miR-335启动子活性鉴定及药物干预  

Identification and intervention of promoter regions of miR-335

作　　者：陈璇[1] 朱璐[1] 徐凛峰[2] 汪悦[3] 

机构地区：[1]南京中医药大学护理学院,江苏南京210023 [2]南京医科大学第二附属医院普外科,江苏南京210011 [3]南京中医药大学第一临床医学院,江苏南京210023

出　　处：《南京医科大学学报（自然科学版）》2015年第1期22-25,共4页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省高校自然科学基金(12KJD360001)

摘　　要：目的：探讨miR-335上游片段是否具有转录活性以及罗格列酮和胰岛素对miR-335启动子区域转录活性的影响。方法：利用PCR技术克隆位于MEST内含子中miR-335上游区域的不同片段,采用双荧光素酶报告基因系统鉴定其转录活性。使用1μmol/L的罗格列酮及不同浓度的胰岛素进行干预并观察转录活性有无变化。结果：不同的miR-335启动子区域转录活性不同,在miR-335上游约600个碱基区域活性最高。未使用药物干预组细胞中,miR-335启动子区域活性的表达与基础值相比差异无统计学意义（P〉0.05）;使用1μmol/L罗格列酮干预后,miR-335启动子区域活性显著升高（P〈0.01）,使用10~12 mol/L的胰岛素干预时,启动子活性升高,但当胰岛素浓度上升后,启动子活性变化并没有统计学差异。结论：miR-335上游约600个碱基为启动子关键区域,同时,miR-335有可能通过自身的转录调控机制参与肥胖与胰岛素抵抗的发生发展。Objective：To explore whether miR-335 has its own transcription unit and effects of rosiglitazone and insulin on transcriptional activity of MiR-335 promoter regions. Methods： We cloned different fragments from upstream sequences of miR-335 in MEST intron by PCR, Then, the lueiferase activity of these different plasmids was detected by dual-luciferase reporter system. After the stimulation of 1 p, mol/L rosiglitazone and insulin of different concentration, the transcriptional activity change of fragments from upstream sequences of miR-335 was examined. Results： Different miR-335 promoter regions had different transcription activity, and the region of about 600 basic groups in miR-335 upstream exhibited the highest activity. The activity of miR-335 promoter in untreated HEK293T cells showed no statistical significance compared to the baseline value. The levels of miR-335 promoter activity showed significant elevation after the intervention of 1 p, mol/L rosiglitazone（P 〈 0.01）. The activity of miR-335 promoter was a little elevated with the stimulation of 10-12 mol/L insulin（P 〈 0.05）. Meanwhile, the levels of miR-335 promoter activity had no statistical significance when the concentration of insulin increased. Conclusion： About 600 basic groups in miR-335 upstream were major promoter regions. Meanwhile, miR-335 may play an important role in the development of obesity and insulin resistance via its own transcription mechanism.

关 键 词：microRNA-335 启动子 罗格列酮 胰岛素 

分 类 号：R589[医药卫生—内分泌]