The Role And Related Mechanisms Of MicroRNA-206 In Targeted Regulation Of EVI-1 On The Biological Behavior Of EGFR~+ Triple Negative Breast Cancer

Objective:Triple negative breast cancer(TNBC)is an important molecular subtype of breast cancer,accounting for about 15%-20%of breast cancer.Compared with other breast cancer subtypes,TNBC patients are young in onset,highly invasive,with high metastasis rate of lung,liver,brain and other organs,early recurrence,rapid progress,short survival period and other characteristics,with poor prognosis.The immunohistochemical characteristics of TNBC were that estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor receptor-2(her-2)were all negative,lacking specific markers as effective therapeutic targets and insensitive to endocrine and targeted therapies.Therefore,to explore new therapeutic targets and carry out multi-site targeted drug therapy is the hot and difficult point in TNBC research at present.Epidermal growth factor receptor(EGFR)is one of the growth factor receptors associated with breast cancer progression and poor prognosis.EGFR is positively expressed in most TNBC,and the positive rate can be as high as 50-80%.Epidermal growth factor(EGF)can be phosphorylated after interaction with EGFR,and then activate its downstream PI3K/AKT and other signal transduction pathways,leading to proliferation,differentiation and metastasis of tumor cells,and promoting the occurrence and development of TNBC.FOXO transcription factor is one of the key downstream targets of the PI3K/AKT pathway.When the PI3K/AKT pathway is activated abnormally,FOXO will lose the activity of tumor suppressor transcription factor and the cell apoptosis process will be blocked.Therefore,EGFR is an attractive potential therapeutic target for EGFR-positive TNBC.Although the molecular targeted drug cetuximab against EGFR has been applied in the clinical treatment of breast cancer,the effective rate of single application is not high,and drug resistance is common,suggesting that other molecules may affect the activation of the EGFR signaling pathway in EGFR-positive breast cancer.At present,the treatment strategy of EGFR-positive TNBC has gradually become one of the important topics in the field of breast cancer research.EVI-1 protein,also known as MDS1 and EVI-1 complex locus protein,is a zinc finger transcription factor.EVI-1 promotes cell proliferation and angiogenesis during normal organism development.Overamplification or overactivation of EVI-1 gene often leads to tumor.EVI-1 is up-regulated in many malignant tumors and is associated with poor prognosis.Studies have found that EVI-1 is highly activated and highly expressed in various solid tumors such as pancreatic cancer,colon cancer,brain glioma and breast cancer,playing the role of proto-oncogene,and its expression level is negatively correlated with prognosis.EVI-1 overexpression can promote cell proliferation and anti-cancer cell apoptosis.In pancreatic cancer,overexpression of EVI-1 promotes the activation of PI3K/AKT pathway,leading to proliferation and invasion of cancer cells and inhibiting apoptosis.In breast cancer,EVI-1 may play a carcinogenic role by promoting the expression of genes related to the growth and invasion of breast cancer cells.miR-206 was initially cloned and identified from human and mouse muscle tissues,with a length of 22nt,and its encoding gene located on chromosome 6.In 2005,miR-206was down-regulated in breast cancer for the first time by mRNA microarray.It was found that for TNBC cell line MDA-MB-231,miR-206 significantly inhibited its proliferation.miR-206 mainly plays the role of tumor suppressor genes in breast cancer and may become a new therapeutic target for breast cancer.In our previous study,through bioinformatics software analysis,we found that miR-206 had a targeted regulatory relationship with EVI-1.Basic experiments confirmed that miR-206 indirectly regulates the biological behavior of breast cancer stem cells by negatively regulating the expression level of transcription factor EVI-1.For EGFR-positive TNBC,the above conclusions need to be confirmed at different levels,such as verification in corresponding TNBC tissues and TNBC cell lines,and elucidation of its mechanism of action.At present,the expression of EVI-1 in EGFR~+TNBC is still