The Research Of IL-13 Effect On EGF Expression In Breast Cancer Tissue And Breast Cancer Growth

ObjectiveTo explore the mechanism of the effects of interlukin-13（IL-13）on breast cancer and try to provide new targets for breast cancer therapy,we studied the effect of IL-13 on epidermal growth factor（EGF）expression in breast cancer tissue and breast cancer growth through establishing a co-cultured model of human breast cancer cells with human fibroblasts in vitro and a breast cancer model in nude mice for simulating the breast tumor microenvironment in vivo and in vitro.Methods1.Cell co-culture in vitro Human breast cancer cell line MDA-MB-231 and human fibroblast line ESF were co-cultured by culture plates and Transwell inserts method.According to the experiment requirements,cells were seeded respectively.The cells seeded on culture plates were used for the experiments.In order to co-culture,the cell seeded-Transwell inserts were put into the culture plates to which cells had adhered.2.RT-q PCR We used Trizol method to extract total cellular RNA,observing A260/A280 ratio and continuous wavelength absorption peaks,judging the quality of RNA extraction.Main steps of c DNA synthesized by reverse transcription:Total RNA solution 11μL（about 0.6～0.8μg）,random primers 1μL,5×Reaction Buffer 4μL,Ribo Lock?RNase inhibitor 1μL（20 U/μL）,2μL（10 mmo L）d NTP（mix）,Revert Aid?MMLV reverse transcriptase 1μL,total volume 20μL,blending,at25℃for 5 min,at 42℃for 60 min,at 70℃for 5 min and then terminating reaction,then saving c DNA at-20℃.Main steps of PCR amplification reaction:IQ SYBR Green Supermix 10μL,consequent primer 1μL（10μmo L）,reverse primer 1μL（10μmo L）,c DNA 8μL（10μmo L）,total volume 20μL;at 50℃for 3 min,at 95℃for 3 min（IQ SYBR Green Supermix）,at 95℃for 6 s,at 60.5℃for 25 s,40 cycles;at 72℃for 7 min for extension;melting curve from 65℃to 95℃.The experimental data were automatically calculated and analyzed by fluorescence quantitative PCR analysis software named Bio-Rad CFX Manager.3.Western-Blotting We washed and collected the cultured cells,and added cell lysis buffer at 4℃for 30 min,12000 rpm centrifuge at 4℃for 5 min.According to the instruction of BCA protein assay kit,protein standart was prepared.Colorimentric determination was performed using a micro plate reader on 562 nm,then a protein standart curve was drawn.The samples were put into 96-well plates and the BCA working liquid was added.The absorbance value of each tested-wells was determined using the micro plate reader on 562 nm and the corresponding protein content of each tested-wells was found based on the standard curve.20μg protein of each lane were put for protein polyacrylamide gel electrophoresis（10%separated gel,10%concentrated gel）at 25 m A electrophoretic current of concentrated gel and 30m A electrophoretic current of separated gel.The electrophoresed protein was transferred to the PVDF membrane.After incubation in a blocking buffer（5%non-fat dry milk）,the membranes were immunoblotted with the first antibody supplemented with PBS,0.1%Tween-20 and 5%non-fat dry milk at 4?C overnight.After washing,the membranes were incubated with the secondary antibody.Finally,the membranes were developed using ECL.The optical density of each electrophoresis band of western blotting was analyzed using Image J software.4.Establishing a breast cancer model in nude mice 100μL cell suspension（cell concentration:2×10⁶MDA-MB-231 cells,1×10⁶ESF cells）were injected into the breast of right side of 6-7 weeks old female nude mice.Every other day we used vernier caliper to measure the length-diameter（a）and the short-diameter（b）of the tumors,calculated the tumor size according to tumor volume formula:V（mm3）=1/6πab2.Length-diameter of tumor was 5 mm which were regarded as tumor occurrence in tumor-burdened nude mice.When the length-diameter of tumor was up to 7 mm,IL-13 was injected into tumor of experimental groups at multiple-points every other day.The total concentration of IL-13 for one time was 1μg/kg,and the total time was nine.200μL normal saline were injected into tumor of control groups.The nude mice were killed by cervical dislocation on the 21^stday of IL-13 injection.The tumor tissues were put into 4%formalin to fix tissues.5.Immunofluorescence detectionTumor tissue was made into paraffin embedding sections.The paraffin sections were labeled by immunofluorescence dye after dewaxing process.Main steps are as follows:repairing antigen in 0.01 M citrate buffer and microwave;blocking with 1%BSA at 37℃for 30 min;incubating sections with primary antibody at 4℃overnight;placing them at room temperature for 30min;incubating sections wiht fluorescent secondary antibody in the dark at 37℃for60 min;staining nuclei with DAPI at room temperature for 10 min;sealing sections.The results were