The Role Of Chemokine CCL18 In The Invasion And Metastasis Of Breast Cancer Cells And Its Mechanism

Objectives:1.To explore the effects of chemokine CCL18 on the proliferation,apoptosis and invasion of human hormone receptor positive(HR)breast cancer cell line BT-474 and triple negative breast cancer(TNBC)cell line MDA-MB-231.2.To investigate the effect of chemokine CCL 18 on epithelial mesenchymal transition(EMT)in BT-474 cells and MDA-MB-231 cells.3.To investigate the activation of chemokine CCL18 on EMT related transcripts in BT-474 cells and MDA-MB-231 cells.4.To explore the effect of chemokine CCL18 on the prognosis of breast cancer patients.Methods:HR+breast cancer cell line BT-474 and TNBC cell line MDA-MB-231 were selected as experimental subjects and cultured routinely.The proliferation of breast cancer cells was detected by sulfororhodamine B(SRB)colorimetry;flow cytometry(FCM)was used to detect the apoptosis of breast cancer cells after ccl18 treatment;transwell migration and invasion assay was used to detect the migration and invasion of breast cancer cells after ccl18 treatment;western blot(WB)was used to detect the expression of E-cadherin and vimentin in cell 8 treated breast cancer cells;real time quantitative polymerase chain reaction(qRT PCR)was used to detect the expression of EMT related transcription factors in cel18 treated breast cancer cells;Kaplan Meier plotter was used to analyze the overall survival rate of breast cancer patients in public data set,and one-way ANOVA was used to analyze the experimental data.Results:1.The result of SRB showed that the proliferation rate of BT-474 cells(F=8.062,P<0.05)and MDA-MB-231 cells(F=9.308,P<0.05)were not significantly changed after ccl18 treatment.The results of flow cytometry showed that the apoptosis rate of BT-474 cells and MDA-MB-231 cells was not significantly changed(P<0.05).The results of Transwell migration and invasion assay showed that the number of BT-474(F=10.666,P<0.05;F=15.212,P<0.05)and MDA-MB-231(F=24.870,P<0.05;F=18.637,P<0.05)migration and invasion into the lower surface of Trans well chamber was significantly increased after treatment with chemokine CCL18.Moreover,the number of cells that migrated and invaded to the lower surface of the Transwell chamber was no longer increased in BT-474 cells(F=44.944,P<0.05;F=18.883,P<0.05)and MDA-MB-231 cells(F=25.655,P<0.05;F=22.374,P<0.05)after using anti-CCL18.2.The result of Western blot showed that after induction of chemokine ccl18 for 48 h,the expression of vimentin,which is associated with mesenchymal phenotype,increased in BT-474 cells(F=10.188,P<0.05)and MDA-MB-231 cells(F=50.398,P<0.01),and vimentin expression was no longer elevated in BT-474 cells(F=19.882,P<0.05)or MDA-MB-231 cells(F=24.123,P<0.05)cultured with the addition of anti-ccl18.At the same time,induction with the chemokine ccl18 for 48 h,the expression of E-cadherin,a marker of epithelial phenotype,was decreased in BT-474 cells(F=31.831,P<0.01)and MDA-MB-231 cells(F=63.076,P<0.01),and E-cadherin expression was no longer decreased in BT-474 cells(F=17.984,P<0.05)or MDA-MB-231 cells(F=60.682,P<0.05)when anti-ccl18 was added to the cultures.3,The result of qRT PCR analysis revealed that the expression levels of snail transcription factors were significantly elevated in BT-474 cells after induction with chemokine ccl18 for 48 h(F=21.57,P<0.05),whereas the expression levels of slug and twist were unchanged.However,the expression level of snail in the experimental group with the addition of anticcl18 was almost flat to that of the control group(F=58.292,P<0.05).MDA-MB-231 cells were also treated with chemokine ccl18 for 48 h,and the expression level of snail was significantly elevated(F=24.36,P<0.05)among the three transcription factors,while the expression levels of slug and twist did not change significantly.Upon addition of anticcl18,the expression level of snail was no longer elevated(F=19.182,P<0.05).4.Kaplan Meier plotter analysis results showed that ccl18 high expression group predicted worse overall survival than low expression group in breast cancer patients(P<0.05),with a hazard ratio(hazard rate,HR)of 1.48.At the same time,the effect of high ccl18 expression on overall survival was greater in patients with lymph node positive or those with high histological grade.Conclusion:1.Chemokine ccl18 promoted the migration and invasion abilities of BT-474 cells(a hormone receptor positive breast cancer cell line)and MDA-MB-231 cells(a triple negative breast cancer cell line),but had no significant effect on their increasing ability or apoptotic process.2.Chemokine ccl18 promotes the migration and invasion of BT-474 cells and MDA-MB-23 1 cells by activating their EMT process.3.Chemokine ccl18 promotes EMT in BT-474 cells and MDA-MB-231 cells by activating snail transcription factor.4.High expression of chemokine ccl18 is associated with poor patient prognosis.