The Effect Of Mitofusin-2 Gene In Cell Proliferation,Apoptosis And Chemotherapy Sensitivity Of Human Breast Carcinoma Cell Line

Objectives Breast carcinoma is one of the common malignant cancers, which incidence rate is the first place in female malignant tumor in many countries. More attentions are paid to the research of breast carcinoma. Cell hyper-proliferation has long been considered as an important etiological factor of cancer.It is hot point how to inhibit hyper-proliferation of carcinoma and induce apoptosis of cancer cell. Recently, a novel hyperplasia suppressor gene was identified and characterized, named HSG (later re-named rat mitofusin-2). This gene expression was markedly reduced in hyper-proliferative vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rat arteries. Mitofusin-2 (mfn2) is human homologue of rat mitofusin-2 (rmfn2). Several studies have demonstrated that human mfn2 and its homologues localize to the mitochondrial outer membrane. Many researches focused on the relationships between mfn2 and mitochondrial morphous, functions, cell respiration, glucose oxidation, energy metabolism, metabolism. Mitochondria are the fetal eukaryotic cell organelles which play important role in cell energy metabolism.But the study on its effect on the carcinoma is seldom seen in related literatures.The aim of my experiment is to study the influence of mfn2 on breast carcinoma cell proliferation and apoptosis in vivo and vitro. To provide the rationales on gene research of breast carcinoma, the effects of mitochondria on carcinoma cell proliferation and apoptosis, and the treatment of breast carcinoma. We want to detect the expression level of mfn2 in breast cancer cell, breast cancer tissue and breast tissue. We construct the eukaryotic expression plasmid including open reading frame (ORF) of mfn2 gene and transduce this vector into breast cancer cells in order to make breast cancer cells express Mfn2 protein.We transduce the eukaryotic expression plasmid pEGFPmfn2 into breast cancer cells in order to study the role and mechanism of exogenous mitofusin-2 gene in cell proliferation of human breast carcinoma cell line MCF-7. To investigate the role of mitofusin-2 gene (mfn2) in cell apoptosis of human breast carcinoma cell line MCF-7 after in vitro transfer, and to evaluate the effect of exogenous mitofusin-2 gene (mfn2) in chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro.To investigate the effect of proliferation and metastatic progression inhibition of breast carcinoma MCF-7 in xenograft implantation model after sofast-mediated gene transfer of mfn2. To detect the effect of proliferation and metastatic progression inhibition of MCF-7 in xenografts implantation model after intratumorally vivo-jetPEITM/pEGFPmfn2 compound.Methods According to the ORF sequence of mfn2 gene in pEGFP-C2 plasmid and multiple clone site(MCS) of pEGFP-C2, we designed the primers used in Polymerase chain reaction(PCR). On the template of cDNA of normal skeletal muscle, we amplified the ORF sequence of mfn2 using PCR technology and purified the PCR product. Make use of the methods of enzyme-cut, PCR and DNA sequence, we successfully constructed pEGFPmfn2. The cells were divided into three groups: non-transfection group as negative control, transfection with pEGFP-C2 group as experimental control group, transfection with pEGFPmfn2 as experimental group. 48h after transfection, and green fluorescence was observed by flow cytometry, and the expression of mfn2 gene by PCR. After transfection, the cell proliferation of MCF-7 was detected by MTT and cell counting.To observe cell cycle of MCF-7 by DNA analytical method,after transfection exogenous mfn2 gene to the cell cycle of MCF-7 cell in vitro.To observe the expression of cyclinA by Cyclins/ DNA flow cytometry, 48h after transfection exogenous mfn2 gene to the cell cycle of MCF-7 cell in vitro.To detect the expression of P21waf1,CDK2 by Western blot, 48h after transfection exogenous mfn2 gene to the cell cycle of MCF-7 cell in vitro. The cell apoptosis of MCF-7 was observed in Annexin-V/PI by flow cytometry. The chondrosome transmembrane potential (△ψm)of MCF-7 marked in JC-1 by flow cytometry.To observe change in form and ultra structure of cell was observed by confocal and electron microscope.As the CAMP was added to the medium, the impacts of mfn2 on chemotherapy sensitivity were evaluated in Annexin- V/PI by flow cytometry in the following days. Fifteen nude mice were randomly divided into 3 groups. The mice were heterograft with three groups cells respectively. Eight weeks after implantation, tumor weight and volume were evaluated respectively after the mice were killed and the tumors were taken. We detect the expression of p21 by PCR in three groups'tumor respectively.To detect the expression of Ki-67,VEGF by immunity histochemistry. To establish nude mice transplanted tumor of breast carcinoma, the MCF-7 were implantation