Effect Of Geminin Overexpression On The Phenotypic Ransformation Of VSMCs

BackgroundDuring the development and progression of atherosclerosis,hypertension, restenosisand other proliferative vascular disease,it is known that the phenotypic transformation ofvascular smooth muscle cells(VSMCs) plays an important role in the above-mentionedprocesses. Proliferation and migration occur in the process from differentiated phenotype todedifferentiated phenotype, in which the quiescent cells regain proliferative capacity andlead to a series of vascular disease. VSMCs usually have two different phenotypes:differentiated phenotype and dedifferentiated phenotype, which could be transformed intoeach other under certain condition. The different phenotype of VSMCs has specialphenotypic markers,α-actin is related to the differentiated phenotype, while osteopontin(OPN)related to the dedifferentiated phenotype. It was reported that Geminin was a cell cycleinitiation repressor,involved in the integration of cell cycle and the process of cellproliferation-differentiation.It was shown that Geminin was helpful for the cells to keep astable differentiated state, to inhibit the reduplication of cellular DNA and ensure thestability of genetic information. Our previous study have shown that Geminin participatedin the phenotypic transformation, its expression can be detected in two phenotypic cells.However, the Geminin level of differentiated phenotypic VSMCs was higher than that ofdedifferentiated cells. Recent studies suggested that the proliferation capacity of cells wasenhanced, even excessive proliferation by interferencing of Geminin gene.It is still unclearhow Geminin gene triggers the phenotypic transformation and what is the mechanism.It is known that polymerization and dissociation of actin is involved in the phenotypictransformation, and helpful for the cells to keep the condition of different phenotype. Theactin-related protein1(ARP1) plays an important role in the process of polymerization anddissociation. The gain of the proliferation ability is a critical step during the transformationprocess from differentiated to dedifferentiated. We hypothesize that the overexpression of Geminin promote the transformation of VSMCs from dedifferentiated phenotype todifferentiated phenotype, ARP1could be involved in the process.ObjectiveThe purpose of this study was to analyze the effect of Geminin overexpression on thephenotypic transformation of VSMCs.Methods1.The culture of two different phenotype VSMCsVSMCs line(A10) is cultured.After being cultivated24hours with culture mediumincluding10%fetal bovine serum(FBS),the dedifferentiated phenotype vascular smoothmuscle cells were obtained. While cultivated24hours with free medium,the differentiatedphenotype cells were obtained. The study was classed into positive group, negative controland blank control (n=3).2.The construction of eukaryotic expression vector pEGFP-N1-Geminin and transfectionof two different phenotype cells.Total RNA was extracted from the VSMCs, then was amplified by RT-PCR technique.Theexpression vector pEGFP-N1and Geminin gene fragment were digested by the restrictionendonucleases HindIII and sacII. The recombinant plasmid was transfected into E. coliDH5α, then PCR and enzyme-digestive method were used to extract and identify, thesequence of Geminin gene was measured. Then pEGFP-N1-Geminin was transfected intoVSMCs of different phenotype using lipofectamine2000. The efficacy of transfection wasdetected with fluorescence microscope and western blotting.3. The examination of phenotypic markersWestern blotting-the semiquantitative analyzing method was used to detect the expressionof mainly phenotypic markers after being overexpressed by pEGFP-N1-Geminin. GAPDHandβ-actin were loading control.Antibodies against α-actin and OPN were used to test theexpression of α-actin and OPN.4. The detection of ARP1expression and analysis of the interaction between Gemininand ARP1The presence of ARP1was tested by western blotting using antibody against ARP1.The interaction between Geminin and ARP1was analyzed by co-immunoprecipitation.Results 1. Two different phenotype cells was successfully obtained.2.Geminin gene was successfully inserted into pEGFP-N1.The transfection efficacywas about60%with lipofectamine2000in all three groups，but the expression of Gemininin positive group was higher than that of negative control and blank control, indicating thatthe eukaryotic vector pEGFP-N1-Geminin was successfully transfected into positivegroup.It was satisfied with the requirement of the study.3. In differentiated VSMCs,the ratios α-actin to GAPDH in three group were1.14±0.03,1.14±0.02,1.12±0.02, while that of dedifferentiated VSMCs were1.14±0.03,0.84±0.03,0.82±0.02.In differentiated VSMCs,the ratios OPN to GAPDH in three groupwere0.31±0.02,0.34±0.03,0.30±0.02, while that of dedifferentiated VSMCs were0.33±0.03,0.65±0.03,0.62±0.03. The mainly phenotypic markers (α-actin, OPN) did not show asignificant difference in differentiated VSMCs among three groups. However,comparedwith two controls, the significant changes of phenotypic markers were found in positivegroup of dedifferentiated cells,α-actin were up-regulated,and OPN were down-regulated.4. In differentiated VSMCs,the ratios ARP1to GAPDH in three group were1.31±0.03,1.31±0.03,1.32±0.03,while that of dedifferentiated VSMCs were1.27±0.03,0.98±0.03,0.96±0.03. ARP1did not show a significant difference in differentiated VSMCsamong three groups. However,compared with two controls, the up-regulation of ARP1werefound in positive group of dedifferentiated cells.The interaction between Geminin andARP1was further confirmed by co-immunoprecipitation.ConclusionOverexpression of Geminin promoted the penotypic transformation of VSMCs fromdedifferentiated phenotype to the differentiated phenotype,ARP1could be involved in thisprocess.