| Objective To study the efftct and the mechanism of Cyclin A DNAmethylation in Homocysteine (Hcy)-induced endothelial progenitor cells (EPCs)dysfunction, and try to explore the possible key target of Hcy-induced AS.Methods This subject intends to extract the target cells from rat blood cells，and detected by FACS calibur flow cytometer and analyzed using Cell-Questsoftware by staining with primary antibodies against CD34， CD133. And theadhesion and migration ability were detected in different Hcy concentrationsgroups respectively. The proliferative activities of cells were assayed by MTT. ThemRNA and protein levels of Cyclin A, DNMT1, MBD and MeCP2were detectedby Real-Time PCR and and Western blotting respectively. The methylation level ofCyclin A were detected by nested methylation-specific-polymerase chain reaction(nMS-PCR) and the SAM, SAH were analyzed by High-performance liquidchromatography (HPLC). To ensure the effect of Cyclin A in the EPCs treated byHcy, an overexpression vector and a RNA silencing vector of Cyclin A were built,then the proliferative activities of cells were assayed by MTT.Meanwhile, wesilenced DNMT1via RNAi to define the role of DNMT1in DNA methylation.Results The result of FACS presented that the percent of CD133positive cells is(20.40±6.92)%and the percent of CD34is (49.17±12.47)%. The result of MTT presented that being treated with100,200,500μmol/L Hcy for72h, theproliferative activities of EPCs were inhibited, especially in500μmol/L(P<0.05). Compared with the control group， Hcy significantly reducedcell adhesion and the adhesion capacity decreased by52%in500μmol/L. And themigration activity of EPCs decreased by (17.23±4.54)%,(11.93%±2.68)%,(9.67±2.08)%,(8.53±1.31)%,(6.1±2.15)%and (11.57±2.38)%in50mol/L，100mol/L，200mol/L，500mol/L groups and antagonist group respectively. Themigration activity of the cells was decreased with the increase of the concentrationof Hcy. After treated with Hcy for72h, the expression of Cyclin A was declinedwith the Hcy concentration increased. Compared with the control group， themRNA expression of Cyclin A decreased by55.2%in500μmol/L group(P<0.05).And the protein level is consistent with mRNA. Meanwhile, we ensured thatCyclinA is a key gene by using recombinant and RNA interference technology. Themethylation status of Cyclin A showed that Cyclin A gradually showinghypomethylation performance with the increase of Hcy concentration. Comparedwith that of control group，our results showed that the500μmol/L group displayedthe most obvious hypomethylation change and its methylation level downregulated15.4%(P<0.05) and upregulated8.2%in folate group respectively. A certain doseof AZC can significantly inhibit Cyclin A methylation status. Meanwhile，compared with control group, the concentrations of SAM and SAH were decreased(P<0.05, P<0.01). Compared with the control group, the SAM/SAH of500mol/Lgroup decreased with49.9%. Compared with500mol/L group, the SAM/SAH ofantagon group and AZC group increased by1.51-and1.89-folds respectively(P<0.05, P<0.01). The protein expression of MBD increased with Hcyconcentrations and the protein expression of MeCP2generally showed a downwardtrend. Compared with the conteol group, the DNMT1mRNA levels in100mol/L, 200mol/L,500mol/L, antagonist group and AZC group decreased45.6%，61.8%，65.5%，51.8%å'Œ63.2%respectively (P<0.05, P<0.01). The result ofDNMT1RNAi displayed that compared with normal group, the methylation statusof Cyclin A deceased by61.7%in Hcy+siRNA group (P<0.05).Conclusion Hcy may inhibit the expression of Cyclin A by changing the DNAmethylation status of Cyclin A, then leading to the dysfunction of EPCs. SAM,SAH, MBD, MeCP2and DNMT1play an important role in the methylationregulation, and DNMT1is a key mechanism in Hcy-induced Cyclin Ahypomethylation in EPCs. |
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