A Study On Phenotypic Effect Of Toxoplasma Gondii ROP16 Protein On Human Breast Cancer MCF-7 Cells And Its Mechanism

Objective The over-expression lentivirus vectors of Toxoplasma gondii ROP16 protein of type I（RH）,type II（ME49）and type III（VEG）were constructed and transfected into human breast cancer MCF-7 cells to explore the effects of Toxoplasma gondii ROP16 protein on the phenotype of MCF-7 cells and its mechanism,and to conduct transcriptional spectrum analysis,in order to provide reference for the clinical treatment of hormone receptor positive breast cancer and the anti-tumor research of parasitic secreted protein.Methods 1.The constructed lentiviral vectors p HBLV-CMV and p HBVV-CMV ROP16_I/II/IIIwere transfected into human breast cancer MCF-7 cells.Real time PCR and Western blot were used to detect the m RNA and protein expression of ROP16.2.CCK-8 and flow cytometry were used to detect cell proliferation,apoptosis and cycle,while cell scratching and Transwell were used to detect cell migration and invasion ability.3.the expressions of apoptosis,cycle-related proteins and JAK-STAT pathway-related proteins were detected by Real time PCR and Western blot,so as to clarify the mechanism of ROP16 in regulating the phenotype of MCF-7 cells.4.Each group of cells is lysed by Trizol and then subjected to RNA-Seq analysis.Results 1.The human breast cancer MCF-7 cells were successfully transfected with the lentivirus vector in the overexpression group,and in the MCF-7-HBLV,MCF-7-ROP16_I,MCF-7-ROP16_II,and MCF-7-ROP16_IIIcells groups were observed to emit strong green fluorescence under a fluorescence microscope;Real time PCR and Western-blot results showed that the expression levels of ROP16 m RNA and protein in the MCF-7-ROP16_I,MCF-7-ROP16II,and MCF-7-ROP16III cell groups were increased.2.CCK-8 results showed that compared with the MCF-7 and MCF-7-HBLV cell groups,the proliferation of MCF-7-ROP16I,MCF-7-ROP16II and MCF-7-ROP16III cell groups was significantly inhibited.The results of flow cytometry showed that in the MCF-7-ROP16Ⅰ,MCF-7-ROP16Ⅱand MCF-7-ROP16Ⅲcell groups,apoptosis was significantly increased and the cell cycle was stalled in stage S;The results of Transwell and cell scoring experiments showed that the cell invasion and migration abilities of MCF-7-ROP16Ⅰ,MCF-7-ROP16Ⅱand MCF-7-ROP16Ⅲcell groups were significantly inhibited.3.The results of Western-blot and Real time PCR showed that compared with MCF-7 and MCF-7-HBLV cell groups,the expression of pro-apoptotic factors Bax,P53,Caspase-9 and Caspase-3 in MCF-7-ROP16I,MCF-7-ROP16II,and MCF-7-ROP16III cell groups was significantly increased（P<0.05）;The expression of anti-apoptotic protein Bcl-2 decreased significantly.Compared with MCF-7 and MCF-7-HBLV cell groups,the expression of cyclin-dependent kinase inhibitor P21（P<0.05）was significantly increased in the MCF-7-ROP16I.MCF-7-ROP16II.and MCF-7-ROP16III.Groups,while the expression of cyclin A1（Cyclin A1）and cyclin-dependent kinase 2（CDK2）decreased significantly.4.Western-blot results showed that compared with the MCF-7 and MCF-7-HBLV cell groups,the expressions of JAK-STAT pathway related proteins JAK1,STAT3,STAT6,p-STAT3,and p-STAT6 in the MCF-7-ROP16I and MCF-7-ROP16III cell groups were significantly increased,while there was no significant change in the MCF-7-ROP16II cell group.5.Cell sequencing results showed that compared with the MCF-7 and MCF-7-HBLV cell groups,the KEGG-enriched Pathway differences in the MCF-7-ROP16I,MCF-7-ROP16II and MCF-7-ROP16III cell groups were manifested in the Pathways in cancer,PI3K-Akt,and MAPK,involving gene changes in JAK1,JAK3,STAT6,CDK6,STAT3,MAP4K3,MAP3K11,and MYD88.Conclusions 1.TypeⅠ/Ⅱ/ⅢROP16 protein overexpression lentivirus vector is successfully transfected into human breast cancer MCF-7 cells.2.TypeⅠ/Ⅱ/ⅢROP16 protein inhibits the cells proliferation,invasion and migration,and induces cell cycle arrest and apoptosis.3.TypeⅠandⅢROP16 protein affects the cell phenotype of MCF-7 cells such as proliferation,apoptosis,cycle,migration and invasion by regulating JAK-STAT signaling pathway.However,type II ROP16 protein has no such effect.4.By transcriptomics analysis,the MCF-7 cells stably expressing ROP16 show gene expression changes in pathways such as Pathways in cancer,PI3K-Akt,and MAPK.