08 January 2017 : Laboratory Research
Upregulation of PIM2 by Underexpression of MicroRNA-135-5p Improves Survival Rates of Skin Allografts by Suppressing Apoptosis of Fibroblast CellsHongtu Zhang1ABCDEFG*
Med Sci Monit 2017; 23:107-113
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.
Keywords: Apoptosis - physiology, Allografts, Blotting, Western, Burns - surgery, Fibroblasts - physiology, Prognosis, Protein-Serine-Threonine Kinases - metabolism, Proto-Oncogene Proteins - metabolism, RNA, Messenger - genetics, Real-Time Polymerase Chain Reaction, Signal Transduction, Skin Transplantation - methods, Survival Rate
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