Study On The Functional Sequence Of Mitofusin 2 Gene And Related Synthetic Peptide In Inhibiting Proliferation Of Vascular Smooth Muscle Cells

Construction, amplification and purification of recombinant adenoviruses containing various sequence structures of Mfn21. ObjectiveTo construct, identify and purify the recombinant adenoviral vector containing various sequence structures of Mfn2. 2. MethodsVarious complementary DNA (cDNA) was first amplified and subcloned into pCR8/GW/TOPO TA cloning plasmid. Subsequently, we performed LR recombination reaction between the pCR8/GW/TOPO-tMfn2 and pAd/CMV/V5-DEST to obtain the adenoviral expression vector. After packaging the pAd/CMV/V5-DEST-Mfn2 in 293 cells, the replication-defective adenovirus was produced. CsCl density gradient centrifugation was used to measure the titer of various adenovirus. Western blot analysis was used to confirm the expression of Mfn2 at the protein level.3. ResultsThe recombinant adenoviruses which carried various sequence structures of Mfn2 (1 A, 2A,3A,4A,5A,6A,7A,8A) was successfully constructed and identified by PCR. The titer of recombinant adenoviruses was 27.8×109 pfu/ml,24×109 pfu/ml,19×109 pfu/ml, 30.8×109 pfu/ml,6.7×109 pfu/ml,12.7×109 pfu/ml,27.6×109 pfu/ml and 11.8×109 pfu/ml, respectively. Western blot analysis showed the expression of Mfn2 protein in infected vascular smooth muscle cells (VSMCs).4. ConclusionsThe recombinant adenoviruses which carried various sequence structures of Mfn2 can be successfully constructed by Recombinant DNA technology, which provides the basis for further study on the effect of various Mfn2 sequence structures on the proliferation and apoptosis of VSMCs. Part IIVarious sequence structures of Mfn2 on proliferation and apoptosis of vascular smooth muscle cells (VSMCs)1. ObjectiveTo investigate the effect of various sequence structures of Mfn2 on proliferation and apoptosis of vascular smooth muscle cells (VSMCs), seek the shortest complementary DNA (scDNA) that can both effectively inhibit proliferation of VSMCs and induce apoptosis of VSMCs and explore the potential molecular mechanisms.2. MethodsVSMCs were infected by recombinant adenoviruses containing various sequence structures of Mfn2 (1A,2A,3A,4A,5A,6A,7A,8A). The effect of various sequence structures on proliferation of VSMCs were tested by cell counting and CCK-8, the cell cycle was observed by flow cytometry; Cell death ELISA and flow cytometry were used to investigate the effect on apoptosis of VSMCs. Western blot analysis was used to detect the expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt).3. ResultsCell counting and CCK-8 both indicated that various sequence structures of Mfn2 significantly inhibited proliferation of VSMCs (P<0.01). Most of the VSMCs were blocked in the stage of G0/G1 and few entered into the S phase in all sequence structures groups (P<0.01). Cell Death ELISA assay and flow cytometry both indicated that 1A,4A and 8A promoted apoptosis of VSMCs remarkably. Western blot analysis showed that 1A, 4A and 8A significantly down-regulated expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt) (P<0.01).4. Conclusions1A is the shortest complementary DNA (scDNA) of Mfn2 that can both effectively inhibit proliferation of VSMCs via Ras-Raf-ERK1/2 signaling pathway and induce apoptosis of VSMCs via Ras-PI3K-Akt signaling pathway.PartⅢMfn2 gene-related synthetic peptide (MRSP) on proliferation and apoptosis of vascular smooth muscle cells (VSMCs)1. ObjectiveTo investigate the effect of Mfn2 gene-related synthetic peptide (MRSP) on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) and the potential molecular mechanisms.2. MethodsMRSP was synthesized according to the shortest complementary DNA (scDNA) of Mfn2 (1A) and TAT-PTD (transactiviting-protein transduction domain). VSMCs were cultured in DMEM containing MRSP of various concentration (1μM,10μM,25μM,50μM, 10μM). The effect of various concentration of MRSP on proliferation of VSMCs were tested by cell counting and CCK-8, the cell cycle was observed by flow cytometry; Cell death ELISA and flow cytometry were used to investigate the effect on apoptosis of VSMCs. Western blot analysis was used to detect the expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt).3. ResultsCell counting and CCK-8 both indicated that MRSP caused a marked dose-dependent and time-dependent proliferation inhibitory effect on VSMCs from 10μM to 100μM (P< 0.01). Flow cytometry showed that most of the VSMCs were inhibited in the stage of G0/G1 and few cells entered into the S phase in all the three MRSP groups (P<0.01). Cell Death ELISA assay and flow cytometry both indicated that MRSP promoted apoptosis of VSMCs effectively. Western blot analysis showed that MRSP significantly down-regulated expression of phosphorylation of Raf (p-Raf), ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt) (P<0.01).4. ConclusionsThe MRSP synthesized according to the shortest complementary DNA (scDNA) of Mfn2 can significantly inhibit the proliferation of VSMCs via the Ras-Raf-ERK1/2 signaling pathway, it can also effectively induce the apoptosis of VSMCs via the PI3K-Akt signaling pathway.