MiR-6X Axis Regulates Herceptin Resistance In Breast Cancer

Background: Breast cancer is one of the most common malignant tumors of females,the incidence of which is increasing and the patients with which is tending to be younger.Herceptin(trastuzumab for injection),a humanized monoclonal antibody and first-line therapy for breast cancer with HER2(Human Epidermal Growth Factor Receptor 2)overexpression,can inhibit the tumor cell's proliferation.Efficiency of Herceptin is limited and Herceptin resistance occurs about a year or two years of treatment.Exploring the mechanism of resistance can prevent resistance and individualize treatment.How Herceptin exerts its function has been largely known,but its resistance mechanism is still to be elucidated.Objective: To explore the effect and mechanism of miR-6X(micro RNA-6X)regulation of Herceptin sensitivity and to better implement the breast cancer's treatment or reduce resistance by providing important theoretical basis and potential molecular targets.Methods: We used a Herceptin drug-resistant cell line and micro RNA chip to screen micro RNAs related to Herceptin resistance and to obtain a previously unreported miRNA(miR-6X).We used the truncated mutant genes and luciferase activity experiments to find miR-6 X promoter regions regulated by Herceptin.We looked through the literature with database to identify the CEBP(CCAAT Enhancer Binding Proteins)transcription factor regulating miR-6X.We used q RT-PCR(Quantitative Real-TimePolymerase Chain Reaction)to detect the effect of CEBP on miR-6X expression,CHIP(chromatin immunoprecipitation)to detect whether CEBP is recruited to the miR-6X promoter and whether this effect is regulated by Herceptin,q RT-PCR to detect the change of miR-6X after using RNA interference to knock down CEBP,Western Blot to detect whether miR-6X affects the protein levels of IGF1R(Insulin like Growth Factor 1 Receptor),cell transfection and luciferase activity experiments to test whether miR-6X directly targets IGF1R,the cell resistance curve and soft agar assays to test whether CEBP/miR-6X increase Herceptin sensitivity through IGF1R,Western Blot to test whether CEBP/miR-6X through IGF1R inhibit p AKT/p ERK(phosphorylation Protein Kinase B/phosphorylation Extracellular Signal-Regulated Kinase)pathways,cell resistance curve,soft agar,and Western Blot assays to test whether CEBP increases Herceptin sensitivity by miR-6X and CEBP and whether miR-6X increases Herceptin sensitivity by p ERK/p AKT pathways,in vivo imaging and HE(Hematoxylin and Eosin)staining to detect whether miR-6X impacts pulmonary metastasis of breast cancer in the body,immunohistochemistry and statistical analysis to test expression of CEBP,miR-6X and IGF1R in breast cancer and the relationship between the three,and immunohistochemistry and statistical analysis to detect relapse-free survival and overall survival with patients who received or did not receive Herceptin therapy.Results: 1.micro RNA chip showed that miR-6X,a previously unreported miRNA,was downregulated in the Herceptin drug-resistant cell line MDA-MB-453/MDA-MB-453 R.2.By using miR-6X truncated mutants and transfecting ZR75-1 cells with and without Herceptin,we found that the transcription factor CEBP regulated miR-6X promoter regions in the presence of Herceptin3.We found that CEBP increased miR-6X expression in a dose dependent manner by extracting total RNA,micro RNA reverse transcription,and q RT-PCR,and by transfecting different doses of Myc-CEBP in ZR75-1/MCF-7/MDA-MB-453 cells.4.After adding Herceptin to the cells of ZR75-1,we found that CEBP increased miR-6X,which was suppressed by Herceptin.5.We extracted total RNA,reverse transcribled micro RNA,and performed q RT-PCR,after transfecting CEBP sh RNA1 or CEBP sh RNA2 and treating with Herceptin in ZR75-1 cell.We found that Herceptin decreased miR-6X expression and Herceptin could no longer reduce miR-6X expression after we knocked down CEBP.6.We transfected miR-6X into ZR75-1 cells to find target genes regulated by miR-6X and found that the most obvious unreported gene was IGF1R.7.By using IGF1R 3'UTR(Universal Training Reactor)expression vector and its mutant,transfecting them with miR-6X into ZR75-1/MCF-7/MDA-MB-453 cells,and detecting luciferase activity,we found that miR-6X controlled the activity of IGF1R 3'UTR and this effect disappeared after IGF1R 3'UTR was mutated.8.By transfecting IGF1R sh RNA and miR-6X into ZR75-1/MCF-7 cells,we found that CEBP/miR-6X increased the Herceptin sensitivity with resistance curve and soft agar assays and this effect was abolished after knocking down IGF1R.9.By transfecting IGF1R shRNA and miR-6X into ZR75-1/MCF-7 cells,we found that CEBP/miR-6X inhibited p AKT/p ERK pathways with Western Blot and this effect was abolished after knocking down IGF1R.10.By transfecting Myc-CEBP and miR-6X inhibitor into ZR75-1 cells,we found that CEBP increased Herceptin sensitivity and this effect was abolished after knocking down miR-6X;CEBP inhibited IGF1R protein levels and p AKT/p ERK pathways and this effect was abolished after knocking down miR-6X using resistance curve,soft agar and Western Blot.11.By transfecting Myc-CEBP and LY(AKT inhibitors)/PD(ERK inhibitors)into ZR75-1 cells,we found that CEBP increased Herceptin sensitivity using resistance curve and soft agar and this effect was abolished after inhibiting the p AKT/p ERK pathways.12.By transfecting miR-6X and LY(AKT inhibitors)/PD(ERK inhibitors)into ZR75-1 cells,we found that miR-6X increased Herceptin sensitivity using resistance curve and soft agar,and this effect was abolished after inhibiting the p AKT/p ERK pathways.13.By injecting miR-6X Ago-miR and Herceptin into the tumor of nude mice,we found that miR-6X Ago-miR inhibited breast cancer metastasis and increased Herceptin sensitivity using in vivo imaging;By dissecting the tissue of its lungs to do the embedding and slicing,we found that miR-6X inhibited pulmonary metastasis of breast cancer in the body and increased Herceptin sensitivity by HE.14.CEBP and miR-6X in carcinoma-adjacent tissue were significantly higher than those in cancer tissue and the IGF1R expression in breast cancer was higher than that in normal tissue by immunohistochemistry and statistical analysis.15.CEBP and miR-6X in breast cancer were positively correlated,CEBP and IGF1R were negatively correlated and miR-6X and IGF1R were negatively correlated by immunohistochemistry and statistical analysis.The P values were meaningful.16.By examing relapse-free survival and overall survival in 103 cases of breast cancer treated with Herceptin therapy,we found that they were longer when CEBP/ miR-6X had higher expression(P value meaning);By examining relapse-free survival and overall survival in 109 cases of breast cancer without Herceptin therapy,we found that they were not longer when CEBP/ miR-6X had higher expression(P value meaningless).Conclusions: 1.miR-6X is one of micro RNAs related to Herceptin resistance.2.Herceptin inhibits miR-6X by CEBP modulation of IGF1R expression,therefore activating p AKT/p ERK pathways and causing Herceptin resistance,breast cancer metastasis and death.