MicroRNA-22 Induces Endothelial Progenitor Cell Senescence By Targeting AKT3

Background and objective Endothelial progenitor cells(EPCs) is vascular endothelial precursor cell, containing in peripheral blood, bone marrow and cord blood. EPCs can participate in angiogenesis in ischemic diseases and repair vessel injury. After vascular endothelial damage, EPCs mobilized from bone marrow, recruited into systemic circulation, homed to sites of damaged vascular endothelial, and differentiated to mature endothelial cells, improved the re-endothelialization of damaged area. A large number of research showed that pure age increasing may influence the quantity and activity of EPCs, reduce EPCs adhesion, proliferation, migration ability and angiogenic capacity. However, the exact mechanism underlying EPC senescence is still unclear. Micro RNAs(mi RNAs) are a new class of endogenous, highly conserved, noncoding and single-stranded RNAs of 22 nucleotides that negatively regulate the stability and translation of target protein-coding m RNAs at the 3′ untranslated region(UTR). mi RNAs often target a cluster of genes, allowing them to regulate a variety of biological processes including cell survival, senescence, proliferation, differentiation and migration. Aberrant expression of mi RNAs is linked to many human disease states including cardiovascular disease(CVD) which are associated with EPCs. Recently, relationship between EPCs and mi RNAs has aroused widespread concern. For expression, Zhao et al. reported that mi R-34 a promoted EPCs senescence and impeded its angiogenesis by directly targeted silent information regulator 1(Sirt1). Zhu et al. reported that mi R-10A* and mi R-21 regulated EPCs senescence, migration, proliferation, self-renewal potential and angiogenesis by suppressing high-mobility group A2(Hmga2), and also reported that mi R-22 expression was significantly higher in mouse aged EPCs than its young EPCs. However,it is not clear that whether mi R-22 is also up-regulation in human aged EPCs, and its roles and mechanisms in EPCs senescence are still unknown. In our study, human samples were divided into 2 groups according to age,21 old and 67 old. EPCs were isolated from them. Subsequently, we performed function assays to research mi R-22 influence on EPCs senescence, proliferation, migration and angiogenesis. Akt3 was predicted as a direct target gene of mi R-22 using bioinformatics analysis. Further studies suggested that mi R-22 promoted EPCs senescence, and inhibited EPCs proliferation, migration and angiogenesis by targeted Akt3. We aimed to elucidate a new regulating mechanism of mi R-22 in EPCs senescence,and provide a new mi RNA and target gene for the clinical application.Methods(1) Using ficoll density gradient centrifugation to isolate peripheral blood derived single nuclear cell of young samples(n=3) and aged samples(n=3), acquire peripheral blood derived EPCs.(2) Using Di I-ac LDL and FITC-UEA-I direct fluorescent staining under a laser scanning confocal microscope to differentiate EPCs. EPCs were further documented by demonstrating the expression of endothelial cell specific antigen,including CD133, CD31 and CD34. Quality real-time PCR(q RT-PCR) was employed to determine mi R-22 expression in young EPCs(Y-EPCs) and aged EPCs(A-EPCs).(3) The lentiviral vectors p LVTHM-mi R-22 and p LVTHM-anti-mi R-22 were constructed. These vectors and the packaging plasmids were co-infected into HEK-293 T cells using lipofectamine 2000 reagent. We then harvested the supernatant containing the lentivirus particles to detect the viral titer.(4) mi R-22 and anti-mi R-22 lentivirus were used to infect young EPCs(Y-EPCs-mi R-22) and aged EPCs(A-EPCs-anti-mi R-22), respectively. And their expression was determined by q RT-PCR. β-galactosidase, MTT, transwell and angiogenesis assays were conducted to assess EPCs senescence, proliferation, migration and angiogenesis, respectively.(5) By using publicly available databases including Target Scan, mi Randa and Pic Tar, we speculated that Akt3 might be a potential target gene of mi R-22 in EPCs. Dual-luciferase reporter assay was used to verify mi R-22 directly regulated Akt3 expression. q RT-PCR and western blotting were conducted to examine Akt3 m RNA and protein expression in Y-EPCs-mi R-22 and A-EPCs-anti-mi R-22.(6) We constructed the lentiviral vectors encoding Akt3 with or without 3′-UTR, which was infected into Y-EPCs-mi R-22 cells. After infection for 48 h, EPCs senescence, proliferation, migration and angiogenesis changes were examined.Results(1) Mononuclear cells(MNC) were got from peripheral blood. At first,the culture cells were small round.After 1 days culture,some cells become large round, oval or irregular. After 7 days,many cells showed as large fusiform, endothelial cells-like morphology.(2) After 7 days culture,EPCs were characterized as Di I-AC-LDL/FITC-UEA-I double positive cells assessed with laser scanning confocal microscopy.(3) The positive rates of CD133, CD31, CD34 and VEGFR-2 expanded EPC at 7 days of culture were 33.3%, 51.7%, 45.4% and 47.2%, respectively.(4) mi R-22 is expressed at low levels in young EPCs(Y-EPCs) and upregulated in aged EPCs(A-EPCs).(5) We overexpressed mi R-22 in Y-EPCs or knocked down mi R-22 expression with anti-mi R-22 in A-EPCs. The results showed that overexpression of mi R-22 in Y-EPCs increased senescence whereas its depletion decreased A-EPC senescence. Moreover, overexpression of mi R-22 in Y-EPCs inhibited proliferation, migration and angiogenesis whereas knockdown of mi R-22 in A-EPCs increased these parameters.(6) Luciferase reporter assays showed that mi R-22 overexpression decreased the relative luciferase activity of the 3′UTR reporter, whereas it had no effect on the mutant 3′UTR reporter, indicating that the intact seed sequence is required for mi R-22 functionality. Furthermore, mi R-22 overexpression in Y-EPCs significantly downregulated AKT3 m RNA and protein levels, whereas mi R-22 knockdown in A-EPCs increased AKT3 expression.(7) The mi R-22 promotion of senescence and repression of cell viability, migration and vascular tube formation depends on the presence of an AKT3 transcript with an intact 3′UTR.Conclusion(1) mi R-22 is upregulated in aged EPCs.(2) mi R-22 promotes EPCs senescence, and inhibites proliferation, migration and angiogenesis.(3) mi R-22 induces EPC senescence by downregulating AKT3 expression.