The Effects Of IL-13 On The Expression Of Autophagosome,Bcl-2 And TIGAR Under The Condition Of Fibroblasts Co-cultured With Breast Cancer Cells

ObjectiveInterleukin-13?IL-13?is a cytokine that is secreted by Th2 cells.They participate in organ fibrosis,inflammation and the occurrence and development of cancer.Studies have shown the presence of IL-13 in human breast cancer tissue.The microenvironment of human breast cancer often displays Th2-associated inflammation and fibrosis whose pathologies can promote tumor development.High levels of IL-13 as a representative of the immune imbalance may lead to the malignant selection of tumor cells.Tumor microenvironment plays an important role on tumor development,and fibroblasts are the main stromal cells in it.The fibroblasts interact with the components of tumor microenvironment through direct cell contact and secreting the informational molecules,and play a fertile soil or battery-operated role on tumor cell growth.In order to explore the molecular mechanism of IL-13 for the breast cancer,this paper studies the effect of IL-13 on anti-apoptosis factors B-cell lymphoma-2?Bcl-2?and Tp53-induced glycolysis apoptosis regulator?TIGAR?and autophagosome under the conditions of human breast cancer cells co-cultured with human fibroblasts.Methods1.Cell culture.After defroster,cells were cultured in Dulbecco's modified Eagles medium?DMEM?with 10%fetal bovine serum at 37?in a saturated humidity and 5%CO₂ atmosphere.Cells were co-cultured by plates and Transwell inserts.The human fibroblast line ESF and the human breast cancer cell line BT-474 were plated according to experimental requirements.This method allowed the ESF and BT-474cells to grow in the same medium without direct contact between them.IL-13 were added according to experimental requirements.2.RT-qPCR experiment.Total RNA was isolated using TRIzol reagent.2?L RNA solution were examined by Merinton SMA4000 equipment,using appropriate solvent as blank control.We observed the ratio and continuous wavelength absorption peak of A260/A280 and A260/A230,calculated the RNA concentration of the solution,then judged the quality of the RNA extraction.RNA between 2.0 and 2.3can meet the requirement of subsequent RT-qPCR experiment.3.Western blot experiment.Cells were lysed in RIPA lysis buffer for 30 min at4?.The cells were collected and placed in-80?fridge.Protein concentrations were determined by BCA method,and the sample was calculated according to standard protein concentration curve.Equal amounts of protein were separated by electrophoresis on an 8%polyacrylamide gel.The electrophoresed proteins were transferred to a nitrocellulose membrane.After incubation in a blocking buffer?5%non-fat dry milk,0.05%tween-20 in TBS?,the membranes were immune-blotted with antibodies overnight at 4?.After washing,the membranes were transfered into the glass dish containing the secondary antibody,at the room temperature for 1-2 h.Finally,the membranes were developed using ECL.The electrophoresis results of western blot were analyzed by Image J software.4.MDC staining and confocal microscopy experiments.Medium was removed.The cells on the cover slips were fixed using the 4%paraformaldehyde.After washing cells with PBS,autophagosomes were stained using MDC method.Cells were covered by 50?mol/L MDC-PBS solution 30 min at 37°C in the dark.After washing cells,autophagosomes were observed by confocal laser scanning microscope,and the fluorescence intensity of each group was analyzed using Image-Pro Plus software.5.Apoptosis experiment Annexin V.The cell quantity of each experiment sample was 5×10⁵.The cells were incubated with biotin-Annexin V for 15min,then incubated with PE-streptavidin-biotin.7-amino-actinomycin?7-AAD?was added before FACScan detection to distinguish the apoptosis from the other types of cell death.6.Cell proliferation experiment CCK8.800?L BT-474 cells were seeded in24-well culture plates and ESF cells in Transwell inserts in 5 copies with blank groups.The cell concentration was 4×10⁴/mL.The final quantity ratio of the co-cultured ESF cells with BT-474 was 4:1.After culture for 24,48,72,96 and 120 h,CCK-8 solution?20??L?were added to each well of the culture plates,and the cells were incubated in incubator for 30 min according to CCK-8 Kit instructions.A 100?L aliquot of the reactive solution was removed from each sample and then placed in96-well