Mitochondrial Fusion Protein 2 Inhibit The Proliferation Of Breast Cancer Cells MCF-7 And Affect The Expression Of EGFR

Objectives (1) The purpose of this study was to lay the basis for the speculation that Mfn2 play a role in invasion and metastasis of breast cancer and to analyze the expression of Mfn2 and EGFR in Normal breast tissue,breast tissue and breast cancer cell lines of human.(2)To regulate the expression of Mfn2 in breast cancer cell lines MCF-7 using the gene transfection technology,and to observe the proliferation in MCF-7 after transfection. (3)It was detected whether there are changes about EGFR in MCF-7 after transfection,to analyse the mechanism that how Mfn2 make the changes of EGFR.. Preliminarily to study how Gene therapy effect the Breast Cancer,in order to explore the possibility of Mfn2 as the therapeutic targets.Methods (1)The Polymerase Chain Reaction(PCR) was used to detect the expression of Mfn2 and EGFR in normal breast tissue, breast cancer and breast cancer cells MCF-7. The Immunocytochemistry was applyed to detect the expression of Mfn2 and EGFR in breast cancer cells MCF-7.(2)To construct eukaryotic expression plasmid including open reading frame (ORF) of mfn2 gene, this vector was transduced into breast cancer cells MCF-7,in order to make breast cancer cells express Mfn2 protein. (3)The method of Polymerase Chain Reaction(PCR) was used to detect the expression of Mfn2 after transfection,and the methord of MTT was used to find out whether it works to the cell proliferation of MCF-7,and then Flow Cytometry was used to detect the transfection efficiency and the effection to the cell cycle of MCF -7 cell in vitro. (4)The methods of Immunocytochemistry and Real-time Quantitative PCR were used o detect the expression of EGFR after transfecting exogenous mfn2 gene into the cell cycle of MCF -7 cell in vitro. Results (1) The Agarose Gel Electrophoresis (AGE) showed that mfn2 was expressed in normal breast tissue, breast cancer and breast cancer cells MCF-7 ,and the expression of mfn2 in breast cancer was obviously lower than in normal breast tissue. (2) The recombinant plasmid pEGFP-N1 successfully loaded full-length mouse Mfn2 gene encoding, DNA sequencing results were basically consistent with the expected design.General PCR Agarose Gel Electrophoresis of transfected MCF-7 cells were in the presence of exogenous Mfn2 expression. (3)The Agarose Gel Electrophoresis (AGE) showed that the expression of Mfn2 in experimental group was significantly higher than in the control group. After transfection, the cell proliferation velocity and the OD value of pEGFP-N1 group and non-transfection group were basically at equal pace. But the pEGFP-mfn2 group'48h was manifestly less than both of pEGFP-N1 group and non-transfection group(P<0.05). The DNA analytical method revealed that there was no significant difference between pEGFP-N1 group and non-transfection group.pEGFP-mfn2 group compared with pEGFP-N1 group and non-transfection respectively ,G1 stage cell ratio was significantly increased 48h after transfection, ,which had extremely significant difference(F=49.299,P<0.05).(4)The Immunohistochemical and Real-time Quantitative PCR were all showed that the number of cells and the expression of EGFR in pEGFP-mfn2 group were lower than in pEGFP-N1 group and cancer cells appeared nuclear fragmentation .Conclusions (1) The expression of mfn2 in breast cancer tissue and breast cancer cells MCF-7 were obviously lower than in normal breast tissue. The expression of EGFR was in a high degree in MCF-7 as a tumor-associated factor. (2)The eukaryotic expression plasmid pEGFP-Mfn2 was successfully constructed, and Mfn2 was expressed in MCF-7 cells after being transfected. (3) Exogenous mfn2 strongly inhibited cell proliferation in MCF-7 cell line, and caused cell arrest at G1 phase.(4)Exogenous mfn2 led MCF-7 to apoptosis,and obviously down-regulated the expression of EGFR .