unclear,and the mechanism of the change of EVI-1 expression on the occurrence and development of EGFR~+TNBC has not been elucidated.In this study,the expression of EVI-1 in EGFR~+TNBC tissues was detected,and the relationship between the expression of EVI-1and its clinicopathological characteristics and prognosis was analyzed.Bioinformatics was used to predict and analyze that EVI-1 was the downstream target gene of miR-206,and the dual luciferase gene reporting system was used for verification.Through in vitro experiments,the influence of miR-206 and EVI-1 on the biological behavior of EGFR-positive TNBC and the related molecular mechanism were further discussed.Research.Methods:1.The expression of EVI-1 in 88 EGFR-positive TNBC tissues and adjacent tissues was detected by immunohistochemical staining method,and its correlation with the clinicopathological factors and prognosis of EGFR-positive TNBC tissues was analyzed.2.Bioinformatics online database was used to preliminarily predict and screen the target genes of miR-206.DAVID and Gene Ontology database were used to analyze gene function enrichment of miR-206 target gene from three aspects:biological process(BP),cell component(CC),and molecular function(MF),and KEGG database was used to analyze biological signal transduction pathway enrichment,so as to preliminarily explore the key signal pathway involved in the regulation of breast cancer by miR-206 target gene.The binding effect of miR-206 with the target gene was verified in vitro by double luciferase reporter system experiment.3.The transfection efficiency was verified by transfection of miR-206 into EGFR~+TNBC cells(MDA-MB-231 and MDA-MB-468 cells),and the dominant cell lines were determined for subsequent experiments.miR-206 mimics and miR-206inhibitors,negative control vector(NC)and EVI-1 gene silencing expression vector(shEVI-1)were used to transfection EGFR~+TNBC cells alone or in combination,and EGFR pathway inhibitor AG1478 was added to treat cells to observe the effect on cell biological behavior and key proteins of EGFR/PI3K/FOXO signaling pathway.CCK-8assay was used to detect the effect of proliferation activity of the above cells.Transwell assay was used to detect the migration and invasion ability of cells.Cell cycle and apoptosis were detected by flow cytometry.Western blot was used to detect the expression of key proteins in the EGFR/PI3K/FOXO signaling pathway and apoptosis-related proteins.4.SPSS 17.0 software was used to analyze the data obtained in the experiment,ImageJ software was used for experimental image processing,and Graphpad software was used to draw charts.The enumeration data were evaluated by the chi-squared test or Fisher’s exact test,the survival analysis was conducted by kaplan-meier and log-rank test,and the multivariate survival analysis was conducted by COX regression analysis.Test level=0.05.Results:1.The high expression rate of EVI-1 in EGFR~+TNBC was 59.1%(52/88),significantly higher than that in the adjacent tissues,and the difference was statistically significant(P<0.05).The expression intensity of EVI-1 was correlated with the number of lymph node metastasis,the expression of p53 and Ki-67(P<0.05).COX risk regression analysis showed that age,histological grade,vascular thrombi,Ki-67 and EVI-1 expression were independent risk factors for EGFR~+TNBC prognosis(P<0.05).Survival analysis showed that the disease-free survival(DFS)and total survival(OS)of patients in the EVI-1 low expression group were significantly prolonged compared with the EVI-1 high expression group(P<0.05).Compared with other subgroups,the double low expressions of EVI-1and Ki-67 were significantly correlated with the prolongation of DFS and OS in patients(P<0.05).2.The target genes of miR-206 were preliminarily predicted by Targetscan7.2 and miRDB,and 544 target genes including EVI-1 were obtained.The predicted results showed that miR-206 could bind to EVI-1 3’UTR,and MECOM(i.e.,EVI-1)gene may be the direct target of miR-206.GO analysis and KEGG pathway enrichment analysis indicated that miR-206 target genes were mainly distributed in Hippo signal pathway,Rap1 signal pathway,adhesion connection and cancer pathway,which play an important regulatory role in the occurrence and development of breast cancer.It is speculated that