culture plates.The optical density was measured at 450 nm using a microplate reader.7.Statistical analysis.The experiment results are expressed mean±SD.The experimental data were analyzed with SPSS Statistics 17.0 software.Compare between two groups was conducted using the t-test.P<0.05 was considered to indicate a statistically significant difference,and P>0.05 was not.Results1.IL-13 upregulates the mRNA of anti-apoptotic factors Bcl-2 and TIGAR of human breast cancer cell BT-474 co-cultured with human fibroblast ESF.Compared with the other groups,Bcl-2 mRNA expression of BT-474 cells was significantly upregulated?P<0.05?in BT-474+ESF+IL-13 group.This was a 2.20-fold increase in Bcl-2 mRNA expression of BT-474 cells in BT-474+ESF+IL-13 group as compared with BT-474 group,and a 1.60-fold increase as compared with BT-474+ESF group,and a 1.26-fold increase as compared with BT-474+IL-13goup.Compared with the other groups,TIGAR mRNA expression of BT-474 cells was significantly upregulated?P<0.05?in BT-474+ESF+IL-13 group.This was a 2.40-fold increase in TIGAR mRNA expression of BT-474 cells in BT-474+ESF+IL-13 group as compared with BT-474 group,and a 1.80-fold increase as compared with BT-474+ESF group,and a 1.30-fold increase as compared with BT-474+IL-13goup.2.IL-13 upregulates the protein expression of anti-apoptotic factors Bcl-2 and TIGAR of human breast cancer cell BT-474 co-cultured with human fibroblast ESF.Compared with the other groups,TIGAR protein expression of BT-474 cells was significantly upregulated?P<0.05?in BT-474+ESF+IL-13 group.This was a 2.89-fold increase in TIGAR protein expression of BT-474 cells in BT-474+ESF+IL-13 group as compared with BT-474+ESF group,and a 1.60-fold increase as compared with BT-474+IL-13 group,and a 3.50-fold increase as compared with BT-474 goup.Compared with the other groups,Bcl-2 protein expression of BT-474 cells was significantly upregulated?P<0.05?in BT-474+ESF+IL-13 group.This was a 2.83-fold increase in Bcl-2 protein expression of BT-474 cells in BT-474+ESF+IL-13 group as compared with BT-474+ESF group,and a 1.51-fold increase as compared with BT-474+IL-13 group,and a 2.13-fold increase as compared with BT-474 goup.These results of mRNA and protein expression confirmed each other.3.IL-13 promotes the formation of autophagosomes of human fibroblasts ESF co-cultured with human breast cancer cells BT-474.The fluorescence intensity of autophagosomes of ESF cells was significantly higher in ESF+BT-474+IL-13 group than other groups?P<0.05?.This was a 1.86-fold increase in the fluorescence intensity of autophagosomes of ESF cells in ESF+BT-474+IL-13 group as compared with ESF+BT-474group,and a 3.78-fold increase as compared with ESF group,and a 1.20-fold increase as compared with ESF+IL-13 goup.These results indicated that IL-13 could upregulate autophagosome of the co-cultured fibroblasts.4.By upregulating Bcl-2,TIGAR and autophagosome,IL-13 inhibits apoptosis of human breast cancer cell BT-474 and promotes proliferation of BT-474 cells.Compared with BT-474+ESF co-culture group,the early apoptotic BT-474 cells of BT-474+ESF+IL-13 co-culture group decreased by 10.85-fold.Compared with BT-474+IL-13 co-culture group,the early apoptotic BT-474 cells of BT-474+ESF+IL-13 co-culture group decreased by 6.49-fold.Compared with BT-474 group,the early apoptotic BT-474 cells of BT-474+ESF+IL-13 co-culture group decreased by14.41-fold.The results of cell proliferation experiment showed that the groups did not significantly differ in BT-474 cell proliferation at 24 h?P>0.05?.But BT-474 cell proliferation was significantly greater in BT-474+ESF+IL-13 co-culture group than in other groups at 48,72,96,and 120 h?P<0.05?.Conclusion1.IL-13 upregulates the expression of anti-apoptotic factors Bcl-2 and TIGAR of human breast cancer cell BT-474.The co-culture of fibroblasts and breast cancer cells can promote the effects of IL-13 on Bcl-2 and TIGAR.2.IL-13 promotes the formation of autophagosomes of human fibroblast ESF.The co-culture of fibroblasts and breast cancer cells can promote the effects of IL-13on the formation of autophagosomes.3.By upregulating the expression of anti-apoptotic factors Bcl-2 and TIGAR of human breast cancer cells BT-474 and promoting the formation of autophagosomes of human fibroblasts ESF,IL-13 inhibits apoptosis and promotes proliferation of human breast cancer cell BT-474.