Distinct Diagnostic And Prognostic Value Of STAT1 In Breast Cancer Patients And Its Effct On The Function Of Tnbc Cell Line

Part I Distinct diagnostic and prognostic values of Signal Transducer and Activator of Transcription genes expression in patients with breast cancerObjective: this study investigated the diagnostic and prognostic values of STAT family in breast cancer patients with TCGA.Materials and Methods: Our data were collected from the cancer genome database TCGA;clinical data were obtained from the UCSC database.GO and KEGG analysis of each member of STAT family were carried out by DAVID,and the interaction between genes and proteins was analyzed using Genemania and STRING.Differentially expressed genes were analyzed using DESeq packets.Patients were divided into high-and low-expression groups according to the 75% expressin values of each STAT gene.Survial data was calculated using multivariate Cox proportional hazard model.Pearson correlation analysis was used to select the genes related to STAT1 gene for pathway enrichment analysis.Result: Bioinformatics analysis showed that the molecular function of STAT family was related to the transcription of genes,and the enriched pathway of this family was related to tumor growth.In addition,gene-gene and protein-protein interaction networks confirmed that the STAT genes had solid homology as well as co-expression with one another at protein and gene level.STAT1,STAT3,STAT4,STAT5 A,STAT5B and STAT6 were differentially expressed in breast cancer and adjacent tissues(p<0.05).The recurrence-free survival rate and overall survival rate of these genes were corrected by multivariate regression analysis.It was found that STAT1 was associated with relapse-free survival rate of breast cancer(p =0.048,p<0.05),but not with the overall survival rate of breast cancer(p = 0.318,p>0.05).DAVID analysis showed that the enriched pathways were cell adhesion molecule(CAMs),natural killer cell-mediated cytotoxicity,T cell receptor signaling pathway,Toll-like receptor signaling pathway,fine cell adhesion molecule-mediated cytotoxicity,Toll-like receptor signaling pathway,Toll-like receptor signaling pathway,Cytokine-cytokine receptor interaction and chemokine signaling pathway.Conclusion: we found that STAT1,STAT3,STAT4,STAT5 A / 5B and STAT6 in were differentially expressed in breast cancer and adjacent tissues.STAT1 and STAT2 have potential prognostic value in the prognosis of breast cancer.Multivariate regression analysis and nomogram showed that the patients with high expression of STAT1 had better prognosis.STAT1 and its related genes were enriched in immunity,cytokine-cytokine receptor interaction and chemokine signaling pathway.Further research needs to be verified by later experiments.Part II The expression of STAT1 in breast cancer patients and its clinical significanceObjective: The aim of this part is to investigate the expression of STAT1 in breast cancer and adjacent tissues,and to analyze the relationship between the expression of STAT1 and clinicopathological features in patients with breast cancer.Materials and Methods: A total of 32 cases of breast tumor resection were performed in the department of Gastrointestinal Gland Surgery,the first first affiliated Hospital of Guangxi Medical University from January 2017 to June2018.Reverse transcriptase polymerase chain reaction(RT-PCR),Western Blot(WB)and immunohistochemistry(IHC)were used to detect the expression of STAT1 in breast cancer and adjacent tissues,and to study the effect of clinical factors on the expression of STAT1 in breast cancer and adjacent tissues.Result: The expression of STAT1 m RNA in breast cancer tissues was higher than that in adjacent tissues(P < 0.05);WB and IHC showed that the expression level of STAT1 protein in breast cancer tissues was significantly higher than that in adjacent tissues(P < 0.01).The relative expression levels of STAT1 m RNA in Ki-67 positive and ER negative patients were lower than those in Ki-67 negative and ER positive patients,respectively(P < 0.05).In addition,there was no significant difference in the expression of STAT1 m RNA among different age,tumor size,nationality,body mass index(BMI),lymph node metastasis,pathological grade and other breast cancer patients(P > 0.05).Conclusion: STAT1 is an important molecular marker of breast cancer.It plays an important role in the molecular mechanism of the occurrence and development of breast cancer,and plays an important role in detecting breast cancer.Part III The in vitro experiment of the effect of Si-STAT1 on breast cancer cell line MDA-MB-231Objective: To investigate the effect of silencing STAT1 on proliferation,cell cycle and apoptosis of triple negative breast cancer cell line MDA-MB-231.Materials and methods: 293 cells were transfected with four STAT1 silencing genes and control plasmids.After 72 ~ 96 hours of lentivirus infection,MDA-MB-231 cells were screened with puromycin and continued to be cultured.After fluorescence microscope observation,the fluorescence of the full screen was 80%-90%.Western Blot was used to detect the four plasmids with the best silencing effect in the cell line.The cell proliferation rate was detected by CCK8,and the changes of cell cycle and apoptosis rate were detected by flow cytometry.Results: Western Blot assay showed that the silencing effect of sh-STAT1-33 plasmid was the best in transfected cell line MDA-MB-231,and the silencing rate was 75%.This stable STAT1 silencing 231 cell line was used for follow-up test.The results of cell proliferation test showed that there was significant difference in OD(450nm)between silent cell line and silent control cell line at12,24,36,48 hours(p < 0.01).There was significant difference in proliferation rate between STAT1 silent cell line and unintervened cell line at 12 and 36hours(p < 0.01).The apoptosis rate was 4.07% ±0.65% in the silent group,10.93% ±3.10% in the normal group and 9.98% ±3.48% in the negative control group.Compared with the normal group and the negative control group,the apoptosis rate of silent group was decreased.The results showed that the silencing of STAT1 gene inhibited the apoptosis of triple negative breast cancer cell lines(p < 0.05),which indicated that the down regulating of STAT1 gene inhibited the apoptosis of triple negative breast cancer cell lines.The results of cell cycle analysis showed that there was no significant difference in the percentage of G2 phase content between the three groups(p > 0.05)in the cell line 231.It showed that silent STAT1 was not associated with the cycle of triple negative breast cancer cells.Conclusion: in vitro experiments showed that in triple negative breast cancer MDA-MB-231,after silencing STAT1,the cell proliferation rate was accelerated and apoptosis was inhibited,which was not related to the cell cycle.Therefore,STAT1 may be a potential therapeutic target for triple-negative breast cancer.