I. Construction And Identification Of GFR?2 Gene Overexpressing Lentiviral Vector 2. Establishment And Functional Verification Of Tamoxifen Resistant Cell Line Of Breast Cancer

Objective: To construct and identify the over-expression lentiviral vector for GFRa2 gene and then investigate the effect of overexpression of GFRa2 gene on the biological behaviors of human breast cancer cell line MCF-7.Methods: GFRa2 gene was cloned through gene recombination technique,and inserted into the retroviral expression vector GV358.The recombinant retroviral expression vector was identified by restriction endonuclease digestion and RT-PCR, then the recombinant vector was infected into the packaging cell line 293T. Western bolt and RT-PCR were used to detect the expression of the mRNA and protein of GFRa2 gene.Results: Restriction endonuclease digestions revealed that the GFRa2gene was cloned into the retroviral expression vector successfully, and the sequences were identified by DNA sequencing. The GFRa2 gene of the recombinant lentiviral vector was successfully packed into 293T cells. The recombinant lentivirus was harvested from 293T cells, and the titer of concentrated virus was 2E+8.Conclusions: The lentiviruses which carrying with GFRa2 and their genen sequence were made correct.Objective: To establish tamoxifen-resistant cell line of MCF-7 human breast cance and investigate the effect of the expression of GFRa2 and HER2 gene on the the tamoxifen-resistant cell line of MCF-7 human breast cancer..Methods : The plasmid for GFRa2 overexpression was constructed .To obtain insight into the genomic basis of endocrine therapy resistance, MCF-7 Tamoxifen-resistant cell line was induced by a high concentration of 4-hydroxytamoxifen (OHT)treatment for a short period. Drug resistance of MCF-7cells after 28 day (MCF-728d) induced by OHT were determined by cell morphology, cell growth curve,drug toxicity tests. We characterized MCF-7 monoclonal sublines that survived 21 -day exposure to OHT. The tamoxifen-resistant cell line MCF-7 human breast cancer was established and indentified.Cell proliferation was dectected by CCK8 method.Thecell aggression and migration was dectected by Transwell assay. Western blot and RT-PCR were used to detect the expression of mRNA and protein of GFRa2 and HER2 gene.Results : Compared with parental cell line, MCF-7 28d cell exhibited a different cell morphology,a similar proliferation ability(P>0.05),a stronger resistance to OHT-mediated cytotoxic effects(P<0.05). The 14th day induced cells have the highest IC50 and RI. Morever, mRNA and protein were both increased highestat 14th day induced cells (P<0.05).For MCF-7 28d cells ,GFR?2mRNA are also overexpressed but not as high as 14th day induced cells(P<0.05).However, the HER2 mRNA in MCF-7 28d cells are lower expressed than MCF-7. But they have the same growth trend.Cell migration also cell aggression were promoted.Conclusion: The tamoxifen-resistant cell line of MCF-7 human breast cance was established and indentified and has been constructed ssuccessfully at 14th day by OHT induced.The results showed the overexpression of GFRa2mRNA and HER2 mRNA may effect the sensitive of MCF-7 cells to tamoxifen. MCF-7 d28 cell lines has a stronger ablity of cell invasion and migration.