Objective: To investigate the effect of miR-155 on immune-factors and its mechanism in mesenchymal stem cells under hypoxia.
Methods: The microRNA sequences targeting miR-155 mimic and mimic NC gene was designed and transfected into MSC by lipofectamineTM 2000. Lipopolysaccharide was used to stimulate the immunity of MSC under hypoxic environment. Transfection efficiency of miR- 155 and immune-related genes (IL-6, IL-8, iNOS, TGF-β, HIF-1α) were detected by real-time RT-PCR. The cell surface antigens (CD29, CD73, CD90, CD105, CD31, CD45) and supernatant cytokines (IL- 6, IL- 8, TGF- β, SDF- 1α) were analyzed by flow cytometry and ELISA, respectively. Western Blot was applied to evaluate related proteins, iNOS and HIF- 1α.
Result: miR- 155 was transfected into MSC effectively (53.447±8.361 vs 1.070±0.174, P<0.01). In miR-155 high-expressed groups, the expressions of IL-6 and IL-8 were up-regulated [(24.201±1.536) vs (1.802±0.058), P<0.01; (24.406±4.611) vs (7.407± 1.553), P<0.01] and iNOS was markedly suppressed [(0.151 ±0.035) vs (32.925±1.632), P<0.01]. Hypoxia up-regulated expressions of HIF-1α [(45.093±3.371) vs (2.210±0.498), P<0.01] and promoted the regulation of miR-155. MiR-155 and hypoxia had effect on mRNA expression of SDF-1α and TGF-β [(5.690±1.655) vs (0.841±0.194), P<0.01; (6.982±1.353) vs (0.632±0.184), P<0.01]. However, there was no influence on cytokines of SDF- 1α and TGF- β [(24.609±2.584) vs (25.359±2.455), P=0.760;(0.568±0.019) vs (0.345±0.037), P=0.002].
Conclusion: Hypoxia environment may promote miR-155 to positively modulate immune factors of MSC through up-regulating the expression of HIF-1α, which down-regulated iNOS protein.
目的: 探讨miR-155在低氧条件下对间充质干细胞（MSC）免疫因子表达的调控以及作用机制。
方法: 通过Lipofectamine™ 2000转染试剂将miR-155模拟物转染至MSC中，构建miR-155高表达的MSC细胞。在水合氯化钴（CoCl2·6H2O）模拟的化学低氧环境中，用脂多糖（LPS）刺激MSC的免疫作用，采用实时荧光定量PCR方法鉴定其转染效果，并检测IL-6、IL-8、诱导型一氧化氮合酶（iNOS）、TGF-β、低氧诱导因子1α（HIF-1α）mRNA的表达。用流式细胞术检测细胞表面抗原，ELISA法检测上清中IL-6、IL-8、TGF-β及SDF-1α的表达水平，Western blot法检测iNOS、HIF-1α蛋白的表达。
结果: 较阴性对照组，miR-155转染组的IL-6、IL-8表达上调（24.201±1.536对1.802±0.058，P<0.01；24.406±4.611对7.407±1.553，P<0.01），iNOS mRNA表达下调（0.151±0.035对32.925±1.632，P<0.01）。同时，HIF-1α在低氧环境下高表达（45.093±3.371对2.210±0.498，P<0.01），且低氧miR-155转染组较低氧对照组上调更为显著（102.965±4.449对45.093±3.371，P<0.01）。低氧miR-155转染组IL-6、IL-8上调较miR-155转染组更加显著（65.670±10.613对24.201±1.536，P<0.01; 35.537±2.285对24.406±4.611，P< 0.01）；miR-155下调iNOS表达（0.235±0.003对0.612±0.043，P<0.01），低氧条件下更为显著（0.087± 0.002对0.235±0.003，P<0.01）。低miR-155组SDF-1α、TGF-β mRNA表达均上调（5.690±1.655对0.841±0.194，P<0.01; 6.982±1.353对0.632±0.184，P<0.01），上清中细胞因子SDF-1α表达水平为24.609±2.584对25.359±2.455（P=0.760）；、TGF-β表达水平为0.568±0.019对0.345±0.037（P=0.002）。
结论: 低氧条件下，通过上调HIF-1α的表达，从而下调iNOS基因及蛋白，由此促进miR-155正向调控MSC免疫因子的表达。