The Study Of NRP-1 Promoting The Stemness Of Breast Cancer Cells And The Related Regulation Mechanisms

Breast cancer is one of the most common tumors that threaten women’s physical and mental health seriously.Data from 2012 show that new cases and deaths of breast cancer among women are the highest in the world.The incidence of breast cancer in women in China increases by 2.5%every year,and the number of cases and the rate of growth are among the highest in the world.Despite significant improvements in the diagnosis and treatment of breast cancer over the past few decades,the recurrence and distant metastasis of tumors remain important threats to the lives of patients.Therefore,it is important to understand the mechanism of breast cancer recurrence and metastasis from the molecular level,and to clarify the regulation mechanism related to it,to improve the prognosis of breast cancer patients..Tumor stem cells(CSCs)refer to a small number of cells in tumor tissue that have self-renewal and infinite proliferation potential,and are closely related to the formation and development of tumors.At present,it is believed that tumor stem cells are resistant to conventional chemotherapy and radiotherapy,and are also the root cause of tumor recurrence and metastasis.Traditional treatment can only kill common tumor cells components in tumor,but it can not effectively kill tumor stem cells.Therefore,the treatment method targeting CSCs may have better clinical prospects.In recent years,many specific markers of CSCs have been identified,but there is still lack of targeted treatment for CSCs.Therefore,there is an urgent need to find specific molecular markers of CSCs and provide new ways for tumor treatment.Neurofibrin 1(NRP-1)is an important member of the Neurofibrin family and was originally found in the nervous system.Until now,studies have found that NRP-1 is closely related to the occurrence,development,proliferation,apoptosis,and migration of tumors.NRP-1 can bind to multiple ligands,enhance the effect of multiple growth factors,and play a role in neurodevelopment,angiogenesis,and tumor invasion and metastasis.More and more studies have confirmed that NRP-1 is a common receptor of VEGF-A.It mainly promotes the formation of new blood vessels and the proliferation and migration of tumors by promoting the binding of VEGF subtypes to their receptors and the formation of NRP-1/VEGF/VEGFR1/2 complexes.In recent years,its role in the pathogenesis of breast cancer has been widely studied.NRP-1 can affect the proliferation,apoptosis,migration,invasion and other biological behaviors of breast cancer cells.However,there is no research on the role of NRP-1 in affecting the sternness of breast cancer cells and related regulatory mechanisms.In our study,the effects of NRP-1 on the the sternness of breast cancer cells were explored by gene silencing and over-expression techniques.The relationship between NRP-1 and the signal pathway of Wnt/β-catenin was further studied..Meanwhile,The regulatory effects of miR-376a on NRP-1 and the sternness of breast cancer cells were also discussed.The purpose of this study was to explore the regulatory and molecular mechanism of NRP-1 for the sternness of breast cancer cells,and to provide new ideas for the targeted treatment of BSCS.The study is divided into three major chapters.Chapter 1:The role of NRP-1 in promoting the stem cell properties of breast cancer cells.Chapter 2:The mechanism of NRP-1 promoting the stem cell properties of breast cancer cells.Chapter 3:The regulation mechanism of NRP-1 in breast cancer cells.Chapter 1:The role of NRP-1 in promoting the stemness of breast cancer cellsObjectiveThe theory of tumor stem cell suggests that targeted tumor stem cell therapy is a promising method for tumor control.Our past studies showed that NRP-1 can promote breast cancer cell proliferation and invasion,and found that over-expression of NRP-1 can promote breast cancer microsphere formation,but the relevant mechanism is not clear.Based on this,we further study the effect of NRP-1 on he sternness of breast cancer cells and provide theoretical basis for targeted treatment of breast cancer stem cell based on NRP-1.Methods1.NRP-1 gene was overexpressed in MCF-7 cell and interfered in MDA-MB-231 cell.The protein and mRNA expression levels of sternness markers OCT4 and Nanog were detected by Western blot and qRT-PCR.In xenograft mice,the effect of NRP-1 interference or overexpression on tumorigenesis in mice was detected.Then,the expression of vascular marker CD34 was detected to determine the effect of NRP-1 on angiogenesis.2.Suspension culture was used to get the cell microsphere.qRT-PCR to explore the expression of NRP-1 and tumor stem cell markers CD44 and ALDH1 in breast cancer cell spheres.In xenograft mice,the tumorigenesis between sphere cells and parent cells was assessed by measuring the weight of the transplanted tumor.The tumors in mice that were induced by sphere cells and breast cancer cells were dissociation to study the expression of CD34 by immunohistochemistry.3.qRT-PCR were carried out to detect the expression of NRP-1 in MCF-7-Adr and MCF-7 cell lines.Mammospheres formation assay was used to detect the sternness of MCF-7-Adr and MCF-7 cells.The sub-population of CD44+/CD24-cells was evaluated in MCF-7 and MCF-7-Adr cells via flow cytometry.4.Apoptosis analysis and Western blot had been taken out to explore the apoptosis that was induced by adriamycin.CCK-8 test was used to detect the alternation of drug resistance in MCF-7-Adr after NRP-1 gene knockout.Results1.Compared with the control group,MCF-7 cell NRP-1 overexpression group had stronger cell spheres formation ability and higher expression levels of mRNA and protein of the stem cell markers OCT4 and Nanog,increased tumorigenesis