ZNF213 Negatively Controls Triple Negative Breast Cancer Progression Via Hippo/YAP Signaling

BackgroundBreast cancer is one of the most common malignancies among women,which can be divided into four types according to molecular typing,among which three-negative breast cancer(TNBC)accounts for 20% of all patients.Compared with estrogen-receptor-positive breast cancer,which can be effectively controlled by endocrine therapy,TNBC is more invasive and has a worse prognosis,and TNBC has a higher rate of genomic mutation,gene amplification and deletion.It contains a variety of cancer-suppressing gene mutations,including p53 and Rb.Therefore,there is still a lack of effective targeted therapy for triple negative breast cancer,and it is urgent to understand the biological effects of TNBC and new therapeutic targets.Zinc finger Domains(ZNF)are small protein motifs containing about 50 residues.The zinc finger domain regulates protein-protein interactions.Although studies have shown that ZNF proteins can directly bind to DNA/RNA and regulate gene expression,some zinc finger proteins can mediate ubiquitination and regulate protein stability.Our current study confirms that ZNF213(zinc finger protein 213)is an important regulator of Hippo signaling.Targeted therapy ZNF213 may be a promising strategy for the treatment of triple negative breast cancer.ObjectiveMolecular biology research is expected to reveal the regulatory mechanism of ZNF213 protein on Hippo signaling pathway in triple negative breast cancer,and to reveal new mechanisms and drug targets for the clinical treatment of triple negative breast cancer.Methods1.qRT-PCR and Western blot were used to detect the silencing effect of ZNF213 in the transfection of triple negative breast cancer cell lines and to clarify the influence of ZNF213 on the target genes in the Hippo signaling pathway.2.The cell scratch test and trans-well test were used to clarify the effects of ZNF213 on the invasion and migration of triple negative breast cancer cells.3.Luciferase reporter gene technology was used to detect whether ZNF213 affected the transcription activity of YAP.4.Through the rescue experiment,YAP was silenced on the basis of ZNF213 silencing,changes of target genes in Hippo signaling pathway and changes of invasion and migration of triple negative breast cancer cells were observed.5.By inquiring TCGA and Kmplot databases and collecting clinical samples for immunohistochemistry,the expression of ZNF213 in the tissues of human triple negative breast cancer patients was determined.6.The cell localization of ZNF213 and YAP was determined by immunofluorescence assay,and the interaction between ZNF213 and YAP was verified by immunoprecipitation assay.To further verify the specific binding regions of ZNF213 and YAP,plasmids in different domains of ZNF213 and YAP proteins were constructed to detect the specific binding regions of the two proteins.7.Western blot experiment was used to verify the stability and stability mode of ZNF213 regulating YAP protein through MG132 assay,CHX assay,ubiquitination assay and other assays.Results1.The silencing effect of ZNF213 was verified by q RT-PCR and Western blot,and it was found that silencing ZNF213 significantly up-regulated several classic target genes in the Hippo pathway,while silencing ZNF213 significantly upregulated the protein level of YAP.2.Through cell scratch test and trans-well test,we found that silencing ZNF213 could promote the invasion and migration of triple negative breast cancer cells,while overexpressing ZNF213 could inhibit the invasion and migration of triple negative breast cancer cells.3.Luciferase reporter gene experiment proved that silencing ZNF213 could significantly promote the transcription activity of YAP,while overexpressing ZNF213 could significantly inhibit the transcription activity of YAP.4.Through rescue experiment,silencing YAP on the basis of silencing ZNF213,we found that the protein level of YAP caused by silencing ZNF213 was up-regulated and downregulated,the up-regulated target gene in Hippo signaling pathway was down-regulated,and the invasion and migration of triple negative breast cancer cells accelerated by silencing ZNF213 also slowed down.5.Through TCGA database,we found that the expression level of ZNF213 in triple negative breast cancer was higher than that in normal tissues,and the expression of ZNF213 was negatively correlated with the expression of target genes CTGF and CYR61 of YAP.Through the Kmplot database,we found that high expression of ZNF213 in triple negative breast cancer was associated with better prognosis,while high expression of YAP was associated with poor prognosis.Immunohistochemical results showed that the expression level of ZNF213 was negatively correlated with that of YAP.6.By immunofluorescence experiments,we found that ZNF213 and YAP main positioning in the nucleus,and through the Co-IP experiment found ZNF213 and YAP has direct interaction,in order to further verify the interaction of the two specific binding site,through building structure Domain of the two respectively,and then through the immune coprecipitation experimental validation of A combination of both parts are respectively in ZNF213 KRAB-A combination and YAP WW paragraphs.7.Through CHX experiment,it was found that silencing ZNF213 could prolong the half-life of ZNF213,while overexpressing ZNF213 could shorten the half-life of ZNF213.Through MG132 experiment and co-IP experiment,with ubiquitin plasmids such as K48 and K63,it was further proved that ZNF213 could promote K48 linked polyubiquitination of YAP,inhibit K63 linked polyubiquitination of YAP,and ultimately affect the stability of YAP protein.ConclusionsIn the triple negative breast cancer cell line,the KRAB-A segment of ZNF213 and the WW segment of YAP interact to promote K48 linked polyubiquitination of YAP,thus affecting the stability of YAP protein.