| Part I.Bioinformatic analysis was used to screen for TTK genes related to cell cycle in TNBC genes associated with rapid proliferation.[Objective]Differential expression genes between triple negative breast cancer(TNBC)and non-TNBC were screened by bioinformatic analysis,and cell cycle-related signaling pathways strongly correlated with cell proliferation were analysed to obtain target genes strongly correlated with rapid proliferation of TNBC.[Methods]1.The Limma method was used to compare differentially expressed genes between TCGA and METABRIC for TNBC and non-TNBC,resulting in the identification of gene clusters with significantly higher expression in TNBC.By overlapping,candidate genes with simultaneous significantly increased expression were discovered;2.The Database for annotation visualization and integrated discovery(DAVID,https://david.ncifcrf.gov/)was used to analyze the biological processes(GO_BP)of candidate genes through enrichment analysis;3.The Pearson correlation analysis was used to investigate the correlation between candidate genes and tumor proliferation markers MKI67,PCNA;4.The STRING(https://string-db.org/)was used to obtain protein-protein interaction(PPI)networks for candidate genes,and the Cytoscape software and the Maximal Clique Centrality(MCC)were used to identify hub genes from the networks;5.Gene Set Enrichment Analysis(GSEA)was used to analyze the enrichment genes of cell cycle and mitotic spindle pathways,to obtain the target gene through comprehensive analysis;6.bc-Gen Ex Miner v4.8 was used to analyze the expression of target genes and their relation to breast cancer patients’ survival;7.The m RNA and protein expression levels of the target genes were determined in various breast cancer cell lines using Real-time Quantitative Polymerase Chain Reaction(RT-q PCR)and Western Blot(WB);8.Graphpad Prism v9 was used for statistical analysis,with three independent repetitions.The results were expressed as mean ± SD(mean ± SD),and p <0.05 was considered statistically significant.[Results]1.TCGA and METABRIC identified 1293 and 199 genes respectively with significant upregulation(Fold change、FC>2,p<0.05)in TNBC,of which 186 were upregulated in both datasets;2.GO_BP enrichment analysis identified 40 TNBC-specific genes associated with five proliferation-related biological processes;3.18 genes highly correlated with Ki-67 were identified using Pearson correlation analysis;4.20 hub genes were identified through PPI screening;5.Analysis of 2,3,and 4 yielded 17 common candidate genes,of which GSEA enrichment analysis determined TTK to be the target gene;6.TTK was significantly higher expressed in TNBC tissue and high Ki-67 subgroup(p<0.05)with poorer survival benefit for TNBC patients(p<0.05);7.TTK expression was significantly higher in TNBC cell lines than non-TNBC cell lines(p<0.05).[Conclusion]1.TTK could promote TNBC cell proliferation via regulation of mitosis;2.TTK has potential as a TNBC-specific biomarker,providing direction for further exploration of TTK’s role in regulating TNBC proliferationPart II.Research into the mechanism of TTK regulating triple negative breast cancer proliferation[Objective]To investigate the relationship between TTK and cell proliferation in different TNBC cell lines;tp examine the effects of TTK inhibitors(TTKi)on the proliferation of different TNBC cell lines and further validate the effect of TTKi in BALB/C Nude mice xenograft model..[Methods]1.si RNA was used to knockdown TTK expression in MDA-MB-231 and BT-549 TNBC cell lines,which was verified by RT-q PCR and WB;2.The IC50 of TTKi(AZ-3146,BAY-1217389)was measured using MTT in different TNBC cell lines;3.MTT,EdU and colony formation assays were used to investigate the proliferation of TNBC cell lines;flow cytometry was used to measure cell cycle,and immunofluorescence cofocalization was used to observe mitotic catastrophe(MC)following TTKi treatment;4.A BALB/C nude mouse xenograft model was constructed using MDA-MB-231 and different drug concentrations were administered orally on a daily basis.At the experiment endpoint,tumor weights and body weight of the mice were monitored along with Immunohistochemistry(IHC)analysis of TTK,MAD1L1,BUB1 B,and Ki-67 expressed in the tissues;5.Graphpad Prism v9 was used for statistical analysis,with three independent repetitions.The results were expressed as mean ± SD(mean ± SD),and p <0.05 was considered statistically significant.