNA Transfection Reagent转染试剂将pLUC空质粒和pLUC-PrLZ 3′-UTR重组质粒分别转染293-T细胞,Promega试剂盒测定荧光素酶的活性。结果得到含PrLZ基因3′-UTR序列的荧光素酶报告重组质粒,并用凝胶电泳和基因测序的方法验证了其序列的正确性。在miR-34a高表达组的293-T细胞中,重组质粒组荧光素酶活性比空质粒组低40%,差异具有统计学意义(P<0.05)。结论成功构建PrLZ基因3′-UTR荧光素酶报告载体,miR-34a可以显著降低荧光素酶的活性,PrLZ基因3′-UTR上可能有miR-34a的调控作用靶点。
Objective To construct the luciferase recombinant vector containing 3＇- UTR of PrLZ gene and to verify its activity. Methods The 3＇- UTR of PrLZ gene was amplified through PCR, and inserted into XbaI/BamHI-modified pLUC control reporter vector. The 3r-UTR of PrLZ which was targeted by miR-34a was predicted by bioinformation method. The recombinant vector or empty vector were transfected into 293-T ceils infected with lentivirus NC or miR-34a by using X- tremeGENE HP DNA transfection reagent. The Luciferase Reporter Assay System was used to evaluate the activity of lucifer- ase. Results The 3＇- UTR of PrLZ gene was successfully cloned into the pLUC vector, which was verified by gel electropho- resis and DNA sequencing methods. Compared with 293-T cells treated with the empty vector, the luciferase activity of 293-T ceils transfected with recombinant vector was decreased by 40%, with statistically significant difference. Conclusions The PrLZ 3＇- UTR luciferase reporter vector was constructed successfully, and the luciferase activity of the recombinant vector can be suppressed significantly by miR-34a. The 3r- UTR of PrLZ gene may have a binding site of miR- 34a.
出处
《现代泌尿外科杂志》
CAS
2014年第5期325-328,共4页
Journal of Modern Urology
基金
国家重点基础研究发展计划(No.2012CB518305)