nude mice in vivo. Eight weeks after implantation, two kinds of compounds: vivo-jetPEITM/pEGFPmfn2 compound and vivo-jetPEITM/pEGFP compound were prepared. Then the experimental group and experimental control group nude mice received an intratumoral injection of Two kinds of compounds respectively 3 times (once/two days).To detect the expression of mfn2 by PCR in three groups tumor respectively.To detect the expression of Ki-67,VEGF by immunity histochemistry.Results The results of agarose gel electrophoresis (AGE) showed that the expression of mfn2 in breast cancer is lower than in breast tissue and the PCR amplification product from skeletal muscle was a specific strand (2274bp) which accords with the positive control and the ORF sequence of mfn2 gene.The produce of enzyme-cutting and T4DNA ligase transform competent DH-5α, and the growed colony are uniform. The results of double enzyme cut and PCR demonstrated that the size of PCR product accords with enzyme-cutting product (2274bp). The successful construction of recombination plasmid pEGFPmfn2 is confirmed tentatively. DNA sequence showed that the sequence of destination gene in recombination plasmid accords completely with the ORF sequence of mfn2 gene in GeneBank. The successful construction of recombination plasmid pEGFPmfn2 is confirmed further.Results The results of AGE showed that the expression of mfn2 in breast cancer cell and breast cance tissue is lower than in breast tissue. Western blot results showed that green fluorescent protein can be observed in pEGFPmfn2 group and pEGFP-C2 group. But no green fluorescent protein can be observed in non-transfection group. After transfection, the cell proliferation velocity and the OD value of pEGFP-C2 group and non-transfection group was basically at equal pace. But the pEGFPmfn2 group was manifestly less than those of pEGFP-C2 group and non-transfection group (P<0.05). PEGFPmfn2 group compared with control groups respectively, S stage cell ratio had extremely significant difference (F=196.7, P<0.05). Mfn2 could induce the expression of cyclinA protein obviously heighten by Cyclins/DNA flow cytometry. pEGFPmfn2 group the expression of cyclinA had extremely significant difference (F=619.2, P<0.05).The correlation of cyclinA and S stage cell ratio was positive, which had extremely significant difference (r=0.983, P < 0. 05).The expression of P21 in pEGFPmfn2 group was higher than control groups respectively (F=202.9, P<0.01).The expression of phosphorylation CDK2 was lower than other two control groups by western blot (F=219.0, P<0.01). Exogenous mfn2 gene significantly induced apoptosis. the apoptotic from 3.6% added to 16.0%(P<0.05). Exogenous mfn2 could decrease chondrosome transmembrane potential. The apoptotic rate of mfn2 transfected MCF-7 induced by CAM for 4h was 69.6%,whereas in control groups were 31.0%,23.4% (P<0.05), respectively. The weight and volume of experimental group were manifestly less than those of experimental control group and negative control group (P<0.05). The expression of P21 in experimental group was higher than experimental control group and negative control group (P<0.05).The expression of Ki67 in experimental group was higher than experimental control group and negative control group (P<0.05). But the expression of VEGF in experimental group was lower than experimental control group and negative control group (P<0.05).The expression of mfn2 in experimental group was higher than experimental control group and negative control group (P<0.05).The expression of Ki67 in experimental group was higher than experimental control group and negative control group (P<0.05). But the expression of VEGF in experimental group was lower than experimental control group and negative control group (P<0.05).Conclusions(1) The expression of mfn2 in breast cancer cell (MCF-7) and breast cancer tissue is lower than breast tissue. We suppose mfn2 may be a novel anti-oncogene.(2) The eukaryotic expression recombinant plasmid pEGFPmfn2 including the whole long mfn2 gene is constructed successfully.(3) Exogenous mfn2 can strongly inhibit cell proliferation in MCF -7 cell line, and result in cell arrest at S phase.(4) Exogenous mfn2 can obviously up-regulation the expression of cyclinA. The correlation of cyclinA and S stage cell ratio was positive.(5) The expression of P21 is up-regulation and phosphorylation CDK2 is down-regulation.(6)Exogenous mfn2 gene can advance the cell apoptosis of MCF-7, and change in form and ultra structure and chondrosome transmembrane potential of cell.(7) Exogenous mfn2 gene can increase chemotherapy sensitivity of camptothecin chemotherapeutics.(8) Exogenous mfn2 can strongly inhibit proliferation and metastatic progression of breast carcinoma MCF-7 in xenograft implantation model. And mfn2 gene was used for the treatment. The mechanisms may be involved in up-regulating the expression of p21 and down-regulating the expression of VEGF.