in naked mice,and increased CD34 positive cells in transplanted tissues.In contrast,MDA-MB-231 cells NRP-1 gene interference group kept lower cell spheres formation ability and lower mRNA and protein expression levels of OCT4 and Nanog,weakened tumorigenesis in nude mice,and decreased CD34 positive cells in transplanted tissues,compared with the control group.2.qRT-PCR showed that the expression of NRP-land tumor stem cell markers CD44 and ALDH1 in spheres cells was higher than that of parental cells.The results of the mouse xenotransplantation model showed that spheres cells had stronger tumorigenic capacity than that of the parent cells.Its proportion of CD34 positive cells in the transplanted tumor tissue was higher than that of the parent cells.3.The mRNA expression of NRP-1 in MCF-7-Adr was higher than that in MCF-7.Tumor sphere formation ability of MCF-7-Adr was stronger than that of MCF-7.Flow cytometry showed that the proportion of CD44 +/CD24-cells in MCF-7-Adr was higher than that of MCF-7.4.The results of CCK-8 and apoptosis analysis of flow cytometry showed that the resistance of MCF-7-Adr cells to dopropoxine was reversed after the NRP-1 gene was knocked out.Conclusion1.NRP-1 can enhance the sternness of breast cancer cell lines.2.NRP-1 can regulates dopropoxine resistance through conferring breast cancer cell sternness.Chapter 2:The mechanism of NRP-1 promoting the stemness of breast cancer cellsObjectiveTo investigate the interaction between NRP-1 and VEGF-A and the regulation of Wnt/β-catenin pathway in breast cancer cells.To explore the mechanism of NRP-1 to promote the characteristics of breast cancer cell stem cells.Methods1.Western blot and qRT-PCR were carried out to detect the expression of VEGF-A and NRP-1 in breast cancer cells and spheres.Immune co-precipitation(IP)was used to detect the combine of VEGF-A and NRP-1 in spheres cells.2.Mammospheres formation assay,soft agar cloning formation test,Transwell test and xenotransplantation test had been carried out to study the effect of VEGF-A/NRP-1 pathway inhibitor(EG00229)on the stemness of breast cancer cells.3.The expression levels of beta-catenin and Wnt3a in breast cancer cells were measured by Western dot and qRT-PCR after over-expression of NRP-1 or interference with NRP-1.4.Transwell experiment and Mammospheres formation assay had been taken out to research the effect of Wnt/β-catenin pathway inhibitor Wnt-C59 on the stemness of breast cancer cells.Results1.Western Blot and qRT-PCR showed that the expression level of VEGF-A and NRP-1 in breast cancer cell spheres cells was significantly higher than in parental cells.The results of immune co-precipitation(IP)confirmed that VEGF-A and NRP-1 could interact directly in breast cancer cell spheres cells.2.Mammospheres formation assay,soft agar colony formation assay,transwell and cell line xenograft assay indicated that EG00229 could inhibit the stemness of breast cancer cells.3.Western blot and qRT-PCR detected that the expression of Wnt3a andβ-catenin were higher in MCF-7/LV-NRP-1 cells than that in MCF-7 cells,and the expression of Wnt3a and β-catenin were lower in MDA-MB-231/LV-NRP-1-shRNA cells than that in MDA-MB-231 cells.4.Mammospheres formation assay,CD44 +/CD24-cell ratio determination and Transwell assay indicate that Wnt-c59 can downregulate the sternness of breast cancer cells.Conclusion1.NRP-1 promotes the sternness of breast cancer cell lines by binding to VEGF-A.2.NRP-1 regulates the stemness of breast cancer cells via downstream signaling pathway Wnt/β-catenin.Chapter 3:The regulation mechanism of NRP-1 in breast cancer cellsObjectiveThe microRNA.org database was used to search for microRNAs that may regulate NRP-1 and was verified by functional experiments.The purpose was to explore the relevant regulatory mechanism of NRP-1 in breast cancer cells.Methods1.Computer software was used to predict the possible microRNAs to regulate NRP-1mRNA in breast cancer cells.2.The effects of miR-376a on NRP-1 in breast cancer cells were detected by Western blot and qRT-PCR experiments.3.Luciferase reporter assay verified whether miR-376a can target and regulate the expression of NRP-1 mRNA.4.Western blot was performed to detect the protein expression of wnt3a andβ-catenin after transfection with LV-miR-376a in MDA-MB-453 and MDA-MB-231 cell lines.5.CCK-8,flow apoptosis analysis,Western blot and Transwell assay were used to detect the effects of miR376a on the proliferation,migration,EMT and stemness of breast cancer cells.6.After transfection of LV-NRP-1 or treatment with Wnt/β-catenin agonist SKL2001,the effects on the proliferation,migration,EMT and sternness of breast cancer cells infected with LV-miR-376a were observed.Results1.The results of computer software prediction indicate that miR-376a may regulate NRP-1 mRNA in breast cancer cells.2.Western blot and qRT-PCR experiments showed that overexpression of miR-376a inhibited NRP-1 expression in breast cancer cells.3.Luciferase reporter assay confirmed that miR-376a can directly target NRP-1 mRNA in breast cancer cells.4.Western blot pointed out that the protein expression of wnt3a and β-catenin were decreased after transfection with LV-miR-376a in MDA-MB-453 and MDA-MB-231 cell.5.CCK-8,flow apoptosis analysis,Western blot and Transwell assay indicated that miR376a could inhibit proliferation,migration,EMT and the sternness of breast cancer cell lines.6.Overexpression of NRP-1gene or treatment with Wnt/β-catenin agonist SKL2001 rescues the inhibitory effects of miR-376a on proliferation,migration,EMT and the sternness of breast cancer cell lines.Conclusion1.NRP-1 is a direct target gene of miR-376a in breast cancer cells.2.MiR-376a affects breast cancer cells proliferation,migration,EMT and sternness by regulating NRP-1 expression.