SLC39A6 Correlate With Tumor Genomic Instability

Part 1 SLC39A6 involve in tumor genomic instabilityBackground and purposeThe major cause of cancer genomic instability is abnormal chromosome division of tumor cells, which Promote tumor progression and deterioration. This study was to observe SLC39A6 involve in tumor genomic instability.MethodsUsing lentivirus siRNA knockdown SLC39A6 in Hela cells, detect colony formation ability after paclitaxel treatment, while the use of siRNA specific for SLC39A6 knockdown variant variant 1 and 2, when knockdown SLC39A6 variants 2 expression in cells detect the toxic drug sensitivity, test the cell cycle changes in drug-induced cycle arrest, by marking the mitosis specific protein by H3P, knockdown SLC39A6 variants 2 expression in cells decreased expression of cell cycle 2 due to toxicity of drugs on the block M Delay change. The cells were synchronized at mitosis after the detection of variant 2 knockdown SLC39A6 expression of the process of post-mitotic rate. X-chromosome fluorescence in situ hybridization detection of knockdown SLC39A6 variants 2 expression in cells chromosomal instability Using real-time quantitative PCR and western detection SLC39A6 variant 2 expression cyclical changes.ResultsLentivirus siRNA knockdown SLC39A6 in Hela cells, paclitaxel treated cells increased colony formation, while the use of siRNA specific for SLC39A6 knockdown variant 1 and variant 2 knockdown the expression of SLC39A6, the cells less sensitive to toxic substances, toxic drug-induced cell cycle to reduce cycle arrest, by marking the mitosis specific protein H3P knockdown SLC39A6 variant 2 expression, reduce drug-induced toxicity reduced M phase arrest. The cells were synchronized at mitosis, knockdown expression of SLC39A6, after knockdown variant 2 accelerated the process of mitosis. X-chromosome fluorescence in situ hybridization detection knockdown variant of SLC39A6 chromosomal instability,in real-time quantitative PCR and western SLC39A6 variant 2 expression in cycles.ConclusionSLC39A6 involved in mitotic checkpoint regulation and promote tumor increased genomic instability.Part2 H2B- RFP fusion protein observe the process of tumor cell mitosis in vivoBackground and purposeThe major cause of cancer genomic instability is abnormal chromosome division of tumor cells, which Promote tumor progression and deterioration. This study was to better observe the process of tumor cell mitosis in vivo use H2B-RFP fusion protein.MethodsPlasmid which Carry H2B-RFP fusion gene was transiently transfected into human cervical cancer cell line Hela. and then screen the positive clone by drug resistant. Using in vivo culture system of laser scanning confocal microscope for recording the cell cycle of Stabilizing Hela cell.ResultsObtain Hela cell line of Stabilizing express H2B- RFP fusion protein., which do not affect the cell cycle compare with the parental Hela cells and can observe the process of tumor cell mitosis.ConclusionH2B- RFP fusion protein can sensitive observe the tumor cell chromosomes And Track the process of tumor cell mitosis in vivo.Part 3 Isolation and identification of Breast cancer associated fibroblastsBackground and purposeIsolation and Identification of breast cancer associated fibroblasts and their paired normal breast fibroblasts. And explore Their biological differences。MethodsUsing collagenase digestion to obtain primary breast cancer associated fibroblasts and their paired normal breast fibroblasts, Cell morphology was observed through microscope、epithelial and fibroblast markers was observed by Immunofluorescence、Their biological differences observed through MTT and western.ResultsObtain breast cancer associated fibroblasts and their paired normal breast fibroblasts, both of them express low level of cytokeratin and high level of Vimentin, and they have some differences in biological properties.Conclusion Compared with the paired normal breast fibroblasts, breast cancer associated fibroblasts express 1 high level of a-SMA, and have much greater growth rate.Part4 Breast cancer associated fibroblasts secrete HGF promote MCF-7 invasion in vitroBackground and purposeExplore the influence of breast cancer associated fibroblasts (CAF) in breast cancer cell line MCF-7 migration and invasion, investigate whether HGF involve in this process.MethodsObtain primary breast CAF and their paired normal breast fibroblasts(NF) by collagenase digestion.On the basis of the co-culture, compare the migration and invasion capacity in breast cancer cell line MCF-7 between CAF and NF by Transwell, detect the difference of HGF expression between them by ELISA, using RNA interference technology to knock down the secretion of HGF in CAF, and then investigate the changes of migration and invasion capacity in MCF-7 by Transwell.Resultswe isolate high-purity CAF and NF, CAF has stronger ability in promoting MCF-7 migration and invasion compare to NF. ELISA results shows that CAF secrete higher HGF, the capacity of MCF-7 migration and invasion has been declined after knock down the secretion of HGF in CAF by RNA interference,ConclusionCAF can promote MCF-7 invasion though HGF in vitro.