Based on TCGA, GEO big data or Creative Proteomics database or customer samples, analyze and verify the new functional genes that have not been reported in the tumors studied, providing a preliminary data basis for the identification of follow-up antineoplastic drug targets and biomarkers.
Through the patent database of key genes of Creative Proteomics disease, the candidate genes which have not been reported in the studied tumors are analyzed. Using lentivirus-mediated RNAi technology, several candidate genes were RNAi, and the infected cells were counted for 4-5 days through the Cellomics high connotation function screening platform, and the genes affecting cell proliferation were found. The clinical correlation of the candidate gene was verified by TCGA, GEO big data or customer samples, and at least three proliferation directions of the gene in one cell line (MTT/ cell cycle (PI/ flow) / apoptosis (Annexin V / flow) / BrdU/ clone formation, etc.) or two metastasis directions (scratch healing / Transwell migration / Transwell invasion, etc.) were completed.
Preliminary screening of functional genes: the candidate gene shRNA lentivirus was infected with target cells, and the expression of candidate genes expressed in target cells was down-regulated. The infected cells were counted for 4-5 days by Cellomics high connotation function screening platform, and the changes of cell proliferation (or scratch healing) of the cells with down-regulated candidate gene expression were observed compared with the control cells. Thus the genes that affect cell proliferation (metastasis) can be inferred.
Objective to verify the clinical correlation of functional genes: to verify the clinical correlation of target genes by TCGA, GEO big data or customer samples (qPCR or immunohistochemistry).
Objective to verify the cellular function of the gene: the target gene was silenced by shRNA lentivirus in the target cell, and the knockdown efficiency of the target gene in the cell was verified by qPCR and WB. The functional experiments of MTT/ cell cycle (PI/ flow) / apoptosis (Annexin V / flow) / BrdU/ clone formation or transfer direction (scratch healing / Transwell migration / Transwell invasion, etc.) were carried out.
Group: NC vs KD, with three multiple holes in each group. Promise the functional positive results of at least 4 proliferation directions (including HCS proliferation test) or 3 metastatic directions (including HCS metastasis test) in tumor target cells after knockout.
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