[Results]1.Knockdown of TTK significantly inhibited proliferation of MDA-MB-231 and BT-549 cells,blocking their cell cycle in the G2/M phase;2.TTKi blocked G2/M phase,induced MC and suppressed the rapid proliferation of TNBC cell lines;3.In vivo,NC group’s tumor volume growth was significantly faster than AZ-3146(p<0.05)and BAY-1217389(p<0.05)groups,and its final weight was significantly heavier than AZ-3146(p<0.05)and BAY-1217389(p<0.05)groups.No significant statistical differences were found in body weights of NC and TTKi groups before and after experiments(p > 0.05);4.The expression of TTK,MAD1L1,BUB1 B,and Ki-67 were significantly lower in AZ-3146 and BAY-1217389 groups than NC group(p<0.05)in IHC of xenograft tumors.[Conclusion]1.Functional inhibition of TTK causes mitotic errors and induces characteristic MC to inhibit the proliferation of TNBC cell lines;2.TTK inhibitor-induced mitotic catastrophe could be a key point of treatment for TNBC;3.Various TTKi applications in animal studies showed anti-tumor effects,making it a promising drug option for TNBC treatment.Part III.Mechanisms of TTK regulation of proliferation in triple-negative breast cancer[Objective]Exploring the mechanisms of the core genes related to the spindle assembly checkpoint(SAC)mitotic checkpoint complex(MCC)and the mechanism of SAC disruption-induced MC,and mining the transcription factors with regulatory roles of TTK.[Methods]1.WB showed TTK and its downstream MCC core genes MAD1L1 and BUB1 B in MDA-MB-231 and BT-549 after knocking down TTK;2.WB detection of TTK,MAD1L1,BUB1 B and MC-related regulatory factors protein expression in different TNBC cell lines after TTKi application.;3.ALLGEN and JASPAR predicted potential TF binding sites in the TTK promoter region,which was confirmed by dual luciferase assay;4.Point mutation of key sites in the TTK promoter region was performed and dual luciferase assay was used to detect the changes in fluorescence before and after the mutation;5.Chromatin Immunoprecipitation(Ch IP)combined with q PCR was used to identify transcription factor regulatory proteins bound to TTK promoter region.6.A total of 203 tumor tissue samples from patients with breast cancer at Sun Yat-sen Memorial Hospital were collected and analyzed by IHC to detect TTK and its upstream regulatory transcription factors in different subtypes of breast cancer,and their expression was correlated with TNBC patient prognosis;7.Graphpad Prism v9 was used for statistical analysis,with three independent repetitions.The results were expressed as mean ± SD(mean ± SD),and p <0.05 was considered statistically significant.[Results]1.TTK depletion resulted in a decrease of MAD1L1 and BUB1 B protein expression(p<0.05);2.TTKi treatment down-regulated protein expression of TTK,MAD1L1,BUB1 B,Caspase2,and Cyclin B1(p<0.05)while inducing up-regulation in γ-H2 AX levels(p<0.05);3.TTK transcript upstream 50 bp region showed significant reduction in dual luciferase intensity(p<0.05);4.After mutation of the CCAAT binding site,the fluorescence intensity of the dual luciferase decreased significantly(p<0.05)5.NFYA specifically binds to the CCAAT region of the TTK promoter(p<0.05);6.ChIP-qPCR was used to identify transcriptional factors and regulatory proteins bound to TTK promoter region;5.Clinical tumor samples showed that NFYA and TTK expressions were significantly higher in TNBC than in other subtypes(p<0.05),bringing worse survival benefits to TNBC patients(p<0.05)..[Conclusion]1.Functional inhibition of TTK downregulates MCC coregulators MAD1L1 and BUB1B,abrogates SAC signaling,causes DNA damage,and leads to proliferation defects;2.NFYA transcription factor regulates TTK transcription initiation through binding to CCAAT sites in the TTK promoter region of-300 to-50. |
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