Effect Of Zinc Finger 3 Of GLIS Family On Malignant Behavior Of Triple Negative Breast Cancer Through NF-κB Signaling Pathway

Breast cancer（BC）is a highly heterogeneous disease,which is considered to be the most common type of cancer among women in the world,and its mortality is only second to lung cancer.Triple negative breast cancer（TNBC）is a special subtype of breast cancer,which is characterized by no expression of estrogen receptor（ER）,progesterone receptor（PR）or human epidermal growth factor receptor 2（HER-2）.This disease accounts for10-20%of all breast cancer.Because TNBC is prone to recurrence,poor prognosis and high risk of death,it poses a great threat to women’s health.Therefore,the selection and research of effective treatment methods for TNBC has always been the focus of our work.GLIS3 is a member of the GLIS subfamily of Krüppel-like zinc finger transcription factors,which can promote or inhibit the expression of target genes.Studies have shown that GLIS3 plays a key role in the regulation of many diseases and is related to the occurrence and development of various diseases,including hypothyroidism and tumors.The relationship between NF-κB signaling pathway and tumor has been partially reported.Studies have shown that the abnormal activation of NF-κB signaling pathway in TNBC leads to the over-expression of downstream targets,such as the over-expressed anti-apoptosis gene,which promotes tumor progression and chemotherapy resistance,and promotes tumor metastasis of TNBC by regulating epithelial-mesenchymal transition（EMT）.However,the role of GLIS3 in TNBC is rarely reported in the literature.To sum up,the purpose of this study is to study the important role of GLIS3 in the development of TNBC tumor and the relationship between GLIS3 regulation and NF-κB signaling pathway.Part one Expression of GLIS3 in triple negative breast cancer and its relationship with clinicopathological characteristicsObjective:This part aims to explore the relationship between the expression of GLIS3 in triple negative breast cancer and clinicopathological features.Methods:1.Case selection:125 patients diagnosed with TNBC by pathology were randomly selected from the Department of Breast Surgery,the Fourth Hospital of Hebei Medical University from January to December 2014.Among them,53 patients were in accordance with medical records,complete follow-up data and complete tissue specimens.2.GLIS3 criteria:3 visual fields were selected under the microscope according to each pathological section,and staining intensity scores were performed for each visual field（grade 5:0=negative,1=weak,2=medium,3=strong,4=obvious）and the rate of positive cells were calculated by quantitative calculation.The final score of all the selected slides was sorted,and the middle digit was selected.Higher than the median was regarded as high GLIS3 expression,lower than or equal to the median was regarded as low GLIS3 expression.Immunohistochemistry（IHC）was assessed while GLIS3 immunohistochemistry was assessed in TNBC and adjacent normal tissues.The relationship between the expression of GLIS3 protein in breast cancer tissues and the age,menstrual status,tumor size,clinical stage,histological grade,pathological type,axillary lymph node metastasis,vascularoma thrombin,TP53,Ki-67 and other clinicopathological indicators of breast cancer patients was analyzed.3.Prognostic analysis:Cox regression model and Kaplan-Meier survival curve were used to analyze the risk factors that might influence disease-free survival and overall survival of triple negative breast cancer patients.4.Follow-up visit:A comprehensive follow-up method combining telephone follow-up,outpatient review and hospitalization was adopted.Follow-up intervals were once every 3 months in the first 1-2 years,once every 6 months in the third to fifth years,and once a year after 5 years.The end point of follow-up was January 31,2023.disease-free survival（DFS）was the time from operation to recurrence,metastasis or follow-up endpoint.overall survival（OS）was the time from pathologically confirmed breast cancer to death or the end point of follow-up.5.Statistical processing:R 4.2.2 software was used to analyze the data,Chi-square test was used to analyze the relationship between GLIS3 and clinicopathological features,and Cox regression model was