Application of TUFT1 gene, protein target and inhibitor in breast cancer
A breast cancer, inhibitor technology, applied to TUFT1 gene and protein targets and inhibitors in the field of breast cancer
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Embodiment 1
[0019] Expression of TUFT1 in samples of breast cancer patients
[0020] Obtain specimens from patients with invasive breast cancer in clinical breast surgery; immunohistochemical experiments and result judgment;
[0021] Bake the paraffin sections in a constant temperature oven at 60°C for 20 minutes—soak in xylene twice for 10 minutes—soak in absolute ethanol for 5 minutes—soak in 95% ethanol for 5 minutes—soak in 75% ethanol—wash in PBS for 3 times, each time for 3 minutes—3% h 2 o 2 Indoor resting for 10 minutes—washing with PBS for 3 times, each time for 3 minutes—antigen restoration, 10 minutes—adding goat serum blocking solution dropwise, room temperature for 30 minutes,—after shaking off excess liquid, add primary antibody dropwise (1:100), refrigerated overnight at 4°C—PBS Wash 3 times, 3 minutes each time—add appropriate amount of secondary antibody, then stand still in the room for 30 minutes—wash 3 times with PBS, 3 minutes each time—DAB color development for 10 ...
Embodiment 2
[0030] Functional cancer-promoting role of TUFT1 in breast cancer cell lines
[0031] In this experiment, three human breast cancer cell lines were used: MCF-7 (Chinese Academy of Sciences), MDA-MB-231 (American Type Culture Collection, USA), T-47D (American Type Culture Collection, USA), and three cell lines were grown in RPMI-1640 ( containing 10% serum and 1% double antibody) culture medium. In addition, a strain of human mammary epithelial cells: MCF-10A (Chinese Academy of Sciences) was used to cultivate in DMEMF12 (containing 5% horse serum) culture medium;
[0032] RNA interference (RNAi) technology is used to select lentiviral vectors with high infection efficiency for breast cancer cell lines T47-D and MDA-MB-231, and to infect breast cancer cell lines with lentiviral vectors to produce persistent TUFT1 gene silencing effects. Real-time-PCR and western blot were used to detect the knockout level of TUFT1, and then Cellomics was used to detect cell growth, MTT was us...
Embodiment 3
[0048] TUFT1 related signaling pathway mechanism experiment in breast cancer cells
[0049] In this experiment, PathArray was used to analyze the human gene expression profile chip, to explore the possible related signaling pathways of TUFT1 in breast cancer, to find its downstream expression differential genes, and to conduct protein verification on the differential genes of related signaling pathways, to further explore its role in breast cancer. mechanism of action;
[0050] Breast cancer MDA-MB-231 cells were infected by lentiviral vectors carrying Scr-siRNA and TUFT1-siRNA;
[0051] The number of genes with significant differential expression, the number of genes up-regulated by TUFT1-siRNA combined with Scr-siRNA is less than 300, and the number of genes down-regulated is more than 800;
[0052] At the same time, through the in-depth analysis of the differential genes related to the MAPK signaling pathway, 5 genes with large differences were selected for protein verific...
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