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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 investigate whether miR-335 upstream fragment has transcriptional activity and the effects of rosiglitazone and insulin on the transcriptional activity of miR-335 promoter region. METHODS: Different fragments of the upstream region of miR-335 in MEST introns were cloned by PCR and their transcriptional activity was identified by dual luciferase reporter gene system. 1μmol / L rosiglitazone and different concentrations of insulin intervention and observe whether there is any change in transcriptional activity. Results: The transcriptional activities of different miR-335 promoter regions were different, and the activity was about 600 bases upstream of miR-335. The expression of miR-335 promoter in the untreated group was not significantly different from the baseline value (P> 0.05). After intervention with 1 μmol / L rosiglitazone, the miR-335 promoter region (P <0.01). When 10 ~ 12 mol / L insulin was used, the promoter activity was increased. However, there was no significant difference in the promoter activity when the insulin concentration was increased. CONCLUSION: About 600 bases upstream of miR-335 is a key promoter region. Meanwhile, miR-335 may participate in the development of obesity and insulin resistance through its own transcriptional regulation mechanism.