The Research Of Effect Of IL-13 On SDF-1 Expression Of Fibroblasts Co-growth With Breast Cancer Cells In Vivo And In Vitro

ObjectiveIn this study,the effect of interlukin-13（IL-13）on stromal cell-derived factor-1（SDF-1）expression in human fibroblasts co-cultured with human breast cancer cells was investigated in vivo and vitro in order to explore the mechanism and effect of IL-13 on occurrence and development of breast cancer.It could provide target molecules for breast cancer treatment and their theoretical and experimental basis.Methods1.The co-culture of human fibroblasts and human breast cancer cells in vitroHuman fibroblast line ESF and human breast cancer cell line MDA-MB-231were co-cultured using plates and tranwell inserts（0.4μm）in vitro.DMEM-H medium was supplemented with 10%fetal bovine serum,10μg/m L streptomycin and100U/m L penicillin.The tested cells（2×10⁵cells/well）were plated at the bottom of 6well culture plates.After the tested cells adhered for 2-4 h,the Transwell inserts plated with cells（1.5×10⁵ cells/Transwell insert）were placed into these wells for co-culture in DMEM-H medium.2.RT-qPCR experimentsTotal RNA was isolated using Trizol method.DEPC-H₂O was used for blank control.2μL RNA solution were examined by Merinton SMA4000 equipment.We examined the ratio of A260/A280 and A260/A230 and continuous wavelength absorption peak,calculated the RNA concentration of the solution,then judged the quality of the RNA extraction.If the ratio of A260/A280 and A260/A230 was between 2.0 and 2.3,it could meet the requirement of subsequent RT-qPCR experiments.3.Immunofluorescence flow cytometry assayCells were digested and collected,then put in 4%paraformaldehyde for 30 min.After washing with PBS for 2 times,0.1%Triton X-100 solution were used for 30min to rupture cell membranes.5%BSA solution were used for 60 min.Cells were incubated in 1:100 rabbit-anti-human polyclonal antibody of SDF-1 at 37℃for 2 h.Then 1:500 goat-anti-rabbit Ig G-FITC secondary antibody solution were used in the dark at room temperature for 60 min.Cells were resuspended using FCM buffer and detected by flow cytometer.4.Cell proliferation experimentThe proliferation of cells was detected using Cell Counting Kit-8（CCK-8）in vitro.The detected cells were seeded in 24-well plates（4×10⁴ cells/well）.The Transwell inserts（3×10⁴ cells/Transwell insert）seeded the co-cultured cells were inserted into the 24-well culture plates for co-culture.Five wells were set for each group.MDA-MB-231 cell proliferation was detected from day 1 to day 5.According to the CCK-8 Kit instructions,20μL CCK-8 solution were added in each well,and incubated for 1 to 3 h in incubator.100μL from each sample were taken and moved into 96-well culture plates to measure the absorbance values at 450 nm wavelength using a microplate reader.5.Establishing the model of human breast cancer in nude miceESF and MDA-MB-231 cells were cultured according to regular method.After washing cells with PBS,100μL cell suspension（2×10⁶MDA-MB-231 cells,1×10⁶ESF cells）were injected into the right side breast of 6-7 weeks female nude mice.Tumor size was measured 3 times each week.When the length-diameter of tumors was 5 mm,we considered that the nude mice had tumorigenesis.When the length-diameter of tumors was about 7 mm,we started IL-13 administration.IL-13was injected into tumors in experimental groups.The mice were killed by cervical dislocation at day 21 of IL-13 injection.The tumor tissues were placed in 4%formalin fixative,then paraffin sections were performed.6.Immunofluorescence confocal microscopy detectionAfter section dewaxing,the sections were placed in 0.01M citrate buffer（pH 6.0）in the microwave to repair antigen.The sections were incubated with 1%BSA in an incubator at 37℃for 30 min,then incubated with the first antibody at 4℃overnight.After sections were put at room temperature for 30 min,the sections were incubated with fluorescent-conjugated secondary antibody in the dark at 37℃for 60 min.Cell nuclei were stained by DAPI at room temperature for 10 min.Sections were sealed by neutral gum.The results were observed using confocal microscope,photographed,and analyzed using Image-Pro Plus software.7.Immunohistochemistry experimentSections were deparaffinized,hydrated,washed with PBS,and incubated with0.3%H₂O₂ at 37℃for 10 min.The first antibody was added to incubate the sections at 4℃overnight.And the sections were incubated with the secondary antibody at 37℃for 15 min.DAB solution was used to color,hematoxylin to stain cell nuclei.The sections were sealed by neutral gum subsequent to dehydration and transparence.The results were observed using microscope,photographed,and analyzed using Image J software.8.Statistical analysisThe experimental results are expressed （?）