| Breast cancer is the most common malignant tumor which Chinese women are suffering from. At present the comprehensive therapy of breast cancer has greatly increased the clinical curative effect. But many patients still die from recurrence, metastasis and treatment resistance. The ultimate goal to beat breast cancer, especially advanced breast cancer is to overcome treatment resistance, recurrence and metastasis. So find the mechanism and related factors of breast cancer biological characteristics, such as proliferation, relapse, resistance,metastasis and treatment resistance, is becoming a top priority.Breast cancer stem cells(BCSCs) are small portion of tumor cells which have the characteristics of stem cells, such as self-renewal, high proliferation and multi-directional differentiation. Multiple evidence indicate that breast cancer stem cells are reason for cancer recurrence, metastasis and treatment resistance. A series of stem cell related pathways control the biological function of BCSCs. The most important is Wnt/β-catenin signaling pathway. Studies have showed that the aberrant activation of Wnt/β-catenin signaling pathway in breast cancer, especially in BCSCs, can trigger the development of a variety of genes’ expression associated with cancer initiation and development, and this is the critical factor in maintaining breast cancer stem cell proliferation. β-catenin is an integral member of adherens junctions that regulates the cellular dynamics of cell attachment or detachment.β-catenin has dual roles within the cell as the main mediator of Wnt signaling and as a junctional protein involved in cell-cell contact. Experiments showed that inducing high expression of Wnt-l and β-catenin can increase the proportion of breast cancer stem cells in the mouse mammary tumor tissue.The researches showed β-catenin and its downstream target genes cyclin D1 and c-my are over expressed in breast cancer. Thus, the specific blocking of Wnt/β-catenin signaling pathway in BCSCs has an important value for the treatment of breast cancer.Micro RNA(micro RNA, mi RNA) is a short chain endogenous small RNA with length of about 22 nt, regulates target genes’ translation and expression by binding with the3’non-coding region(3’UTR) of target m RNA. Therefore, micro RNA and it’s target m RNA which encode structural and regulatory proteins form a complicated network and regulate individual development, cell differentiation, proliferation, apoptosis, and tumor development.Nowadays several micro RNAs has been found regulating Wnt/β-catenin signaling pathway activity by inhibiting β-catenin expression and then regulating BCSCs, while Wnt /β-catenin signaling pathway is an important pathway in the regulation of stem cell properties of cancer stem cells in breast cancer.But there is no research about the function of mi R-1 regulating Wnt/β-catenin signaling pathway in BCSCs.Thus, in this study, we aimed to study the action mechanism of mi R-1 regulating Wnt/β-catenin signaling pathwy and breast cancer stem cells, as well as the effects of breast cancer related genes to breast cancer biology behaviors. The detail work is as follows:In this experiment, first we investigated the profile of the Wnt/β-catenin signal-related mi RNA expression in BCSCs and non-CSC tumor cells by mi RNA microarray and showed that there are 14 mi RNAs up-regulated, 13 mi RNAs down-regulated in BCSCs compared with non-CSC tumor cells, with mi R-1 down-regulated. The levels of mi R-1 were determined by Quantitative RT-PCR analysis in different subtypes of breast cancer tissues and their corresponding serum samples, confirmed that mi R-1 expression was associated inversely with the aggressiveness of breast cancer. To understand the role of mi R-1 in the function of BCSCs, MCF-7, SKBR3 cells, MCF-7/CSCs and SKBR3/CSCs were transfected with, or without, mi R-1mimic or mi R-1inhibitor to increase or reduce the expression of mi R-1. Flow cytometry analysis revealed that enhanced mi R-1 expression in SKBR3 cells decreased the percentages of SKBR3/CSCs and mi R-1 inhibition in MCF-7 cells increased the percentages of MCF-7/CSCs. Thus mi R-1 overexpression reduced breast cancer cell lines of cancer stem cell ratio. The potential targets of mi R-1 in the Wnt/β-catenin signaling were characterized by bioinformatics analysis and luciferase assay. Dual luciferase assay and weastern blot revealed that enhanced mi R-1 expression significantly reduced the Frizzled 7and TNKS2-regulated luciferase activity in 293 T cells and decreased Frizzled 7, TNKS2,c-Myc, Oct4 and Nanog expression and the ratios of nuclear to cytoplasmic β-catenin as well as β-catenin-dependent luciferase activity in breast CSCs in vitro. Thus mi R-1 inhibits the Wnt/β-catenin signaling and reduces the stemness of breast CSCs. Proliferation and migration assay revealed mi R-1 inhibited proliferation, migration of breast CSCs in vitro.Xenograft breast tumor model revealed enhanced mi R-1 expression inhibited the growth of implanted MCF-7/CSCs while mi R-1 inhibition promoted the growth of implanted MCF-7/CSCs in vivo. These data(Research 1.1) indicate that mi R-1 down-regulates BCSCs stemness, proliferation and migration by targeting the Frizzled 7 and TNKS2 to inhibit the Wnt/β-catenin signaling. In view of that cancer stem cells are considered to be the source of breast cancer genesis, metastasis, and drug resistance, mi R-1 blocking therapy may play an important role in preventing and blocking breast cancer relapse and metastasis。By studying correlations between biological behavior of breast cancer and β-catenin,cyclin D1, c-myc, the key factors of Wnt/β-catenin signaling pathway(Research 1.2), we found β-catenin abnormal expression rate, cyclin D1 and c-myc positive expression rate in breast cancer group was significantly higher than the corresponding index in hyperplasia group. Lymph node metastasis is related with the expression of cyclin D1 and c-myc. The abnormal expression of β-catenin in breast cancer is related with cyclin D1 and c-myc expression. Western blot indicated that the expression of β-catenin in breast tissue, cancer cell cytoplasm and nucleus was significantely higher than breast hyperplasia. The level ofβ-catenin in nucleus was higher than cytoplasm. These data suggest that the occurrence of breast cancer may be associated with abnormal expression of β-catenin, positive expression of cyclin D and c-myc, cyclin D1 and c-myc may be activated by β-catenin.Lipase member H protein is a crucial tumor regulating factor just like Wnt/β-catenin signaling pathway. It is an enzyme encoded by LIPH genes in human, involved in several biological processes, including platelet aggregation, smooth muscle contraction, the stimulation of cell proliferation. Present studies have showed LIPH m RNA expressed in intestines, lungs and pancreas, but the expression in breast is unclear. To find the new target blocking the proliferation of breast cancer, we studied relations between the biological behavior of breast cancer and Lipase member H(Research 1.3), we found that comparedwith adjacent tissues, breast cancer tissue is of high proportion of Lipase member H protein expression, and the expression significantly correlated with tumor size, histological grade,lymph node metastasis and postoperative distant metastasis. Further analysis detecting the expression of Lipase member H protein was identified as an independent prognostic factor for breast cancer. So Lipase member H protein is a poor prognostic factor for breast cancer,and it is hopeful to be a potential target for breast cancer management. |
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