Based on TCGA, GEO big data or customer samples, we carried out a variety of experiments to verify the cellular, in vivo and molecular mechanisms of the selected new functional genes, in order to provide a preliminary data basis for the determination of follow-up antineoplastic drug targets and biomarkers.
The function of the target gene was verified at the tumor cell line and animal level, and the molecular mechanism of the target gene was discussed from downstream molecular discussion, interaction protein screening, DNA methylation detection, acetylation, ubiquitin, autophagy, dryness, senescence and other hot mechanisms.
1. CRISPR-CAS9 knockout target gene for functional verification: the target cells infected by sgRNA and CAS9 lentivirus were constructed, the knockout targets were screened by mismatch enzyme method, and the knockout efficiency was verified by WB. The corresponding experiments were selected from MTT, BrdU, EDU, apoptosis, caspase3/7 activity detection, proliferation or apoptosis marker WB detection, clone formation, cell cycle and other proliferation-related functional experiments or scratch healing, Transwell migration, Transwell invasion and other metastasis-related experiments.
2. Objective to verify the function of gene in vivo: the target gene knockout tumor cells and control tumor cells were prepared, and the tumor models were constructed by subcutaneous tumor formation in nude mice or by tail vein injection. record the tumor growth curve, wait for the tumor to grow to a certain extent, kill the mice, take the tumor tissue, measure and analyze the data.
3. Downstream molecular mechanism study 1: Whole gene expression profiling microarray to detect downstream differential genes in target gene knockout and control groups and bioinformatics analysis of differential genes. The downstream pathways of the target genes were analyzed by qPCR and WB validation.
4. Downstream molecular mechanism study 2: after knockout of the target gene in tumor cell lines, panel, a classical pathway molecule related to proliferation or metastasis, was selected for WB detection, and the classical molecular mechanism downstream of the target gene was analyzed.
5. Screening of gene interaction proteins: screening the interaction proteins by co-IP mass spectrometry, constructing a vector fusing 3 x FLAG tag (3 x FLAG-bait) at the N-terminal of the target gene (bait), preparing lentivirus, infecting cells, screening over-expressed stable strains and control stable strains, collecting cell proteins, IP,SDS-PAGE electrophoresis and Coomassie brilliant blue staining with Anti-Flag antibody, cutting the differential bands of the target group compared with the control group. The peptides extracted by enzymolysis were tested and analyzed by LC-MS/MS, and the proteins were identified by database search. The interacting proteins were analyzed and screened, and the co-IP binding was verified by the specific antibodies of candidate interacting proteins.
6. Intracellular localization of the target gene: the distribution of the target gene in the cell was detected by immunofluorescence.
7. Study on the mechanism of DNA methylation direction: the methylation detection of the target gene and the mechanism of downstream methylation of the target gene.
8. Research on dry mechanism: the effect of target gene knockout on tumor cell dryness was detected by 3D culture stem cell ball forming experiment, and the difference of marker expression of several tumor stem cells under the condition of target gene knockout was detected by immunoflow fluorescence.
9. Research on the mechanism of aging: the effect of target gene knockout on aging was explored by SA-b-gal staining, and the expression differences of several aging-related marker under the condition of target gene knockout were detected by qPCR.
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