Construction of Recombinant Plasmid of Human RIP3 Gene and Its Effects on the Death of Breast Cancer MCF7 Cells / 天津医药

ABSTRACT

Objective To construct the recombinant RIP3 over-expressed plasmids and transfect them in breast cancer MCF7 cells, and identify the expression and localization of fusion protein, as well as its effect on the death way of MCF7 cells. Methods The expression levels of RIP3 mRNA in four breast cancer cell lines and normal mammary epithelial cells were detected by reverse transcription polymerase chain reaction (RT-PCR). The RIP 3 coding sequence was amplified by polymerase chain reaction and subcloned into mCherry vector to construct recombinant plasmids. The plasmids were transfected into MCF7 cells by lentivirus after DNA sequencing, then screened by basticidin (4 mg/L) for 1 week. The efficiencies of RIP3 expression were validated by Western blotting assay. The death way of mCherry-RIP3-MCF7 cells was observed under the treatment of TNF-αand Z-VAD-FMK. Results The lowest expression of RIP3 mRNA was found in MCF7 cells. The sequencing results validated the well recombinant plasmids. The expression of mCherry-RIP3 fusion pro-tein with a molecular weight of 85 ku was detected by Western blot assay. The mCherry-RIP3 expression enhanced the sensi-tivity of MCF7 cells to TNF-αand Z-VAD-FMK induced cell death. Conclusion The recombinant RIP3 over-expressed plasmids were successfully constructed, and the stable MCF7 cells with ectopic RIP3 transfection were obtained. The mCher-ry-RIP3 fusion protein was expressed in the cytoplasm and was conformed to mediate TNF-αinduced necroptosis.