Correlation And Mechanism Of ZMIZ2 With Malignant Degree Of Triple-Negative Breast Cancer

Breast Cancer is the malignant tumor with the highest incidence in the world,among which Triple Negative Breast Cancer(TNBC)has a high malignant degree,which is a difficult problem in the field of breast cancer research.TNBC lacks molecular targets for other breast cancers,such as Progesterone receptors(PR),Estrogen receptors,and progesterone receptors.ER)and Human Epidermal Growth Factor Receptor-2(HER2),do not benefit from endocrine therapy and targeted therapies.Despite the rapid development of new targeted drugs in the field of breast cancer,drug development and targeted therapy are significantly hindered due to the high heterogeneity and uncertainty of targets in triple-negative breast cancer.Therefore,finding new molecular targets is the core of TNBC research.ZMIZ(Zinc Finger MIZ-Type Containing,ZMIZ)protein is a transcriptional co-regulator with zinc finger structure,which has transcriptional co-regulation effects on steroid hormone receptors and p53 signals.This paper systematically studied the correlation between the expression of ZMIZ2(Zinc Finger MIZ-Type Containing 2,ZMIZ2)in TNBC and tumor progression,and revealed the correlation between ZMIZ2 and malignant proliferation and migration of TNBC through a variety of functional experiments.The downstream target genes of ZMIZ2 regulating TNBC were analyzed and established,and the effect and signaling mechanism of ZMIZ2 on the occurrence of tumor in vivo were investigated by human specimens and nude mice.Firstly,the breast cancer RNA sequencing data in the TCGA database was analyzed by bioinformatics method.A total of 3833 differentially expressed m RNAs were found in the triple-negative breast cancer samples,among which 1729 m RNA expressions were up-regulated.Compared with normal tissues,the expression level of ZMIZ2 in triple-negative breast cancer was significantly increased.Then,clinical samples were collected to detect the expression of ZMIZ2 in clinical breast cancer tissues.Fluorescent quantitative PCR and Western-Blot experiments proved that,compared with normal tissues,HER2-positive cancer tissues and LUM cancer tissues,the expression of ZMIZ2 was the highest in triple-negative breast cancer TNBC tissues.The expression level of ZMIZ2 in all types of cancer tissues was significantly higher than that in normal tissues.Immunohistochemical results further demonstrated that ZMIZ2 was highly expressed in triple negative breast cancer tissues.Furthermore,a variety of human breast cancer cell lines were selected to prove that the expression level of ZMIZ2 was the highest in triple-negative breast cancer cell lines,significantly higher than that of normal breast cancer cell lines or HER2 type breast cancer cell lines.Combined with the results of biogenic analysis and breast histology and cell detection,it was proved that high expression of ZMIZ2 is an expression feature of triple-negative breast cancer.At the cellular level,ZMIZ2 overexpression plasmid and interfering sh RNA were constructed to detect the effects of ZMIZ2 expression level on multiple cell functions of triple-negative breast cancer.Two kinds of triple negative breast cancer cell lines,MDA-MB-231 cells and MDA-MB-468 cells,were tested by CCK8 test and Transwell test,and it was proved that the ZMIZ2 expression level in two kinds of triple negative breast cancer cells was knocked down,which significantly inhibited the proliferation,migration and invasion of triple negative breast cancer cells.Annexin V-FITC/PI staining demonstrated that knockdown of intracellular ZMIZ2 expression in two triple negative breast cancer cells can significantly promote cell apoptosis.Overexpression of ZMIZ2 in two types of triple-negative breast cancer cells promoted cell proliferation,migration and invasion,but inhibited apoptosis.The above results confirmed that ZMIZ2 was specifically highly expressed in triple-negative breast cancer,and ZMIZ2 promoted cell proliferation,migration and anti-apoptosis of triple-negative breast cancer.On this basis,we explored and studied the cancer-promoting mechanism of ZMIZ2 in triple-negative breast cancer.By using protein interaction network analysis and gene enrichment analysis(GSEA),it was found that ZMIZ2 may be involved in the regulation of proliferation-related signaling pathways and may directly regulate target genes MCM3,CCL5,E2F4 and DHX38.Quantitative PCR and Western-Blot experiments showed that the transcription and expression of four genes,MCM3,CCL5,E2F4 and DHX38,were also decreased after ZMIZ2 was knocked down in MDA-MB-231 cells.Choosing MCM3 as the representative,we systematically studied the regulatory effect of ZMIZ2 on MCM3 in triple-negative breast cancer.First,it was found and proved that MCM3 