the EGFR/PI3K/FOXO pathway may be a signal pathway closely related to EGFR~+TNBC.The dual luciferase reporter gene assay system verified the direct targeting of the complementary sites of EVI-1 3’UTR and miR-206.3.1)CCK-8 results showed that compared with the control group,the proliferation activities of MDA-MB-231 and MDA-MB-468 cells were significantly reduced after 24h of transfection with miR-206 mimics,and MDA-MB-231 cells showed stronger inhibitory effect on proliferation and higher transfection efficiency(P<0.05).MDA-MB-231 cell lines were selected for subsequent experiments.2)Western blot results confirmed that up-regulation of miR-206 inhibited the expression of EVI-1protein in MDA-MB-231 cells(P<0.05).3)Transwell results showed that,compared with the control group,the number of cells transfected with miR-206 mimics or shEVI-1decreased slightly after transfection(P<0.05).Compared with the miR-206 mimics group,the number of transfected miR-206 mimics and shEVI-1 combined with AG1478had the lowest number of transfected cells(P<0.05).4)flow cytometry results showed that compared with the control group,G0/G1 cell cycle arrest occurred in the miR-206mimics or shEVI-1 group,and apoptosis rate increased(P<0.05).Compared with the miR-206 mimics group,the above effect was enhanced by AG1478,which was strongest after the co-transfection of miR-206 mimics and shEVI-1 with AG1478(P<0.05).5)Western blot analysis showed that compared with the control group,the expression levels of EGFR,PI3K,p-PI3K,AKT and p-AKT in MDA-MB-231 cells in the miR-206overexpression group or the EVI-1 silencing group were significantly decreased,p-foxo expression was decreased,p-FOXO1/FOXO1 ratio was decreased,anti-apoptotic protein bcl-2 expression was decreased,pro-apoptotic protein Bax expression was increased,and bcl-2/Bax ratio was decreased(P<0.05).Compared with miR-206 mimics group,miR-206 overexpression or EVI-1 silencing plus EGFR pathway inhibitor AG1478further enhanced the inhibitory effect on proliferation and apoptosis induction of MDA-MB-231 cells,and the co-transfection of miR-206 mimics and shEVI-1 combined with AG1478 showed the most significant inhibitory effect on proliferation and apoptosis induction of MDA-MB-231 cells(P<0.05).Conclusion:1.EVI-1 is mostly highly expressed in EGFR~+TNBC tissues,and is mostly low or absent in the corresponding paracancer tissues.EVI-1 expression was significantly correlated with pathological type,angiocarcinoma thrombus,lymph node metastasis,pathological stage,and increased ki-67.Low EVI-1 expression was associated with prolonged DFS and OS in TNBC patients,and low EVI-1 expression was an independent prognostic protective factor of EGFR~+TNBC.Combined detection of EVI-1 and Ki-67 expression can be used as an important prognostic indicator of EGFR~+TNBC.2.EVI-1 3’UTR can bind directly to miR-206,and EVI-1 is the downstream target gene of miR-206.miR-206 is involved in the regulation of multiple target genes and plays an important role in the occurrence,development,proliferation and invasion of breast cancer.The target gene of miR-206 is involved in the regulation of multiple signaling pathways,and the EGFR/PI3K/FOXO pathway may be closely related to EGFR~+TNBC.3.Specifically enhanced miR-206 down-regulated the expression of EVI-1 in EGFR~+TNBC cell lines.miR-206 overexpression or EVI-1 silencing can inhibit the proliferation,migration and invasion of MDA-MB-231 cells,induce cell cycle arrest,promote apoptosis,inhibit the expression of key proteins in the EGFR/PI3K/FOXO signaling pathway,reduce the phosphorylation level of PI3K/AKT protein,reduce the phosphorylation level of FOXO1,and increase the transcriptional activity of FOXO1,ultimately affecting the expression of apoptosis-related proteins.In addition,EGFR pathway inhibitors showed similar synergistic effects,and the group of miR-206 mimics and shEVI-1 combined with AG1478 showed the strongest synergistic effects.The molecular mechanism of miR-206’s tumor inhibition may be that EGFR/PI3K/FOXO signaling pathway and apoptosis-related pathway are negatively regulated by EVI-1,so as to regulate the expression of its downstream genes and exert corresponding biological functions.The key factor of the above intervention may be the potential therapeutic target of EGFR~+TNBC.