| Research background: Breast cancer is one of the most common cancers affecting women worldwide,with distant metastases causing significant mortality.According to statistics,the number of female invasive breast cancer reported by the United States in 2015 was 231,840,of which 40290 were dead.In breast cancer secondary metastases,85% of metastases are considered osteolytic.Breast cancer metastases form visible metastatic bone lesions,the process of which involves the preferential transfer of breast cancer cells to bone tissue leading to osteolytic lesions;wherein breast cancer cells undergo recruitment to bone tissue,survive in the bone microenvironment and clone expansion,As well as osteoclast activation and absorption of bone tissue.There is growing evidence that the abnormal expression of bone remodeling genes in in situ breast cancer cells increases the risk of bone metastases.In the meantime,in recent years,the results show that breast cancer stem cells(BCSCs)with self-renewal and regenerative tumor are the major causes of distant metastasis and recurrence of breast cancer and Chemotherapy resistance.However,the underlying molecular mechanisms are largely unknown.Runx2 is a transcription factor that is a member of the Runt gene family and plays a key role in osteoblast and bone development.It regulates the expression of several genes involved in bone matrix remodeling by binding to the osteoblast-specific cis-acting element 2(OSE2).In breast cancer cells,Runx2 is involved in the regulation of bone metastasis-related genes such as bsp,opn,mmp-9 and mmp-13.Runx2 and its target genes are highly expressed in breast cancer tissues and play an important role in bone metastasis of breast cancer.In normal epithelial cells,Runx2 binds to DNA.In the meantime,Runx2 is also involved in the prolactin action of mammary gland lactation.Normal human mammary epithelial cells(MCF-10A)obtained tumor phenotype after Runx2 induction.Most current mechanistic studies suggest that Runx2 causes osteolytic metastatic disease by promoting the interaction between tumor cells and the bone microenvironment.However,It has not been reported yet whether Runx2 regulates BCSCs that play an important role in the metastasis of breast cancer.It have been widely confirmed that common features of malignant tumors,namely the loss of epithelial traits and certain stromal cell traits obtained by epithelial-mesenchymal transition processes.A variety of signaling pathways are involved in the development of EMT,and TGF-? signaling pathway is widely recognized to play an important role in the EMT process.The EMT process involves the generation of stem cell-like tumor-initiating cells,spread of cancer cells,metastasis and chemotherapy resistance.There is some evidence that CSCs are phenotypically distinct cell types with unlimited proliferation potential and ability to regenerate tumors.In breast cancer,these cells usually have EMT characteristics.In addition,the EMT stage in breast cancer is associated with cancer stem cell properties such as self-renewal,resistance to conventional therapies,and posttreatment relapse.Studies have also shown that EMT may promote stem acquisition and maintenance and initiate angiogenic mimicry(VM).Therefore,we speculate whether Runx2 can affect the biological function of BCSCs,and whether this is through the role of EMT.In this study,the effect of Runx2 expression on the biological behavior of breast cancer cells was examined.Up-regulated or down-regulated Runx2 expression on the expression level of BCSCs marker CD44 and mammosphere formation are detected by In vitro experiment.The effect of inhibition of Runx2 expression on high tumorigenicity of BCSCs is detected in Immunodeficient Mice.By detecting the expression of EMT-related marker,whether Runx2 can affect the occurrence of EMT,which can regulate breast cancer stem cells.By exploring the role of Runx2 in breast cancer stem cells,it provides a new direction for clinical eradicating breast cancer.Research Objective: To investigate the biological role of Runx2 in breast cancer stem cells and to find out its regulatory mechanism.Research content: Biological Role of Runx2 in Breast Cancer Stem Cells and Mechanisms of Runx2 Regulation of BCSCS.Previous studies have shown that Runx2 and breast cancer stem cell marker CD44 expression in breast cancer cell lines and human breast cancer tissue specimens showed correlation.1.MCF-7 breast cancer cells were infected with lentivirus that up-regulated Runx2 expression.The breast cancer cells MB-231 were infected with lentivirus with Runx2 down-regulation,and puromycin was used to screen the cells stably expressing GEP fluorescence.2.The expression of Runx2 in lentivirus-infected breast cancer cells MB-231 and MCF-7 were detected by RT-PCR and Western blot.3.Using the cell lines constructed above to study the biological function of Runx2 in breast cancer cells.(1)The effect of Runx2 on the activity of breast cancer cells MB-231 and MCF-7 by MTT assay.(2)The effect of Runx2 on the colony formation ability of breast cancer cells MB-231 and MCF-7 was detected by plate clone assay.The effect of Runx2 on the non-anchored growth ability of breast cancer cells MB-231 and MCF-7 was detected by agar colony formation assay.(3)The influence of Runx2 on invasion ability of breast cancer cells MB-231 and MCF-7 by Transwell assay.4.The effect of up-and down-regulated Runx2 expression on the expression of BCSCs marker CD44 by Western Blot and mammosphere formation.The effect of inhibition of Runx2 expression on high tumorigenicity of BCSCs in Immunodeficient Mice.5.Western blot was used to detect the effect of Runx2 on EMT-related marker.Research result:1.We successfully constructed Runx2-knockdown MB-231 and Runx2 overexpressing MCF-7 stable cell lines.2.The effect of Runx2 on the viability of breast cancer cells: The results of MTT showed that the viability of Runx2 was significantly decreased after knockdown of Runx2 in MB-231 cells;however,the viability of MCF-7 cells was significantly increased after Runx2 was overexpressed.3.The effect of Runx2 on the ability of colony-forming and non-anchored growth of breast cancer cells.The results of plate clone showed that the size and number of clones formed were significantly decreased after Runx2 was low expression in MB-231.It indicates that inhibition of Runx2 can inhibit the ability of breast cancer cell cloning and non-anchored growth.After overexpression of Runx2 in MCF-7,the size and number of clones formed were significantly increased.The size and number of colony formation of soft agar also increased significantly,indicating that Runx2 can promote the colony-forming ability and non-anchored growth ability of cells.4.Effect of Runx2 on invasion ability of breast cancer cells: The results of tranwell showed that the invasion ability of cells was reduced after knockdown of Runx2 in MB-231 cells;however,the invasion ability of cells was significantly increased after overexpression of Runx2 in MCF-7 cells increase.The above results show that Runx2 can promote the invasiveness of breast cancer cells.5.Overexpression of Runx2 increased the expression of CD44,a marker of BCSCS,and mammosphere formation.Knockdown of Runx2 expression inhibited the high tumorigenicity of BCSCS against immunodeficient mice.6.The relationship between Runx2 and EMT: By WB,the expression of Marker in MB-231 Mesenchymal reduced and the expression of epithelial-like Marker increased when Runx2 expression was knocked down.and overexpression of Runx2 in MCF-7,the result is the opposite.Conclusion: Down-regulation of Runx2 expression in breast cancer cells can inhibit breast cancer stem cell viability,invasion,clonality and in vivo nude mice tumorigenicity.Runx2 promotes the population of BCSCS through EMT to affect the transformation of common cancer cells into BCSCs. |
PDF Full Text Request