The Effect Of Mitofusin2 Gene On Suppressing Proliferation Of Rat Vascular Smooth Muscle Cell Line A7r5

Objective Vascular smooth muscle cells'(VSMCs) abnormal proliferation play a key role in vascular proliferation diseases. This kind of proliferation is the pathological basis of atherogenisis, hypertension, coronary artery disease and restenosis. Cell growth factors, cytokines and hemodynamic disorder can result in VSMC hyperproliferation via signaling transconduction pathway, especially Ras-Raf-MAPK and Ras-PI3K-Akt. So, blocking these pathways and suppressing VSMC hyperproliferation are effective measurements for preventing vascular proliferation diseases. Mitofusin2 (Mfn2) is a new gene which can suppress proliferation. It was confirmed recently. Not only suppresses many tumor cells proliferation, but also Mfn2 participates in mitochondrial fusion, metabolism regulation and maintaining its network structure. Mfn2 expressions of aortic VSMC are decreased in hypertension rats and balloon-damaged rats. Increased expression of Mfn2 maybe useful for preventing hypertension and restenosis. In the present study, we plan to observe the effect of Mfn2 on VSMC proliferation by means of gene clone and gene recombination. We also want to discuss its molecular mechanism and hope to provide proof that Mfn2 might be regarded as a target for preventing vascular proliferation diseases.Methods The Mfn2 cDNA was obtained from the A7r5 RNA by reverse transcription polymerase chain reaction (RT-PCR) and was amplified, purified and retrieved.Recombined pGEM-T and the Mfn2 cDNA with ligase.Recombinant cloning vector pGEM-T-mfn2 was transformated into DH5αand was screened with ampicillin.To digest pEGFP-N1 plasmid and pGEM-T-mfn2 with dual-endonuclease, we retrieved a big fragment from pEGFP-N1 and a 2.2 kb fragment from pGEM-T-mfn2. Recombined the big fragment and 2.2 kb fragment. Then the correctness of the pEGFP-mfn2 was evaluated by using restriction enzyme and sequencing analysis. The recombinant plasmid containing either the Mfn2 cDNA fragments or GFP gene was transfected into the A7r5 cells with lipofectin.A7r5 cells were divided into 3 groups:control group A7r5 (untransfected cells), A7r5-GFP group(transfected with empty vector pEGFP-N1) and A7r5-Mfn2-GFP group(transfected with recombinant plasmid).The transient expression of green fluorescent protein(GFP)was observed under fluorescence microscope after 24 hours. 48h after transfection,the expression of Mfn2 gene in A7r5 cells was examined by RT-PCR and Western blot.After transfection,the cell proliferation of A7r5 was detected by MTS and cell counting.Cell cycle progression and distribution were determined by flow cytometric analy- sis.The expression of several signal transducers relevant to VSMC proliferation including phospha-Raf ,phospha-ERK1/2 and phospha- AKT was detected by Western blot. ANOVA was used to make data analysis.Results We have successfully cloned Mfn2 gene from A7r5 cells and constructed a sort of recombinant plasmid containing either the Mfn2 cDNA or GFP gene.By digesting the 6.9kb fragment with dual-endonuclease, we obtained two fragments, one is 4.7 kb, the other is 2.2 kb.It was identified by DNA sequencing that the sequence of 2.2 kb fragment accords completely with the CDS sequence of Mfn2 gene in GeneBank.The successful construction of recombination plasmid pEGFP-mfn2 is confirmed further.70% A7r5 cells transfected with lipofectin emitted out GFP under fluorescent microscope after 48h.The result of RT-PCR and Western blot showed that Mfn2 gene effectively was expressed in the transfected A7r5 cells.Cell Counting and MTS showed that the cell number in A7r5-Mfn2-GFP group became slower than those of A7r5-GFP group and non-transfection group(P<0.05).Flow cytometry analysis also showed the cell growth in A7r5-Mfn2-GFP group was arrested in G1 phase, resulting in a declination in s phase(F=109.8,P<0.05).The result of western blot indieated that phosphorylation of Raf-l,ERK1/2 and AKT protein in A7r5-Mfn2-GFP group was Inhibited effectively compared with A7r5-GFP group and non-transfection group(P<0.05).Conclusions1. Overexpression of Mfn2 can significantly inhibit A7r5 cells proliferation.2. Down-regulating the expressions of Raf, ERK1/2 and AKT phosphorylation is the possible mechanism of Mfn2 suppressing A7r5 cells proliferation.