Effect Of TMfn2 Gene On Inhibiting The Proliferation Of Vascular Smooth Muscle Cells And Its Molecular Mechanisms

Partâ… Packing,Identification and Expression of the RecombinedAdenovirus containing tMfn2 gene1.ObjectiveTo construct the recombinant adenovirus vector containing the cDNA for Mfn2 with thetransmembrane domain deleted (tMfn2) using the Lipofecamine^TM 2000 Packing System,and examine its ability to express the tMfn2 gene in VSMCs.2.MethodsHEK 293A cells were transfected by using Lipofectamine^TM 2000 and packaged for therecombinant adenovirus particles.Replication-deficient recombinant adenovirus whichcarried the tMfn2 gene were identified by PCR and amplified.The titer of adenovirus wasmeasured by cesium chloride (CsCl) gradient ultracentrifugation.Finally,the expression oftMfn2 gene in VSMCs was determined by Western blot analysis.3.ResultsThe replication-deficient recombinant adenovirus which carried tMfn2 gene wereconstructed successfully and identified by PCR.The titer of adenovirus particles was66.424×10⁹ pfu/ml.The result of Western blot indicated that VSMCs can express highlevel of tMfn2 protein efficiently when transfected.4.ConclusionsThe method of packing in vitro is a shortcut for construction of replication-deficientrecombinant adenovirus with tMfn2 gene,which provides a basis for the further research onthe effect of tMfn2 gene on the proliferation and apoptosis of VSMCs. Partâ…¡Effects of tMfn2 gene on inhibiting the proliferation of VSMCs1.ObjectiveTo investigate the effect of tMfn2 on inhibiting the proliferation of VSMCs.2.MethodsVSMCs were infected by adenovirus-mediated tMfn2 (Adv-tMfn2) or adenovirus-mediated Mfn2 (Adv-Mfn2).Western blot was used to testify the expression of these twoproteins.Cell counting and MTT were used to investigate the effect on proliferation ofVSMCs and Flow cytometry to observe the cell cycle.Before collected,the cells werestimulated by ET-1 for 2～5min.Western blot were used to analyze the expression ofp-ERK1/2 and p-Raf-l.ANOVA was used to make data analysis.3.ResultsAccording to the results of cell counting and MTT,they both indicated that Adv-tMfn2had more remarkable effect on inhibiting the proliferation of VSMCs than Adv-Mfn2 (P＜0.01).Flow cytometry showed that most of the cells infected by Adv-tMfn2 or Adv-Mfn2were blocked in the stage of G0/G1 and few cells entered into the S phase;tMfn2 hadstronger effect than Mfn2 (P＜0.01).The result of Western blot indicated that tMfn2 caninhibit phosphorylation of ERK1/2 and Raf-1 protein more effectively compared with Mfn2(P＜0.01).4.ConclusionsOverexpression of tMfn2 reduces ET-1-stimulated VSMCs proliferation by inhibitingERK phosphorylation and Raf activity,then leading to the arrest of cell cycle.What's moreimportant,tMfn2 gene is superior to Mfn2 gene in attenuating the proliferation of VSMCsvia the Ras-ERK1/2-Raf signaling pathway. Partâ…¢tMfn2 gene promotes apoptosis of VSMCs via mitochondrialpathway1.ObjectiveThe aim of the study is to investigate the effect of tMfn2 gene on promoting theapoptosis of VSMCs.2.MethodsVSMCs were infected by adenovirus-mediated tMfn2 (Adv-tMfn2) or adenovirus-mediated Mfn2 (Adv-Mfn2).FACS analysis,cell death ELISA and TUNEL staining wereused to investigate the role of tMfn2 on VSMCs apoptosis.Western blot was used toanalyze the expression of p-Akt,Bcl-2,Bax and cleaved capase-9.3.ResultsFACS analyses and ELISA assay showed tMfn2 was superior to Mfn2 in promoting theapoptosis of VSMCs and had a robust time-dependent increase in apoptosis (P＜0.01).TUNEL staining displayed that there were more positive-apoptotic VSMCs in tMfn2 groupthan that in Mfn2 group (P＜0.01).The results of Western blot indicated that the proteinexpression of phosphorylated Akt and Bcl-2 remarkably decreased,whereas Bax andcleaved capased-9 protein highly expressed in tMfn2-infected group.4.ConclusionsThe proapoptotic effect of tMfn2 was mediated by inhibition of PI3K-Akt signallingand subsequently activated the mitochondrial apoptotic pathway.To compare with Mfn2gene,tMfn2 has more remarkable effect on inducing VSMCs apoptosis.