examined by laser confocal microscope,photographed,and analyzed by Image-Pro Plus software.6.Statistical analysisData are presented as mean±SD.SPSS Statistics software（17.0）was used to analyze the data.Statistical analysis of the data was performed by Student’s t-test.P values of<0.05 were considered to be statistical significance,P values of>0.05 were considered to be statistical no-significance.Results1.The results of EGF m RNA expression of the co-cultured human fibroblasts in vitro using RT-q PCR detection Compared with the single culture group and the co-culture group without IL-13,adding IL-13 could increase EGF m RNA level in ESF cells in the MDA-MB-231+ESF+IL-13 group（P<0.05）.The expression of EGF in the ESF group is lower than the other groups（P<0.05）.The effect of IL-13 on expression of EGF m RNA could be enhanced under the condition that breast cancer cells and fibroblasts were co-cultured.2.The results of EGF protein expression of the co-cultured human fibroblasts in vitro using Western-Blotting assayThe experimental results showed that the expression of EGF protein in MDA-MB-231+ESF+IL-13 group was higher than the other groups（P<0.05）.It indicated that IL-13 could promote the expression of EGF protein of fibroblasts under the condition that breast cancer cells and fibroblasts were co-cultured.It also showed that tumor microenvironment played a synergistic role in function of IL-13.3.The results of EGF expression of breast cancer tissues in tumor-burdened nude mice using immunofluorescence confocal microscopy assayConfocal microscopy was used to observe the expression of EGF in breast cancer tissue of tumor-burdened nude mice.EGF is marked by green fluorescence.The tumor tissue appeared strong green fluorescence in MDA-MB-231+ESF+IL-13 group,but the fluorescence intensity was weak in other groups.Image-Pro Plus software was used to analyze fluorescence intensity.The fluorescence intensity in MDA-MB-231+ESF+IL-13 group was much stronger than the other groups（P<0.05）.The fluorescence intensity was no significantly different between MDA-MB-231+IL-13group and MDA-MB-231 group.It indicated that IL-13 could not up-regulate the expression of EGF in MDA-MB-231 cells.The main target cells of IL-13 were fibroblasts and IL-13 could up-regulate the expression of EGF in fibroblasts.We observed the expression of EGF in MDA-MB-231 group which suggested that there was the basal expression of EGF in breast cancer cells,but IL-13 had no significant effect on the expression of EGF in breast cancer cells.4.The effect of IL-13 on the tumor growth of breast tumor-burdened nude（1）HE staining of the sections proved to be cancerous tissue.（2）Before the injection of IL-13,the tumor volume of MDA-MB-231+ESF+IL-13 group was no significantly different compared with MDA-MB-231+ESF group（P>0.05）.From the 7th to the21th day after injection of IL-13,the tumor volume of MDA-MB-231+ESF+IL-13group was significantly larger than MDA-MB-231+ESF group（P<0.05）.It indicated that IL-13 could promote the growth of transplanted tumor.（3）The tumor growth in MDA-MB-231+ESF+IL-13 group,as seen in the transplanted tumor growth curve diagram,was faster than MDA-MB-231+ESF group.（4）The tumor growth index was 5.73 in MDA-MB-231+ESF group and 11.43 in MDA-MB-231+ESF+IL-13group.The tumor growth index in MDA-MB-231+ESF+IL-13 group was 1.99 times more than MDA-MB-231+ESF group.The experimental results showed that IL-13could promote the growth of transplantated tumor.5.The results of IL-13 immunohistochemistry in human breast cancer tissue sectionsBrown grains in cytoplasm meant IL-13 positive.Image-pro-plus 5.1software was used to measure optical density of IL-13 immunohistochemistry in breast cancer group and beast no-cancer group.The results showed that the mean optical density value of IL-13 in breast cancer tissues was higher than breast no-cancer tissues（P<0.05）.It indicated that the expression of IL-13 in breast cancer tissues was higher than breast no-cancer tissues.6.The results of EGF immunohistochemistry in human breast cancer tissue sectionsBrown grains in cytoplasm meant EGF positive.Image-pro-plus 5.1software was used to measure optical density of EGF.The results showed that the mean optical density value of EGF in breast cancer tissues was higher than breast no-cancer tissues（P<0.05）.It indicated that the expression of EGF in breast cancer tissues was higher than breast no-cancer tissues.Conclusion1.IL-13 up-regulates the expression of EGF in human fibroblasts co-cultured with human breast cancer cells.2.IL-13 up-regulates EGF expression of fibroblasts in breast cancer tissues of tumor-burdened nude mice.3.IL-13 promotes the growth of breast tumor in tumor-burdened nude mice.4.Overexpression of IL-13 and EGF are detected in human breast cancer tissues.