±SD.SPSS Statistics 17.0 software was used to analyze experimental data.The differences between two groups were analyzed using t-test or analysis of variance.P<0.05 was considered to indicate a statistically significant difference,and P>0.05 was the reverse.Results1.IL-13 up-regulates the expression of SDF-1 m RNA in fibroblasts co-cultured with the breast cancer cells in vitroCompared with the other groups,SDF-1 m RNA expression of ESF cells was significantly up-regulated（P<0.05）in ESF+MDA-MB-231+IL-13 group,which was1.98 times more than ESF group,1.40 times more than ESF+IL-13 group,1.47 times more than ESF+MDA-MB-231 group.SDF-1 m RNA expression of ESF cells was significantly lower in ESF+IL-13 group than ESF+MDA-MB-231+IL-13 group（P<0.05）.These results indicated that IL-13 could up-regulate SDF-1 m RNA expression of ESF cells and the co-culture of fibroblasts with breast cancer cells could enhance the up-regulaton effect of IL-13 on SDF-1 expression.2.IL-13 up-regulates the expression of SDF-1 protein in fibroblasts co-cultured with the breast cancer cells in vitroCompared with the other groups,the peak of SDF-1 protein expression moved to the right,which indicated that the expression of SDF-1 protein was up-regulated.The results suggested that IL-13 could up-regulate the expression of SDF-1 protein of fibroblasts co-cultured with breast cancer cells.And the results were consistent with PCR results.3.IL-13 promotes the proliferation of breast cancer cells and fibroblasts in vitroThe proliferation of MDA-MB-231 cells was no significant difference among groups on the first day（P>0.05）.The proliferation of MDA-MB-231 cell was significantly faster in ESF+MDA-MB-231+IL-13 group than other groups（P<0.05）from day 2 to day 5.This showed that IL-13 could promote the proliferation of breast cancer cells co-cultured with fibroblasts.The proliferation rate of breast cancer cells in the co-culture group was faster than breast cancer cells cultured alone.The proliferation of ESF cells was no significant difference among groups on the first day and the second day（P>0.05）.The proliferation of ESF cell was significantly faster in ESF+MDA-MB-231+IL-13 group than ESF+MDA-MB-231 goup（P<0.05）from day3 to day 5.4.IL-13 up-regulates SDF-1 expression of breast cancer tissue in tumor-burdened nude miceAfter immunofluorescence labeling,slices were observed using confocal microscopy to examine the effect of IL-13 on the expression of SDF-1 in tumor tissue.Strong green fluorescence emerged in the tumor tissue of ESF+MDA-MB-231+IL-13group,and weak green fluorescence emerged in the tunor tissue of ESF+MDA-MB-231 group.The fluorescence intensity was analyzed by Image-Pro Plus software.The results showed that green fluorescence intensity was significantly higher in ESF+MDA-MB-231+IL-13 group than ESF+MDA-MB-231 group.The significant expression of SDF-1 was not detected in MDA-MB-231+IL-13 and MDA-MB-231groups,suggesting that IL-13 could promote SDF-1expression of fibroblasts in breast cancer tissue of tumor-burdened nude mice.5.Overexpression of IL-13 and SDF-1 in clinical human breast cancer tissuesThe results of immunohistochemistry showed that IL-13 and SDF-1 in clinical human breast cancer tissue displayed high expression.However,IL-13 and SDF-1were not significantly expressed in clinical human breast no-cancer tissues.Conclusion1.IL-13 up-regulates SDF-1 expression of human fibroblasts co-cultured with human breast cancer cells in vitro.The co-culture of fibroblasts and breast cancer cells could promote the effect of IL-13 on SDF-1 expression.2.IL-13 promotes the proliferation of human breast cancer cells co-cultured with human fibroblasts.And IL-13 accelerates the proliferation of co-cultured fibroblasts.3.IL-13 interference targets SDF-1 of fibroblasts in breast cancer tissue of tumor-burdened nude mice,and up-regulates SDF-1 expression.4.IL-13 and SDF-1 are highly expressed in clinical human breast cancer tissues.