MicroRNA-34a Decreases Breast Cancer Stem Cells By Modulating DLL1

Background:Breast cancer is one of the most common malignant tumor in women in recent years.The incidence of Breast cancer in women ranks the second place in malignant tumor around women.Although methands of breast cancer treatment concludes surgical operation, radiotherapy, chemotherapy and so all, approximately 1/3patients will eventually develop relapse and metastasis clinically.Recent studies show that cancer stem cells(cancer stem cells, CSC) play an very important role in the process of metastasis and relapse. Various of genes and proteins have close relations to breast cancer stem cells. Among them, mi R-34 a plays really important roles in the proliferation, invasion,metastasis and apoptosis of breast cancer.As a result,only to kill breast cancer stem cells can we improve the prognosis of breast cancer.DLL1(Delta-like 1) protein,acting as a ligand of Notch1 receptors in the Notch signal pathway is a single transmembrane glycoprotein.DLL1 can induce the activition of the Notch signaling pathway,which can regulate cell differentiation and a variety of tissue growth. Another study shows that DLLl is also involved in the regulation oftumor proliferation, differentiation, apoptosis, metastasis, recurrence process. Notch signaling pathway can not only help to the embryo and the normal tissue growth but also plays a important role in cancer stem cells.For example,it plays a very important role in the regulation of stem cell proliferation, differentiation etc.The corresponding biological characteristics of the Notch signaling pathway must be reflected by the downstream target genes such as Hey, cyclin D1, Hes, NRARP, p21, pre-T, NF- kappa B and so on.Objective: To investigate the role of Mi R-34 a in its regulation of DLL1 protein,then to discuss the function of Mi R-34a/DLL1 on Notch signaling pathway and the breast cancer stem cells.Method:1.The breast cancer cell lines MCF-7 are the main objection, using Macs technology to sort CD44+/CD24- breast cancer stem cells,q RT-PCR and Western blot technique detect the expressions of Mi R-34 a and DLL1 in breast cancer stem cells.2.Using instant transfection technology to transfect mi R-34 a mimics and inhibitors into MCF-7 cell lines,q RT-PCR and Western blot technique detect the expressions of m DLL1 and DLL1、HEY1、Caspase3 protein.3.Using si RNA interference to knock down DLL1 gene,q RT-PCR and Western blot technique detect the expressions of m DLL1 and DLL1、HEY1、Caspase3 protein.4.Using CCK-8 experiments and Hochest33342 staining to verify the rugulation of mi R-34a/DLL1 on the proliferation and apoptosis of breast cancer stem cells.Results:1.Using Macs technology to select CD44+/CD24- breast cancer stem cells,then to detect the expression of mi R-34 a and DLL1 protein in breast cancer stem cells.The results show that the expressin of mi R-34 a is inhibited, while the DLL1 has a relatively high level.2.Transfecting the mi R-34 a mimics and inhibitors into MCF-7 cells,Western Blot detects the expression of designated protein, results show that over expression of mi R-34 a inhibited the expression of DLL1. The expression of HEY1 protein, ALDH1 protein have a positive correlation with DLL1,,while the expression of Caspase3 protein has a negative correltion with DLL1.3.Knocking down DLL1 gene by the si RNA interference technology, Western Blot detects the expression of HEY1, Caspase3, ALDH1 protein,finding that their expression tendency are in consistent with the expression level of mi R-34 a mimics group.4.Using CCK-8 to detect the proliferation of MCF-7(the cells has been transfected by Mi R-34 a mimics and inhibitors),results show that the down-regulation of DLL1 protein inhibits the proliferation of MCF-7 cells,while upregulation of DLL1 protein has a promoting effect on cell proliferation.Hochest33342 staining shows that downregulation of DLL1 protein increases cell apoptosis.The apoptosis may have a closely relationship with Inhibition of cell proliferation.Conclusion:1.Mi R-34 a can downregulate the expression of DLL1 protein and promote the apoptosis of breast cancer cells.2.Dll1 can regulate apoptosis of cancer cells and breast cancer stem cells by activating the Notch signal pathway.