Thesis (Ph.D.) - Cairo University - Faculty of Pharmacy - Department of Biochemistry

Background: TNFRSF13B, TACI, is a member of the TNF receptor superfamily; it plays a key role in cancer cell proliferation and progression. Method: Influence of silencing of human cytokine receptors on cell viability was screened by Luminescent Cell Viability Assay, after transfection with the siRNA library to find the maximum cell death superhits in both triple- negative MDAMB- 231 and triple-positive MCF-7 breast cells. The mode of cell death was investigated by dual DNA fluorescence staining. The expression of mRNAs of TACI, BAFF, BAFF-R, and APRIL was explored by qPCR. Immunocytofluorescence analysis was used to evaluate changes in TACI, Bcl-2, TNFR2, cyclin-D2, and PCNA. NF-kB p65, cell cycle, and necrosis/apoptosis (late and early) were analyzed by flowcytometry. Results: TACI was the most potent cytotoxic superhit resulting from highthroughput screening of the siRNA library, in both types of cells. Our findings indicated that silencing TACI receptor in both types of breast cancer cells led to significant cell death, after different intervals from siRNA transfection. Cell death mediators (TNF-R2, Bcl-2, and NF-mB p65) were significantly decreased after TACI silencing. The key factors for cell division (cyclin-D2 and PCNA) were significantly increased in silenced cells of both types but the cell cycle was arrested before the completion of mitosis. Expression of BAFF, BAFF-R and APRIL mRNA in TACI-silenced cells showed significant upregulation in MDAMB- 231 cells, while only BAFF-R and APRIL showed significant downregulation in MCF-7 cellsConclusion: TACI silencing can be a new and promising therapeutic strategy for mesenchymal-stem like triple- negative breast cancer subtype

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