The Expression Of AGR3 In Breast Cancer And Its Function And Mechanism Study

Objective: We analyzed the Gene Expression Omnibus(GEO)database and found that the expression of AGR3(Anterior Gradient protein3)in ER(Estrogen Receptor)-positive breast cancer(BC)was significantly higher than that in ER-negative breast cancer.However,the function and mechanism of AGR3 have not been reported in breast cancer yet.The purpose of this study was to investigate the expression of AGR3 in ER-positive and ER-negative breast cancer and in different breast cancer subtypes,and to further clarify the relationship between AGR3 and ER and its potential regulation mechanism in breast cancer cells,as well as prediction and clarify the specific functions of AGR3 in breast cancer,so as to provide a new theoretical basis for the diagnosis and treatment of breast cancer.Methods: We collected a total of 44 pairs of ER-positive and 28 pairs of ER-negative fresh frozen clinical breast cancer patient specimens and matched para-cancerous tissues,and the tissue was stored in liquid nitrogen for subsequent RNA extraction.The mRNA expression of AGR3 in these72 pairs of breast cancer tissue specimens and paired para-cancerousspecimens was detected by qRT-PCR.We also analyzed the expression of AGR3 in ER-positive and ER-negative breast cancer patients and the expression between breast cancer patients and normal patients through chip data from different GEO databases.The effect of AGR3 on survival in patients with ER-positive and ER-negative breast cancer was also analyzed online using the Kaplan-Meier Plot curve.RNA and protein expression level of AGR3 in each human breast cancer cell line was also evaluated by qRT-PCR and Western Blotting(WB).We treated cells with estradiol in cell lines that have AGR3 protein expression,and extracted RNA from different estradiol treatment time,and reversed RNA to cDNA and detected the change of AGR3 mRNA level.We also treated cells with4-hydroxyoxymoxifen(4-OH-TAM)in high expression of AGR3 protein cell line and tested the effect of different concentrations of 4-OH-TAM on cell viability.Simultaneously,the RNA and protein were extracted and changes in ESR1 and AGR3 RNA level and protein level were detected.Finally,AGR3 was knocked down in AGR3 high expression cell line,and its biological behavior of breast cancer cells and underling molecular mechanism were explored by qRT-PCR,WB and flow cytometry.Results: The expression of AGR3 in ER-positive breast cancer was significantly higher in ER-positive breast cancer than in ER-negative breast cancer,and AGR3 had a significant positive correlation with ESR1.The expression of AGR3 in breast cancer tissues was also significantly higherthan that in normal breast tissues,and the differential expression of AGR3 in different ER states was only found in breast cancer tissues,but not in adjacent ER-positive and ER-negative normal tissues.In ER-positive breast cancer,the high expression group of AGR3 had a poor relapse-free survival(RFS)relative to the low-expression group,while in ER-negative breast cancer,the high expression group of AGR3 had a poor recurrence-free survival rate and overall survival(OS)relative to the low expression group.In the ER-positive luminal A and luminal B breast cancer patients,the expression of AGR3 in cancer was significantly higher than that in matched normal tissues,while in ER-negative breast cancer(TNBC)patients,the expression of AGR3 in cancer tissues was significantly lower than that in matched adjacent normal tissues.We also analyzed paired cancer and para-cancerous tissues in TCGA and found similar differential expression of AGR3 in ER-positive and ER-negative breast cancer tissues.While in HER-2(Human Epidermal Growth Factor Receptor-2)breast cancer tissue,there was no significant difference between the paired adjacent tissues.GSEA(Gene Set Enrichment Analysis)analysis showed that AGR3 was significantly enriched in estrogen response and other cancer-related signaling pathways.When adding estradiol into ER-positive breast cancer cell lines,the RNA level of AGR3 was significantly up-regulated.And the treatment of ER-positive breast cancer cell lines with 4OH tamoxifen significantly reduced cell proliferation activity,and the RNA and proteinlevel of AGR3.Finally,knocking down AGR3 in AGR3 high expression breast cancer cell line showed a significant decrease in cell proliferation activity,and increase cell proliferation activity.Conclusion: Our study showed that AGR3 was highly expressed in ER-positive breast cancer,estrogen could up-regulate AGR3 expression and 4-OH-TAM can decrease AGR3 expression.AGR3 can promote the proliferative activity of breast cancer cell lines,which may affect the biological behavior of breast cancer by estrogen-dependent pathway and may be a potential therapeutic target for ER-positive breast cancer.