MiR-7 Inhibits The Breast Cancer Stem Cell Subpopulation By Downregulating RELA To Decrease CD44 And ESAM Expression

Breast cancer is one of the most common malignant tumors in women.With the improvement of diagnosis and treatment technology,the survival rate of breast cancer patients has been significantly improved.However,breast cancer stem cells(BCSCs)often lead to metastasis and recurrence of breast cancer patients and become the main cause of patient death.Accordingly,targeting BCSCs is critical to the treatment of breast cancer.Studies have shown that miR-7 plays an important role in many tumors,and our group found that overexpression of miR-7 can significantly downregulate BCSCs subpopulation,but the mechanism is unknown.Therefore,elucidating the regulatory effect and mechanism of miR-7 on BCSCs will be a good strategy to find new and effective targets for treatment of breast cancer by targeting BCSCs.In this study,we found that there may be regulatory effects between miR-7 and the CD44,ESAM and RELA by bioinformatics prediction and protein array detection.CD44 is a transmembrane glycoprotein that plays roles in cell-cell interactions,cell adhesion,migration and lymphocyte homing.The high expression of this molecule in many tumors is related to the invasion and metastasis of tumor cells.And CD44 is also one of the surface markers of BCSCs.Endothelial cell-selective adhesion molecule(ESAM)has been identified as a novel hematopoietic stem cell marker and can promote tumor metastasis by regulating angiogenic processs.In addition,RELA,as a member of the nuclear transcription factor NF-κB,can be activated by intracellular and external factors and transferred to the nucleus to induce the expression of tumor-associated genes.At present,the mechanisms of miR-7regulating these molecules have not been reported in breast cancer and BCSCs.Based on the research background,we focused on the molecular regulation between the miR-7 and the CD44,ESAM and RELA in this study,to elucidate the mechanisms of miR-7 affecting BCSC subpopulation,which may provide a new strategy for radical treatment of breast cancer by targeting BCSCs.PurposeTo explore the molecular mechanisms of miR-7 inhibiting the BCSC subpopulation and metastasis,and to provide experimental evidence for targeting BCSCs to treat breast cancer.Methods and Contents1.The effects of miR-7 on the BCSC subpopulation and related molecules expression in human breast cancer cell lines: Transfection of miR-7 mimic and miR-7 inhibitor in four human breast cancer cell lines,respectively.The proportion of BCSC subpopulation and the expression levels of related molecules in the cells were detected by q PCR,FCM and WB.Mi R-7 agomir was injected into the situ tumor in BCSCs xenografts bearing mice and the tumor tissues were collected for detection to confirm the experimental results in the cell lines.2.Identification of miR-7 targets and exploration of the molecular mechanisms of miR-7 downregulating BCSCs subpopulation: We used bioinformatics methods to predict the target of miR-7,and the interactions between molecules were verified by dual-luciferase reporter assay,Ch IP-PCR assay and si RNA interference assay.3.Detection of the relationship between the miR-7 and the related molecules in breast cancer clinical specimens: Collection of breast cancer clinical specimens and detection of the molecular expression by q PCR and WB after extracting total RNA and protein from the tissues.4.Isolation and identification of human breast cancer primary cells from the cancer tissues: Human breast cancer primary cells were isolated and cultured from clinical specimens of breast cancer by tissue block culture method.Comparison of the BCSC subpopulation proportion and the expression levels of related molecules in the established primary cells and the other three cell lines of human breast cancer cell lines by q PCR,FCM and WB experiments.5.The effects of miR-7 overexpression on the tumorigenicity of BCSCs in NOD/SCID mice: The lentiviral recombianant overexpressing miR-7 were packaged to infect human breast cancer primary cells,and monoclonal cells stably overexpressing miR-7 were screened and cultured to detect the molecular expression level in the cells by q PCR,FCM and WB.The BCSCs xenografts bearing mice model were established,and the general condition and tumor growth of the tumor-bearing mice were monitored.The tumors,lungs and brain tissues of the tumor-bearing mice were taken for q PCR,FCM,WB,IHC and IF experiments.6.Screening of differentially expressed proteins in tumor tissues by protein array technique: The total proteins of tumor tissues in the control group and miR-7agomir treatment group were analyzed by protein array to screen out the differentially expressed proteins,especially in the interested proteins.7.Investigation on the molecular mechanisms of miR-7 inhibiting differentially expressed protein ESAM: Prediction of targets using bioinformatics methods,and validation by Ch IP-PCR,miRNA transfection and si RNA interference experiments.As well as detection of the related molecules expression in breast cancer clinical specimens to evaluate the regulatory role of miR-7 on the ESAM molecule.8.The effects of miR-7 on the biological function of breast cancer cells and BCSCs by targeting ESAM: Down-regulating ESAM expression by miR-7 mimic and si ESAM in MDA-MB-231 cells,and detecting the changes in abilities