used to analyze the risk factors that may affect the prognosis of triple negative breast cancer patients.Kaplan-Meier method was used to map the relationship between GLIS3 and disease-free survival and overall survival of patients with triple-negative breast cancer,and the survival rate was calculated by Log-rank.Results:1.A total of 53 patients met the requirements of the experiment,including 7 cases of recurrence,metastasis and death,and 44 cases of disease-free survival.2.GLIS3 expression in TNBC tissues and adjacent normal tissues:Compared with adjacent normal breast tissues,GLIS3 protein was highly expressed in TNBC tissues（Fig.1）.3.The relationship between GLIS3 expression in breast tissue and clinicopathologic parameters:The results showed that GLIS3 expression was significantly increased in higher tumor stage and larger tumor volume（P<0.05）.GLIS3 expression level was not significantly correlated with age,menstrual status,lymph node metastasis,pathological type,vascular tumor thrombus,Ki-67,TP53,and recovery（Table 1）.There was no significant correlation between GLIS3 expression level and histological grading of infiltrating ductal carcinoma（Table 2）.4.Survival analysis:Cox regression model was used to analyze the disease-free survival of patients.Univariate results showed that lymph and metastasis,TNM staging,GLIS3 expression were correlated with disease-free survival of triple-negative breast cancer patients（P<0.05）.According to the analysis of multi-factor results,lymph node metastasis,TNM staging and GLIS3 expression level were not significantly correlated with disease-free survival of patients with triple-negative breast cancer（P<0.05,Table 3）.Cox regression model was used to analyze the overall survival of patients,and the single factor results showed that the TNM stage was correlated with the overall survival of patients with triple-negative breast cancer（P<0.05）.According to the analysis of multi-factor results,the TNM stage was correlated with the overall survival of triple negative breast cancer patients（P<0.05,Table 4）.According to the survival curve plotted by Kaplan-Meier method,the disease-free survival of patients with high GLIS3 expression was significantly lower than that of patients with low GLIS3 expression（P<0.05,Fig.2）.However,there was no significant difference in overall survival between patients with high GLIS3 expression and patients with low GLIS3expression（P<0.05,Fig.3）.Conclusions:1.GLIS3 protein was highly expressed in TNBC tissues.2.In clinicopathologic analysis,tumor size and TNM stage were correlated with high GLIS3 expression.3.High expression of GLIS3 protein predicts poor prognosis.Part two Effect of GLIS3 on biological behavior of triple negative breast cancer cellsObjective:Through the first part of the study,it has been confirmed that GLIS3 protein is highly expressed in tumor tissue of TNBC patients,and it is significantly related to tumor stage and tumor size of patients.This part will analyze the influence of GLIS3 on the malignant behavior of TNBC cells by silencing or overexpressing GLIS3 gene in different TNBC cells.Methods:1.TNBC cell lines（MDA-MB-436,MDA-MB-468,MDA-MB-231,Hs578t）and human normal mammary epithelial cells（MCF-10A）were selected,and the expression level of GLIS3 protein in these cell lines was detected by Western blot.The expression level of GLIS3 protein is the highest in Hs578t cells and the lowest in MDA-MB-468 cells.2.Three GLIS3 silencing fragments GLIS3 si RNA-1,GLIS3 si RNA-2,GLIS3 si RNA-3 and control NC si RNA were constructed according to GLIS3gene sequence and transfected into Hs578t cells;GLIS3 overexpression plasmid GLIS3-OE and empty control vector（vector without fluorescence）were constructed and transfected into MDA-MB-468 cells.The experimental groups of Hs578t cells were Control,NC si RNA,GLIS3 si RNA-1,GLIS3si RNA-2,GLIS3 si RNA-3;MDA-MB-468 cells were divided into Control,Vector and GLIS3-OE.After 48 hours of transfection,the expression levels of GLIS3 gene and GLIS3 protein were detected by Real-time PCR and Western blot,respectively,to verify the influence of interference sequence and overexpression sequence on GLIS3 gene expression.3.Select the two fragments with the best silencing effect from the three GLIS3 silencing fragments for the experiment.MDA-MB-468 cells were