was also specifically highly expressed in triple-negative breast cancer.In 142 clinical tissue samples,it was proved that the expression of MCM3 was closely related to the malignancy of TNBC.The strongly positive expression rate of MCM3 in the sections of patients with tumor diameter >2.0cm was significantly higher than that of patients with tumor diameter≤ 2cm.The expression intensity of MCM3 increased with the increase of TNM grading.Both Western-Blot and immunohistochemical results demonstrated that MCM3 was specifically highly expressed in triple-negative breast cancer.Then,after overexpression and interference with the expression level of ZMIZ2 in triple-negative breast cancer cells MDA-MB-231,it was proved that ZMIZ2 has a positive regulatory effect on the transcription and expression of MCM3.In order to further study the effect of target gene MCM3 on the function of ZMIZ2,we constructed MCM3 interference sh RNA.Rescue experiment results showed that inhibition of MCM3 expression in triple-negative breast cancer cells could remove the regulatory effect of ZMIZ2 overexpression on cell function.The results of double luciferase reporter gene detection showed that ZMIZ2 activated the transcriptional activity of the downstream target gene MCM3.The Ch IP-q PCR results of triple-negative breast cancer cells showed that ZMIZ2 was enriched in the promoter region of MCM3.The above experimental results prove that ZMIZ2,as a transcription factor,directly binds to the promoter region of MCM3 and activates the transcriptional activity of MCM3 promoter,thus opening the gene transcription and protein expression of MCM3.Therefore,the study results established the ZMIZ2-MCM3 regulatory signal of triple-negative breast cancer.In order to analyze the effect of ZMIZ2 and its target gene MCM3 on tumor growth in vivo,we established a tumor transplantation model in nude mice.MDA-MB-231 cells with ZMIZ2 overexpression,MDA-MB-231 cells with MCM3 interference,and MDA-MB-231 cells with ZMIZ2 overexpression of cotransmutation MCM3 sh RNA were injected into the subcutaneous groin of mice,and the growth of transplanted tumors was observed.The results showed that overexpression of ZMIZ2 promoted tumor growth and malignant degree.Inhibition of MCM3 expression after overexpression of ZMIZ2 significantly reduced the tumor growth rate.ZMIZ2 regulates the proliferation of transplanted tumors through MCM3.Through the experiments of Ki-67 staining,Tunel staining and EMT index in nude mice bearing tumor,we proved that Ki-67,a proliferating marker of tumor tissue,was strongly expressed in nude mice overexpressing ZMIZ2,the apoptosis level detected by Tunel staining was significantly reduced,and the expressions of MCM3,BCL2 and N-cad were increased.The expressions of cyclin A,BAX and E-cad were down-regulated.The overexpression of ZMIZ2 interferes with the tumor tissue of MCM3 nude mice and reverses the above histological changes.Therefore,animal tumor transplantation has proved that ZMIZ2-MCM3 signal is an important signal of malignant proliferation,anti-apoptosis and malignant transformation of EMT in triple negative breast cancer.In order to explore the signaling pathway that may be involved in the regulation of tumor growth by ZMIZ2/MCM3,we conducted high-throughput sequencing of transplanted tumors in nude mice in the ZMIZ2 overexpression group,MCM3 knockout group and control group,and performed KEGG annotation on their differentially expressed genes.In the results,MAPK signaling pathway,m TOR signaling pathway,and m TOR signaling pathway were found.Many pathways related to malignant proliferation,such as Wnt signaling pathway and Ras signaling pathway,were enriched by differential genes.It is suggested that the pathway related to malignant proliferation of tumors may be regulated by ZMIZ2/MCM3 and participate in the genesis and development of tumors.Finally,using human triple-negative breast cancer samples,we analyzed the relationship between ZMIZ2-MCM3 expression levels in clinical samples,and proved that ZMIZ2 and MCM3 are specifically highly expressed and positively correlated in triple-negative breast cancer,and are closely related to tumor size,TNM grade and poor prognosis of patients with triple-negative breast cancer.Thus,the ZMIZ2-MCM3 regulatory signal in triple negative breast cancer was identified at the tissue level.Therefore,this study revealed the abnormal expression of ZMIZ2 on triple-negative breast cancer,analyzed and established the downstream target gene MCM3 of ZMIZ2 regulating TNBC,and proved the promotion significance of ZMIZ2-MCM3 signal on triple-negative breast cancer through a variety of in vivo and in vitro experiments.This study provides a possible novel molecular target for the pathogenesis and treatment of triple-negative breast cancer.