| Background: Malignant neoplasms are one of the most important diseases that threaten human health. The incidence of malignancy is increasing year by year. Breast cancer has become the first malignancy of women. In 2008, there were 1.38 million new cases of global breast cancer, death 453,000, so breast cancer is widely studied around the world. With the human genome research has decoded human gene, breast cancer etiology related genes are also found, the rapid development of breast cancer treatment, significantly improved the overall survival of patients with breast cancer and disease-free survival. However, recent studies have shown that there are a small number of cells with particularly strong tumorigenic ability and poorly differentiated cells in tumor tissues, including breast cancer, with self-renewal and multi-directional differentiation of stem cells, called "cancer stem cells,(CSCs) ". Cancer stem cells play a decisive role in initiating tumor formation and growth and are the leading cause of tumor recurrence, metastasis and chemotherapy resistance. In vitro and in vivo experiments have shown that breast cancer stem cells have resistance to radiotherapy and chemotherapy. Traditional tumor therapy is aimed at ordinary tumor cells, but can not fundamentally eliminate cancer stem cells, after treatment, cancer stem cells to re-form tumor tissue and lead to tumor recurrence and metastasis. More effective tumor therapy requires the removal of tumor cells while removing common tumor cells. So the new treatment and new target discovery is imminent, is the focus of research in recent years.The majority of transient receptor potential (TRP) channels are a cationicnonselective channel consisting of the alpha helix domain of the six transmembrane domains and the N-terminal and C-terminus of the cells, Six transmembrane domain constitutes the pore area. TRP channels form functional tetrameric or isomeric tetramers that function in signal transitions. Recent studies have found that TRP channel family and gastric cancer, prostate cancer, leukemia and lymphoma and other malignant tumors related. TRPM7 (transient receptor potential malestatin7) belongs to the LTRP subfamily, and has a protein kinase structure and a cationic channel structure. It has been reported that TRPM7 is highly expressed in lung cancer, bladder cancer and breast cancer and is associated with cell proliferation and metastasis. But TRPM7 is how to regulate breast cancer cell function and whether it can breast cancer stem cells, so far no relevant research. Based on previous research results, we make the assumption that TRPM7 may play an important role in the proliferation and migration of breast tumor cells and may have a modest effect on breast cancer stem cells, which affects the tolerance of breast cancer to chemotherapeutic drugs.Objective: To study the effect of TRPM7 on the proliferation, invasion and metastasis of breast cancer cell lines and to explore possible regulatory mechanisms.Methods: QPCR, Western blot and immunohistochemistry were used to evaluate the m RNA and protein expression of TRPM7 in breast cancer patients and breast cancer cells. The effects of TRPM7 on the function of breast cancer cells were studied by MTT, transwell and scratches healing. Flow cytometry is used to detect cell cycle and changes in breast cancer stem cells. Finally, Western blot was used to detect changes in related signaling pathways. Then, TRPM7 non-specific ion channel inhibitor 2-APB was used to treat the cells, and the related function and signal pathway were detected. The specific functional differences of TRPM7 double active protein were clarified.Result:1, TRPM7 expression in breast tumor tissue, and elevated expression of TRPM7 in tumor tissue after chemotherapy, and the high expression of TRPM7 was associated with poor prognosis of breast cancer patients.The expression of TRPM7 in tumor tissue and its corresponding adjacent tissues was detected by immunohistochemistry. The expression of TRPM7 in tumor tissue was higher than that in adjacent tissues. QPCR showed the same trend of QPCR. QPCR was the same The expression of TRPM7 m RNA in the samples before and after chemotherapy showed that the expression of TRPM7 was increased after chemotherapy. At the same time further retrieval of the database found that high expression of TRPM7 breast cancer patients overall survival is poor.2, knock low TRPM7 expression can inhibit breast cancer cell proliferation, migration and invasion.The use of lentivirus transfection system to establish stable knockdown of TRPM7 expression in MDA-MB-231 and MCF7 breast tumor cell lines. MTT assay showed that the ability of cell proliferation was decreased after knockdown of TRPM7 expression. Scratch repair and transwell experiments showed that knockdown of TRPM7 decreased cell migration and invasion.3, knock down the TRPM7 cell cycle arrest and MET process.The expression of E-cadherin was decreased and the expression of E-cadherin was increased by the results of Western blotting. The results showed that the expression of E-cadherin was decreased in the cell cycle after flow cytometry. , Thereby reducing the migration and invasion of cells.4, TRPM7 in breast cancer stem cells in the high expression of knockdown TRPM7 will reduce the proportion of breast cancer stem cells, thereby enhancing the sensitivity of breast cancer cells to chemotherapy drugs.The expression of TRPM7 in ALDH + tumor stem cell subsets was higher than thatin the control group. The results showed that TRPM7 could reduce the proportion of ALDH + tumor stem cells. In addition, compared with the control group, the proportion of ALDH + tumor stem cells after knockout group was significantly decreased compared with the control group.5, knock low TRPM7 significantly affected STAT3 and AKT signaling pathways.Western blot was used to detect the phosphorylation of major signal transduction pathways. The results showed that knockdown of TRPM7 significantly reduced the phosphorylation of STAT3 and AKT in MDA-MB-231 cells. Suggesting that TRPM7 may regulate tumor stem cells through these two signaling pathways.6, Inhibition of TRPM7 ion channel activity can inhibit breast cancer cell proliferation, migration and invasion, cycle arrest, increased apoptosis and down-regulation of breast cancer stem cell ratio, but only affect the AKT signal pathway.The results showed that inhibition of TRPM7 ion channel activity inhibited the proliferation, migration and invasion of breast cancer cells, increased cell cycle arrest, increased apoptosis and down-regulation of breast cancer stem cells and the effect of TRPM7 on non-specific ion channel inhibitor 2-APB was performed. The proportion, the trend and knockdown TRPM7 results consistent, but in addition to proliferation inhibition, the rest are not as low knockdown TRPM7. Signal pathway only inhibit the AKT pathway, no significant effect on STAT3.Conclusion: (1) TRPM7 is highly expressed in breast cancer and has significant correlation with the development and prognosis of breast cancer. (2) Low knockdown of TRPM7 can down-regulate the proliferation of breast cancer by promoting G1 phase arrest and MET process (3) knockdown of TRPM7 can down-regulate the proportion of breast cancer stem cells and enhance the sensitivity of tumor cells to paclitaxel;(4) TRPM7 mainly through STAT3 and AKT signaling pathway to play a function,(5) TRPM7 ion channel activity is involved inmost of the regulation of tumor function, but only affects the AKT pathway, STAT3 pathway may only be regulated by kinase activity. |
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