miR-335在急性缺血性脑卒中表达下调及对钙调蛋白的调控作用

Negative Regulation of Calmodulin by miR-335 in Acute Ischemic Stroke

目的检测微小RNA-335在急性缺血性脑卒中(AIS)患者血清中的表达,并探讨其对钙调蛋白(Ca M)调控的作用机制。方法采用实时荧光定量聚合酶链反应(qRT-PCR)法测定106例AIS患者及98例健康对照组血清中miR-335表达情况;用生物信息学数据库查询分析miR-335对钙调蛋白编码基因CALM1的靶向结合关系;脂质体转染法将miR-335模拟物、抑制物以及相应阴性对照物分别转染至人脐静脉内皮细胞(HUVECs)中并进行细胞培养,采用逆转录PCR(RT-PCR)和Western blot分别检测干预后HUVECs中钙调蛋白mRNA和蛋白的表达水平。结果与健康对照组相比较,AIS组患者血清miR-335表达明显下调(P<0.01);生物信息学软件的查询分析显示CALM1存在miR-335潜在靶向结合位点;RT-PCR和Western blot检测结果显示miR-335模拟物组Ca M mRNA和蛋白的表达水平较对照组显著下降(P<0.01),而miR-335抑制物组Ca M mRNA和蛋白的表达水平较对照组显著增高(均P<0.05)。结论急性缺血性脑卒中患者血清miR-335表达明显下降,表达下调的miR-335可能通过上调钙调蛋白的表达从而发挥重要作用。

Objective The miRNA dysfunction is suspected to be a contributing factor for many CNS pathologies in- cluding ischemic stroke. The present study investigates the expression of serum miR-335 in acute ischemic stroke （AIS）, and explores the underlying mechanisms of miR-335 in acute ischemic stroke （AIS）. Methods Blood samples were ob- tained from AIS patients （n = 106） and healthy controls （n = 98）. MiR-335 was measured by using a quantitative real-time PCR. Bioinformatics database assay was used to determine whether calmodulin gene （ CALM1 ） was the direct target of miR- 335. The miR-335 mimics and inhibitors were transfected into human umbilical vein epithelial cells （HUVECs） with lipo- fectamine respectively. Calmodulin expression was determined by Western blot and reverse transcription-PCR （RT-PCR） in the cultured HUVECs. Results Compared with the healthy control group,the expression of serum miR-335 was signifi- cantly lower in patients with AIS group （P 〈 0.01 ）. In addition, bioinformatics database assay show that the CALM1 present the binding sites,which was potentially targeted by miR-335. Finally, Transfection of the miR-335 mimic inhibited the ex- pression of CaM mRNA and protein in the HUVEC compared to the control group （ P 〈 0.01 ）, while transfection of the miR-335 inhibitors increased the expression of CaM mRNA and protein （P 〈 0.05）. Conclusion MiR-335 down-regu- lation may increase the ischemic brain injury through up-regulating the expression of CaM.