of cell proliferation,colony formation,and migration.Detection of metastasis-associated molecules expression levels in tumor tissues from the BCSCs xenografts bearing mice treated with miR-7 mimic and si ESAM.Results1.Transfection with miR-7 mimic could significantly increase the expression of miR-7,and effectively downregulate the proportion of BCSCs as well as the expression of CD44,RELA,EGFR and HAS2,in MDA-MB-231,MCF-7,SK-BR-3 and LD cell lines;while inhibition of miR-7 increased the proportion of BCSCs and promoted the expression of the above related molecules.In addition,the proportion of BCSC subpopulation and the expression levels of CD44,RELA and other molecules in the tumor tissues of mice treated with miR-7 agomir were also significantly lower than those of control,which were consistent with the results in cell lines in vitro.2.Using bioinformatics methods predicted that the RELA 3’-UTR region contains two miR-7 binding sites and it also has seven binding sites in the promoter region of CD44.The results of dual-luciferase reporter assay showed that the miR-7mimic reduced the relative luciferase activity of the wild-type vector,which contained the putative miR-7 binding sites in the RELA 3’-UTR,but did not decrease the relative luciferase activity of the mutant A or B vector,or the A plus B vector,indicating miR-7 can target the RELA 3’-UTR region and inhibit RELA expression.Subsequently,the Ch IP-PCR experiment result also confirmed that RELA could bind to the-1234 to-1243,-1654 to-1663,and-2073 to-2082regions in the CD44 promoter region in MDA-MB-231 cells,that is,RELA can directly regulate the transcription of CD44.To further confirm these findings,we investigated whether silencing RELA could decrease CD44 expression in MDA-MB-231,MCF-7,SK-BR-3 and LD cells.The results showed that CD44 transcriptional and translational expression levels were significantly decreased after transfection with si RELA recombinants in comparison with the control cells,suggesting the RELA has regulatory role on CD44 expression.3.The detected results in clinical specimens of breast cancer showed that,the expression of miR-7 was lower in patients with high expression of RELA and CD44 compared with the normal breast tissues.Moreover,miR-7 expression was negatively correlated with the expression of RELA and CD44,while the expression between the RELA and the CD44 was positively correlated.4.A human breast cancer primary cell line was successfully isolated and identified from the clinical surgical specimens and it was named LD cells.It was found that MCF-7 cells with relatively high expression of miR-7 had the lowest expression levels of RELA and CD44 and the proportion of BCSC subpopulation;while in the other three cell lines with low expression of miR-7,the expression levels of RELA and CD44 as well as the BCSCs proportion were increased.5.Cultured monoclonal LD cells stably overexpressing miR-7 were successfully screened and expanded.In vitro and in vivo experiments showed that overexpression of miR-7 by lentiviral system could effectively reduce the expression levels of RELA and CD44 and downregulate the subpopulation proportion of LD-CSCs in LD cells,and also significantly inhibit the abilities of LD-CSCs oncogenicity and lung metastasis in NOD/SCID mice.6.The results of protein array in tumor tissues from the BCSCs xenografts bearing mice showed that compared with the control group,the expression of 34 protein molecules was downregulated,while the expression of 21 protein molecules was upregulated in the tumor tissues from the mice with miR-7 agomir treatment.The expression level of ESAM protein was downregulated by 3.2 times compared with the control group.The results of q PCR were similar to those of protein array,showing a decrease of 3.6 times expression level of ESAM.7.The results of bioinformatics prediction showed that there are four RELA binding sites in the promoter region of ESAM.It was demonstrated that the RELA could bind to the-1232 to-1243 region of the ESAM promoter to directly regulate ESAM transcription level by Ch IP-PCR assay.Subsequently,the miR-7 mimic transfection assay,the si RELA interference assay,and the detection of molecular expression in breast cancer clinical specimens further validated the regulatory effects of miR-7 and RELA on ESAM.8.In vitro transfection with miR-7 mimic or si ESAM could effectively downregulate the expression of ESAM in MDA-MB-231 cells and significantly reduce the abilities of cell proliferation,colony formation and migration.In vivo experiments showed that the tumor growth,the angiogenesis and the metastasis of the BCSCs driven xenografts in miR-7 overexpression and si ESAM downregulated groups were significantly inhibited compared with those of the control group,which were statistically significant.ConclusionsThe findings from this study demonstrate that miR-7 can significantly downregulate the BCSC subpopulation in MDA-MB-231,MCF-7,SK-BR-3 and LD cell lines,and effectively inhibit the tumorigenicity and metastasis of BCSCs driven xenografts in NOD/SCID mice.The molecular mechanism investigation suggests that miR-7indirectly inhibits the expression of CD44 and ESAM by targeting RELA,thereby reducing the proportion and stemness of BCSCs and tumor metastasis,and that miR-7and its downstream molecules RELA,CD44 and ESAM can be used as effective targets for treatment of breast cancer by targeting BCSCs.