Involvement Of TRPM7 In MDA-MB-435s Breast Cancer Cells Migration And Invasion

BackgroudBreast cancer is one of common malignant tumors in women, which induces a serious of damage to the quality of life. Invasive breast cancer accounts for 22.9% of females and 16% of all tumors all over the world. Breast cancer incidence in developed countries is higher than developing countries, which developed the diet and living habits. In recent years, as people gradually raise the standard of living, breast cancer has shown a high incidence of multiple trends. According to the statistics, the incidence of breast cancer accounts for 7-10% of the body in China. Breast cancer is great impact on people’s health through the high metastasis and high invasion. Metastatic breast cancer is a complex multi-step process involving the expansion of cancerous cells from the breast to other areas of the body. It is a serious complication of breast cancer, as metastatic disease in breast cancer is often fatal.Breast cancer primarily metastasizes to the bone, lungs, regional lymph nodes, liver and to the brain, with the most common site being the bone. Breast cancer in women has been ranked the second cause of death, and the upward trend continues in china. Breast cancer has seriously affected the people’s living standard and life quality, so the breast cancer metastasis has the important practical significance.Metastatic disease is the spread of a disease from one organ or part to another non-adjacent organ or part, so this is being reconsidered due to new research. Cancer occurs after a single cell, cancer cells acquire the ability to penetrate the walls of lymphatic and/or blood vessels, after which they are able to circulate through the bloodstream to other sites and tissues in the body. After the tumor cells come to rest at another site, they re-penetrate through the vessel or walls, continue to multiply, and eventually another clinically detectable tumor is formed. This new tumor is known as a metastatic (or secondary) tumor. Metastasis and invasion are the important biological characteristics in tumors, and these are the main fetal factors. Metastasis and invasion are the same process in tumors, but different stage.Invasion throughout the entire process, and it is the prelude of the metastasis.The metastasis is the final result of invasion.Migration experiment is mainly to test the metastasis ability in tumor cells.Therefore, metastasis and invasion are closely related each other. Bone metastasis of breast cancer studies have shown that tumor cells can synthesize osteopontin and can be such a limitation and separation of osteopontin in vivo. These are closely related to migration and invasion of tumor. Metastasis of breast cancer cells interact with the surrounding tissue, but extracellular matrix (ECM) as a barrier to prevent cancer cell migration, so the breast cancer cells must break through the ECM barrier. Migration of breast cancer also depends on tumor angiogenesis, new blood vessels is an important condition of the tumor cell migration, invasion and proliferation, which can provide nutrition for the breast cancer cells and migration.Genetic factors, tumor suppressor genes and oncogenes play an important role in breast cancer, when some oncogenes tumor suppressor gene is missing or increased, it is possible to produce cancer. Some of the current study and breast cancer-related genes including:BRCA、MsH2、MsH6、p53、ERK、JNK、TRPs. TRPs (transient receptor channels) are a kind of important cation channels in the cell membrane, many of its subtypes, belonging to seven sub-families, different mechanisms, related to pain, temperature, mechanical feel, smell, taste, functionalities are very complex. TRPM7 was discovered and reported by Loren W in 2001.The beginning of TRPM7 named:TRP-PLIK, the protein molecular weight in the 220KDa, after the study found that, TRPM7 molecule is 6 transmembrane channels, the main physiological and biochemical functions in paragraphs 5 and 6 cross-between membranes. Has now been confirmed, TRPM7 is the main permeability of calcium and magnesium ions, divalent cations between 5 and 6 membrane-spanning structure domain, and then regulate the cells biological behavior. Some recent studies have shown that, TRPM7 molecule plays an important role in cancer cell proliferation, adhesion, which has also been reported, TRPM7 molecule involved in cancer cell migration and invasion. The occurrence of physiological and biochemical activities of these cells must involve in cells signal transduction. This study discussed that the TRPM7 molecule, in breast cancer cells MDA-MB-435s, in the growth and further by studying the TRPM7 molecule involved in cell migration, invasion, and up and downstream in the pathway of MAKPKs the possibility of phosphorylation and downstream MAPKs pathway.The expression of phosphorylated pathway to elucidate the molecular breast cancer MAPKs phosphorylation, as well as expected by changing the phosphorylation of MAPKs into