MDA-MB-435 Cell Apoptosis Induced By Recombinant Retroviral Vector Mediated CED-4 Gene Transfection

AIM: It is to investigate that apoptosis of MDA-MB-435 cell, a human breast cancer cell line, was induced by recombinant retroviral vector mediated CED-4 gene of cadherin 5 N terminal and its apoptosis mechanism. METHODS: This experiment is succeeding to the former work.The methods as following.⑴Restriction endonucleases analysis of the plasmids: the plasmids pVSV-G,pGAG-POL,pMSCV,pMSCV-CED4 were obtained from the former work,and they were cut by the restriction endonucleases BspH Ⅰ, Bgl Ⅱ, AflⅡrespctively. These plasmids cut by restriction endonucleases were examined by agar electrophoresis.⑵Packaging of the retrovirus in 293 cells: the 293 cells were transferred with the plasmids pVSV-G , pGAG-POL , pMSCV ,pMSCV-CED4 plus lipofectamine respectively.The recombinant retrovirus containing target gene were packaged by 293 cells and secreted from cells.At the same time,the control group was established. ⑶The recombinant retrovirus transfection of MDA-MB-435 cells: the experiment group and the experiment control group cells were tranfected with the recombinant retrovirus respectively.EGFP(enhanced green fluorescent protein) gene inserted in pMSCV was the positive label of the positive transfected cells.The green fluorescence in the transfected cells was detected after 3 days of transfection and the morphology of the transfected cells was intensively observed after 2 weeks of transfection. ⑷Verification of apoptosis:the morphology of cell apoptosis was observed using microscope.The characters of apoptosis were verified by annexin V-boitin apoptosis detection kit.⑸RT-PCR:the total RNA of three groups was extracted.The CED-4 mRNA,integrinβ1 mRNA and c-fos mRNA were amplified by RT-PCR and detected by agar electrophoresis.⑹Westhern blot: The c-fos protein was analysed by westhern blot.RESULTS:①The results of restriction endonuclease cutting and agar electrophoresis indicated that the size of plasmids pVSV-G,pGAG-POL,pMSCV,pMSCV-CED4 were normal and intact.②The recomninant retrovirus were packaged by 293 cells. Green fluorescence was detected in 293 cells after 48 hours of transfection.③Green fluorescence was detected in the transfected MDA-MB-435 cells after transfection of 3 days.This result indicated that MDA-MB-435 cells transfected by CED-4 gene were obtained.④The mRNA expression of transfected target gene was verified by RT-PCR in MDA-MB-435 cells. ⑤The MDA-MB-435 cells apoptosis was induced by exogenous CED-4 gene. ⑥The expression of integrinβ1 subunit mRNA and c-fos mRNA were decreased in the experiment group in the apoptosis process. ⑦The expression of c-fos protein were greatly decreased in the apoptosis process. CONCLUSION: ①The exogenous CED-4 gene induced the MDA-MB-435 cells to apoptosis. ②The exogenous CED-4 gene down-regulated the expression of integrinβ1 subunit mRNA. ③The exogenous CED-4 gene down-regulated the expression of c-fos mRNA. ④The exogenous CED-4 gene down-regulated the expression of c-fos protein.