Zhang Hongtu
Department of Burn and Plastic Surgery, Jining Number 1 People's Hospital, Jining, Shandong, China (mainland).
Med Sci Monit. 2017 Jan 8;23:107-113. doi: 10.12659/msm.897613.
BACKGROUND It has been reported that miR-135-5p is involved with many diseases. In this study, we aimed at define the relationship between miR-135-5p level and burn patient survival after skin transplantation. MATERIAL AND METHODS Expression of miR-135-5p and PIM2 was measured using real-time PCR and Western blot analysis in the skin samples collected from burn patients who received skin graft or in the fibroblast cells transfected with miR-135-5p mimics or inhibitors. The regulatory association between miR-135-5p and PIM2 was verified using bioinformatics analysis and luciferase assay. RESULTS The expression level of miR-135-5p was determined in 60 tissue samples divided into 2 groups based on the presence of rejection (long survival n=30, and short survival n=30). We found that miR-135-5p was substantially downregulated in the long survival group. We then searched the miRNA database online with the "seed sequence" located within the 3'-UTR of the target gene, and then validated PIM2 to be the direct gene via luciferase reporter assay system. We also established the negative regulatory relationship between miR-135-5p and PIM2 via studying the relative luciferase activity. We also conducted real-time PCR and Western blot analysis to study the mRNA and protein expression level of PIM2 among different groups (long survival n=30, short survival n=30) or cells treated with scramble control, miR-135-5p mimics, PIM2 siRNA, and miR-135-5p inhibitors, indicating the negative regulatory relationship between MiR-135-5p and PIM2. We also conducted experiments to investigate the influence of miR-135-5p and PIM2 on viability and apoptosis of cells. The results showed miR-135-5p reduced the viability of cells, while PIM2 negatively interfered with the viability of cells, and miR-135-5p inhibited apoptosis and PIM2 suppressed apoptosis. CONCLUSIONS MiR-135-5p is involved with the prognosis of burn patients after skin transplantation. PIM2 is a virtual target of miR-135-5p, and there is a negative regulatory relationship between miR-135-5p and PIM2. MiR-135-5p and PIM2 interfered with the viability and apoptosis in cells.
背景 据报道，miR-135-5p与多种疾病相关。在本研究中，我们旨在确定miR-135-5p水平与皮肤移植后烧伤患者生存率之间的关系。
材料与方法 使用实时PCR和蛋白质免疫印迹分析，检测接受皮肤移植的烧伤患者皮肤样本中，或用miR-135-5p模拟物或抑制剂转染的成纤维细胞中miR-135-5p和PIM2的表达。使用生物信息学分析和荧光素酶测定法验证miR-135-5p与PIM2之间的调控关系。
结果 在60个组织样本中测定了miR-135-5p的表达水平，根据是否发生排斥反应将其分为两组（长期存活n = 30，短期存活n = 30）。我们发现长期存活组中miR-135-5p显著下调。然后我们利用位于靶基因3'-UTR内的“种子序列”在在线miRNA数据库中进行搜索，随后通过荧光素酶报告基因检测系统验证PIM2为直接作用基因。我们还通过研究相对荧光素酶活性建立了miR-135-5p与PIM2之间的负调控关系。我们还进行了实时PCR和蛋白质免疫印迹分析，以研究不同组（长期存活n = 30，短期存活n = 30）或用乱序对照、miR-135-5p模拟物、PIM2 siRNA和miR-135-5p抑制剂处理的细胞中PIM2的mRNA和蛋白质表达水平，表明MiR-135-5p与PIM2之间存在负调控关系。我们还进行了实验，以研究miR-135-5p和PIM2对细胞活力和凋亡的影响。结果显示miR-135-5p降低细胞活力，而PIM2对细胞活力有负向干扰作用，miR-135-5p抑制细胞凋亡，PIM2也抑制细胞凋亡。
结论 miR-135-5p与皮肤移植后烧伤患者的预后相关。PIM2是miR-135-5p的直接靶点，miR-135-5p与PIM2之间存在负调控关系。miR-135-5p和PIM2干扰细胞的活力和凋亡。
