ABSTRACT
Objective To study the effects of RNA interference(RNAi)-mediated silencing of mitofusin-2 (Mfn2) gene on glycornetabolism and insulin resistance in BALB/c mice. Methods Mfn2 short hairpin RNA (shRNA) and negative control green fluorescent protein(GFP) -expressed plasmid vectors were constructed. 44 mice were randomly divided into transfection group (Mfn2) and negative control group (HK). 1.5 ml GFP-expressed plasmid(negative control or Mfn2 shRNA,75 μg for each mouse)was injected into the mice in 5 seconds through vena caudalis. Five days later, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test(IPITT)were performed to evaluate glycometabolism and insulin sensitivity. D-3-[3~H]-glucose in PBS buffer were injected via the tail vein. Blood samples were taken at specific time points. Radioactivity was measured in all samples with liquid scintillation counter. The rates of hepatic glucose production in vivo were calculated. Mfn2 protein expression in hepatic tissue was detected by Western blot. Results Compared with HK mice, the Mfn2 expressions of Mfn2 mice decreased markedly(8.05±0.15 vs 8.56±0.01 ,P<0.05). The fasted blood glucose leves [(6.95±0. 83 vs 4.68±0. 29) mmol/L,P<0. 05] in Mfn2 mice were higher than those in HK mice. The insulin sensitivity of Mfn2 mice decreased markedly compared with HK mice. The rate of hepatic glucose production was significantly elevated in Mfn2 mice [(49.53±16.31)μmol·kg~(-1)·min~(-1)],compared with negative control mice[(24.91±4.07)μmol·kg~(-1)·min~(-1),P<0.05].Conclusion The down-regulatd expression of Mfn2 induces glycometabolic disorder and insulin resistance in BALB/c mice. Mfn2 plays an important role in maintaining glucose homeostasis in vivo.
ABSTRACT
In order to evaluate the effect of mitofusin-2 gene (mfn2) on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-V/PI double labeling method was em- ployed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 eDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the per- centage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively (P<0.05). The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin (CAMP) followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively (P<0.05). It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and pro- mote their sensitivity to CAMP with a synergic effect.