divided into Vector and GLIS3-OE groups;Hs578t cells were divided into NC si RNA,GLIS3 si RNA-1,GLIS3 si RNA-2 groups.Inoculate cells in each group into 96-well culture plate,and the number of cells inoculated in each well is 3×10³cells,5 wells were set up,and after transfection for 0 h,24 h,48 h and 72 h,the proliferative ability of cells in each group was tested by CCK8 experiment.After 48 hours of transfection,the expression level of PCNA in each group of cells was detected by WB assay;The distribution of cell cycle in each group was analyzed by flow cytometry.4.Hs578t cells were selected and divided into NC si RNA,GLIS3si RNA-1 and GLIS3 si RNA-2 groups.48 h after transfection,the apoptosis level of cells in each group was analyzed by flow cytometry.The protein expression levels of pro-caspase-3,cleaved caspase-3,Bax,and B lymphoblastoma-2 gene（BCL2）were detected by WB assay.5.MDA-MB-468 cells were divided into Vector and GLIS3-OE groups.Hs578t cells were divided into NC si RNA,GLIS3 si RNA-1 and GLIS3si RNA-2 groups.24 h after transfection,Transwell assay was performed to detect the invasion and migration ability of cells in each group.48 h after transfection,the expression levels of E-cadherin,Vimentin and N-cadherin,which are related to cell invasion and migration,were detected by WB assay.Results:1.WB assay analyzed the expression level of GLIS3 protein in MCF-10A cells and TNBC cell lines MDA-MB-436,MDA-MB-468,MDA-MB-231,and Hs578t.The results showed that the level of GLIS3 in TNBC cell line was significantly higher than that in MCF-10A cells（P<0.05）.Therefore,MDA-MB-468 cells were selected for overexpression of GLIS3gene and silenced in Hs578t cells.2.The expression level of GLIS3 gene and protein in cells was detected by q RT-PCR and WB assay.The results of q RT-PCR showed that the expression level of GLIS3 gene in the GLIS3-OE group was significantly higher than that in the Vector group（P<0.01）;In Hs578t cells,GLIS3 gene expression level in GLIS3 si RNA group was significantly lower than that in NC si RNA group（P<0.01）.The experimental results of WB were consistent with the results of q RT-PCR,indicating that the transfection effect was ideal and the experiment could be continued.3.The results of CCK8 experiment showed that after overexpression of GLIS3 gene in MDA-MB-468 cells for 48 hours and 72 hours,the proliferation ability of cells was significantly enhanced（P<0.05）;On the contrary,the expression of GLIS3 gene was silenced in Hs578t cells,and the proliferation ability of cells was significantly reduced（P<0.05）.WB test results showed that overexpression of GLIS3 promoted the high expression of PCNA protein,while inhibition of GLIS3 expression significantly reduced the content of PCNA protein（P<0.01）.The cell cycle distribution of each group was detected by flow cytometry.The results showed that the number of cells in MDA-MB-468 cells was significantly increased in S phase（P<0.01）and decreased in G1 phase（P<0.05）;However,the inhibition of GLIS3 expression showed the opposite result.The number of G1 phase cells in Hs578t cells increased significantly（P<0.01）,while the number of S phase cells decreased（P<0.01）.4.The apoptosis level of Hs578t cells was detected by flow cytometry（Annexin V/PI staining）.The results showed that compared with NC si RNA group cells,interference with GLIS3 expression could significantly promote cell apoptosis,with a significant difference（P<0.01）;WB results showed that inhibiting the expression of GLIS3 significantly increased the expression level of Cleared-capase-3 and Bax,and decreased the expression of BCL2（P<0.01）.The expression level of pro-capase-3 did not change.5.Transwell experiment showed that the number of cell migration and invasion in GLIS3 overexpression group was more than that in vector group;On the contrary,the number of cell migration and invasion in GLIS3down-regulated group decreased significantly（P<0.01）.The results of WB experiment showed that the up-regulated GLIS3 significantly decreased the expression of epithelial marker E-cadherin and increased the expression of mesenchymal marker N-cadherin and Vimentin（P<0.01）.On the contrary,down-regulated GLIS3 enhanced the expression of E-cadherin and significantly inhibited the expression of N-cadherin and Vimentin（P<0.01）.Conclusions:1.The expression