The Research On CCL5 Expressive Modulation In Mouse Breast Cancer Cell Line 4T1

Objective: The breast cancer as a threat to women’s physical and mental health has been the first malignant tumor in women, there is an upward trend in the incidence. Because the cause has not been fully elucidated, prevention,early screening and diagnosis has become the key to cure this tumor,this makes the molecular biological techniques become more importent. CCL5 chemotactic cytokines is a kind of inflammatory cytokines and can chemotaxis T cells and mononuclear cells, with multiple effect in tumor growth and metastasis, including the immune effect of inhibiting tumor cell, promote the growth of tumor, the creation of new blood vessels and transfer, etc, are the major chemokine expressed in breast tumor cells and is closely related to the progress of breast cancer. In this experiment, we try to find the mechanism which LPS increased CCL5 expression in breast cancer cell line 4T1.Methods:1 We checked TLR4 expression in breast cancer cell lines 4T1 by RT-PCR and Western Blot method with or without LPS stimulation.2 LPS stimulated 4T1 cells, extracting total RNA, detected MCP-1,VEGF and CCL5 expression at the genetic level using RT-PCR method.At the same time, we also used the method of real-time quantitative PCRto detect the expression of MCP- 1, VEGF and CCL5.3 We checked T-p38 and p-p38, T-Erk and p-Erk,T-JNK and p-JNK,T-p65 and p-p65 change after stimulation with LPS in 4T1 cell line usingWestern Blot method.4 We checked LPS induced CCL5 expression in 4T1 cells after blocking signaling pathways using real time quantitative PCR method.5 We built recombinant plasmid with purpose gene and transfected intomice macrophage RAW264.7 cell line using lipofectamine. luciferase data were determined on the luciferase detector machine.6 We checked the effects of p38 inhibitor on 4T1 cells migration and invasion by scratch and Transwell experiments.7 We detected EMT related protein expression in 4T1 cells after using p38 inhibitor by western blot method.Results:1 There were TLR4 expression in 4T1 cells at gene and protein level with or without LPS stimulation.2 At the genetic level, LPS stimulation significantly increased CCL5 expression in 4T1 cells compared with no LPS stimulation group, but the expression of VEGF and MCP- 1 there were no obvious change.3 LPS stimulation can induced obvious phosphorylation on p38, Erk,JNK and p65 in 4T1 cells.4 The expression of CCL5 were changed after using p38 and p65 inhibitors, but there were no obvious change of CCL5 expression after using Erk and JNK inhibitors.5 The luciferase detection results showed that LPS induced CCL5 high expression in 4T1 cells were implemented by increasing CCL5 promoter.6 The p38 inhibitors can inhibit 4T1 cells migration ability and has no impact on the invasive ability7 The p38 inhibitors can inhibit the EMT related protein beta- catenin and the expression of Vimentin, and no influence on the expression of Snail.Conclusion:1 LPS stimulation can increase CCL5 expression in 4T1 cells and this increase were implemented through enhancing CCL5 promoter.2 The p38 inhibitors can inhibit 4T1 cells migration and EMT related protein expression.