lipofectamine and Hypoxia

To investigate the effect of miR-155 on immune-factors and its mechanism in mesenchymal stem cells under hypoxia.. 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.. 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].. 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.

Topics: Cell Hypoxia; Chemokine CXCL12; Down-Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunologic Factors; Interleukin-6; Lipids; Mesenchymal Stem Cells; MicroRNAs; Real-Time Polymerase Chain Reaction; Transfection; Transforming Growth Factor beta; Up-Regulation