level of GLIS3 protein in TNBC cell line was significantly higher than that in normal breast cells.2.Intervention of GLIS3 expression in TNBC cells can significantly reduce the cell proliferation ability,make the cell cycle more stagnant in G1phase,promote cell apoptosis,reduce the migration and invasion ability of cells,and thus prevent the malignant behavior of tumor cells.Overexpression of GLIS3 has the opposite effect and can significantly promote the malignant behavior of cells.Part three GLIS3 phosphorylates P65 in triple negative breast cancer and NF-κB signaling pathway.Research on relationshipObjective:There is a correlation between NF-κB signaling pathway and tumor.Abnormal activation of NF-κB signaling pathway in TNBC leads to overexpression of downstream signaling targets,promoting tumor progression and chemotherapy resistance.Combined with the experimental results of the first two parts,this part will focus on exploring whether GLIS3 plays a regulatory role in TNBC through the NF-κB signaling pathway,so as to clarify the mechanism of GLIS3.Methods:1.MDA-MB-468 cells and Hs578t cells were grouped as in the second part.48 h after transfection,cell slides were prepared,and the nucleation of p65 in cells was detected by immunofluorescence.The levels of p65 and p-p65^Ser536in cells were detected by WB assay,and the p-p65/p65 values were calculated.DNA binding activity of NF-κB in nuclear was analyzed by EMSA assay.2.After transfection of MDA-MB-468 cells for 24 h,NF-κB inhibitor BAY11-7082 with concentration of 5μM was added for 24 h,and cell viability was detected by CCK-8 assay.Transwell assay was used to detect cell invasion ability.Results:1.Immunofluorescence staining showed that NF-κB p65 mainly exists in the cytoplasm of the carrier cell,while obvious NF-κB p65 staining can be observed in the nucleus of GLIS3 overexpression group.Compared with the negative control,the down-regulation of GLIS3 gene can significantly reduce the NF-κB p65 staining in the nucleus（P<0.01）.Western blot results showed that the expression level of p-p65 Ser536 protein was significantly increased after GLIS3 up-regulated compared with Vector group（P<0.01）,and the expression level of p-p65 Ser536 protein was significantly decreased after inhibiting GLIS3 expression（P<0.01）,while the expression level of p-65protein was not affected.2.The EMSA results showed that GLIS3 overexpression could significantly increase the band strength of NF-κB-DNA oligonucleotide complex,and confirmed that the binding of NF-κB to common DNA sequence increased when GLIS3 was up-regulated（P<0.01）.On the contrary,inhibition of GLIS3 gene expression could significantly reduce the band strength and binding of NF-κB-DNA oligonucleotide complex（P<0.01）.3.The biological behavior of the cells was detected by adding NF-κB inhibitor BAY11-7082.The results showed that upregulated GLIS3 could significantly activate the NF-κB signaling pathway,but the addition of pathway inhibitor BAY11-7082 reduced cell proliferation and invasion caused by upregulated GLIS3,both of which reached a significant difference level（P<0.05）.Conclusions:1.Overexpression of GLIS3 can significantly increase the expression level of p-p65Ser536,and also increase NF-κB binding activity of with common DNA sequence.2.GLIS3 affects the malignant behavior of TNBC cells by regulating the NF-κB signaling pathway.Summary:1.GLIS3 protein was highly expressed in TNBC tissue.In the analysis of clinicopathological indexes,the high expression of GLIS3 was related to tumor size and TNM staging.High expression of GLIS3 protein indicates poor.2.The expression level of GLIS3 protein in triple negative breast cell line was significantly increased.Interfering with the expression of GLIS3 in triple negative breast cancer cells can obviously weaken the cell proliferation ability,stop the cell cycle in G1 phase,promote cell apoptosis,reduce the cell migration and invasion ability,and prevent the malignant behavior of tumor cells.These malignant behaviors can be improved by overexpressing GLIS3.3.Over-expression of GLIS3 can significantly increase the expression level of p-p65Ser536 in NF-κB signaling pathway and increase the binding activity of NF-κB to DNA.GLIS3 affects all kinds of malignant behaviors of TNBC cells through NF-κB signaling pathway.