IL-13Effect On Autophagy Genes Beclin1and LC3Ⅱ Expression Of Human Breast Fibroblasts Co-cultured With Human Breast Cancer Cells

Objective: Carcinoma-associated-fibroblasts (CAFs) is a recent widespread concernstromal cell. As it is the most important component of the tumor stroma, bycontinuously secreting a large number of cytokines and other substances to itsexternal environment, directly or indirectly affecting its surrounding tumor cells. Thisnot only promotes CAFs and its surrounding tumor cell proliferation, but alsoaccelerates the invasion and metastasis of tumor cells. There is evidence that CAFsdegrade their own macromolecules by high level autophagy and secrete nutrients tothe external environment to provide nutrition to cancer cells in the microenvironment.Interleukin13(IL-13) is a multifunctional cytokines secreted by Th2cells. Recentstudies show IL-13is expressed abnormally high in breast cancer and mastitis andinvolved in the regulation of the NF-kappaB pathway in fibroblast subtypes cells.NF-kappaB pathway is an important signaling pathway involved in autophagyregulation. Creating a tumor microenvironment by culturing human breast fibroblastswith human breast cancer cells, we try to explore the effect of IL-13on proliferationand autophagy gene expression of human breast fibroblasts in the tumormicroenvironment model.Methods:1. Cell culture. Cells were cultured in DMEM medium containing10%fetalbovine serum,105U/L penicillin and105U/L streptomycin in a37℃,5%CO2andhumidified incubator.2. Experiment groups. The cells were divided into four groups:Control group, the human breast fibroblast line Hs578Bst were cultured in this group.IL-13group, the human breast fibroblast line Hs578Bst were cultured with IL-13(final concentration of20ng/mL) in this group. Co-culture group (also called435group), the human breast fibroblast line Hs578Bst with the human breast cancer cellline MDA-MB-435were co-cultured in the Transwell suspension chamber.Co-stimulatory group, the human breast fibroblast line Hs578Bst with the humanbreast cancer cell line MDA-MB-435were co-cultured in the Transwell with a finalconcentration of20ng/mL IL-13.3. Cell viability assay. Cell viability was determinedby trypan blue staining in each group of Hs578Bst cells.4. Cell proliferation assay:Hs578Bst cells (1×105cell/mL) were placed in six-well plates adding a finalconcentration of20ng/mL IL-13or co-cultured with MDA-MB-435cells for72h. Theproliferation of Hs578Bst cells was measured by CCK-8kit.5. Immunocytochemistryanalysis: Autophagy proteins Beclin1and LC3II expressions were detected byimmunocytochemistry method in Hs578Bst cells cultured for72h. Optical density ofBeclin1and LC3II expressions was measured by Image-Pro Plus6.6. Real-time PCR.Beclin1and LC3II mRNA expressions were detected by real-time PCR in Hs578Bstcells cultured for72h. Results:1.The percentage of living cells of Hs578Bst cells was hardly changed afteradministration of IL-13to the medium.2. Cell proliferation assay results: Comparedwith the control group or IL-13group, the number of cells increased significantly (P<0.05) in435group and co-stimulatory group. No significant difference was observedbetween the control group and IL-13group (P>0.05).No significant difference(P>0.05) was also observed between the435group and co-stimulatory group.3.Immunocytochemistry analysis results: The expressions of Beclin1and LC3II inco-stimulatory group increased significantly (P<0.05) as compared with other groupsand the expressions of Beclin1and LC3II in435group also increased significantly (P<0.05) as compared with the control group or IL-13group. No significant difference(P>0.05) was observed between the control group and IL-13group.4.Determinationof autophagy gene mRNA expression: The expressions of Beclin1and LC3II mRNAin co-stimulatory group increased significantly (P<0.05) as compared with othergroups and the expressions of Beclin1and LC3II RNA in435group also increasedsignificantly (P<0.05) as compared with the control group or IL-13group. Comparedwith control group, expressions of Beclin1and LC3II mRNA were not significantlyenhanced (P>0.05) in IL-13group.Conclusions:1. Hman breast fibroblast line Hs578Bst proliferation is promoted inco-culture condition of Hs578Bst cells with human breast cancer cell lineMDA-MB-435.2. IL-13has no significant effect on the proliferation of human breastfibroblast line Hs578Bst.3. IL-13and human breast cancer cell line MDA-MB-435have a synergistic effect on human breast fibroblast line Hs578Bst,which can increasethe Beclin1and LC3II expression in Hs578Bst cells.4.There was no effect on theexpression of Beclin1and LC3II in human breast fibroblast line Hs578Bst with IL-13alone.