therapeutic molecules to provide a new method for breast cancer treatment. Src protein is mainly upstream MAPKs, which is a cancer gene and a histidine kinase network in the growth of cancer cells play an important role in differentiation. So the regulatory mechanism of TRPM7 molecules can provide the new theoretical basis of mechanisms of tumor cells.Src as a proto-oncogenic tyrosine kinase may play a role in the regulation of embryonic development and cell growth. The protein encoded by this gene is a tyrosine-protein kinase whose activity can be inhibited by phosphorylation by Src kinase.Rui zheng showed that it could inhibite the migration and invasion of A549 lung cancer cell line by joining the inhibitor of Src-tyrosine.Hiscox showed that it could Elevate Src activity promotes cellular invasion and motility in tamoxifen resistant breast cancer cells.Hauck CR showed that stable expression of the FAK C-terminal domain (termed FRNK) in v-Src-transformed NIH 3T3 fibroblasts inhibited cell invasion through Matrigel and blocked experimental metastases.All these researches showed that Src plays an important role in cell migration and invasion.Mitogen-activated protein (MAP) kinases are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens, osmotic stress, heat shock and proinflammatory cytokines) and regulate various cellular activities, such as gene expression, mitosis, differentiation, proliferation, and cell apoptosis.Krueger indicated that MAPK was upregulated in estrogen receptor (ER)-negative, invasive human breast cancer cell lines, compared with noninvasive, ER-positive lines. Thus, they speculated that MAPK may be involved in cell migration and invasion. Demuth T research suggested that MKK3 and p38 could increase the glioma invasion, and predict the prognosis by detecting the abundance of MKK3 and p38 in patients.Saika S research suggested that phosphorylation and nuclear translocation of p38MAPK were markedly evident in migrating epithelium during healing, but not in uninjured epithelium of mouse, and addition of p38 MAPK inhibitors blocked cell migration In organ culture.All evidents suggested that MAPK family is trongly correlated with tumor cells migration and invasion. ObjectiveThe experiment is to investigate the role of TRPM7 in migration and invasion of breast cancer cell and investigate the MAPKs pathway protein content and protein phosphorylation after TRPM7 interference.Methods1. Routinely cultured breast cancer cells MDA-MB-435s, and Growth of the number of passages on cells.2. Acidic phenol-guanidine thiocyanate-chloroform extracted and purificated the breast cancer cells total RNA, and detected interference effect of TRPM7-siRNA by the fluorescence quantitative PCR.3. Optical microscope detected cancer cells morphology, spreading the situation, change adhesion before and after the TRPM7-siRNA interference.4. Wound healing assay and Transwell chamber assay detected the breast cancer cells migration changes after the the TRPM7-siRNA interference.5. Transwell chamber assay detected the breast cancer cells invasive ability.6. Western Blot Detected Src, AKT, ERK, p38, JNK protein abundance changes after TRPM7-siRNA interference.Results1. Liposomes can be effective in breast cancer cells MDA-MB-435s transfection, the transfection efficiency is 93.24%.There were no significant difference of cell number between normal group and transfected group (n=4, P>0.05)2. qRT-PCR showed that it’s very high transfected effect at the RNA level in breast cancer cells MDA-MB-435s, the transfection efficiency is 76.70%,and there no significant difference between siRNA1 and siRNA2,but significant difference vs NC group (n=3, P<0.05). Western Blot also showed that it’s very high transfected effect in breast cancer cells MDA-MB-435s at the protein level, significant difference vs NC group (n=3, P<0.05).3.46.50% decline in breast cancer cells adhesion after TRPM7-siRNA interference, significant difference vs NC group (n=3, P<0.05).4. Breast cancer cells migration and invasion were both decline, significant difference vs NC group (n=3, P<0.05).5. There is no changes in the expression of AKT and p-AKT after TRPM7-siRNA interference, no significant difference vs NC group (n=3, P>0.05).6. There is no changes in the expression of Src, but p-Src expression declined, significant difference vs NC group (n=3, P<0.05).7. There is no changes in the expression of p38, ERK and JNK; but the expression of p-p38, p-ERK and p-JNK were declined, significant difference vs NC group (n=3, P<0.05).Conclusion1. TRPM7 plays an important role in adhesion, migration and invasion of breast cancer cell MDA-MB-435s.It can decline the cells adhesion, migration and invasion after TRPM7-siRNA interference.2. According to the experiment of MAPK pathway protein phosphorylation, it suggested that TRPM7 participates in the migration and invasion of MDA-MB-435s cells, and may play the key role in migration and invasion. Whether TRPM7 correlates with another pathway still needs more experiments.