Bmi1/miRNAs Regulatory Loop Modulates Chemotherapy Response In Breast Cancer Cells

Backgound and ObjectiveBreast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females world. Although the treatment of this disease is development, there are over450thousand people died of breast cancer. Chemo-resistance and invasion are critical reasons in cancer patients’death. The mechanism of chemo-resistance is not clear yet. In order to study the mechanism, the5Fu resistance cell line MCF-7/5Fu was established. It showed not only resistance to therapeutics, but display an EMT phenotype and Stem cell Ratio up-regulated. There are many reports about the stem cell marker Bmil. It plays an important role in stem cell and cancer stem cell maintain. This study is focus on the role of Bmil in breast cancer chemo-resistance and it’s regulation network, based on the experiment of primary drug-resistance cancer cell MDA-MB-231, and obtained drug-resistance cancer cell MCF-7/5Fu. We want give a supplement to understand the mechanism of chemo-resistance in breast cancer. Give a new target to explorer drug to cancer therapy.Method(1) RT-qPCR was applied to measure the lever of Bmil mRNA, Western Blot was used to semi-quantify the protein lever in MCF-7> MCF-7/5Fu、MDA-MB-231and MDA-MB-453cell. The sensitivity of each cell line was test by MTS assay.(2) Overexpress Bmil is mediate the vector pMSCV-Bmil and knockdown Bmil was through the pRNAT-shBmil, transfect cells with lipofectamine2000. Puromycin or Blasticidin was used to screeing clones who had been transfected successfully. Western Blot detect the Bmil protein lever after overexpression and knockdown Bmil. MTS assay was applied to evaluate the sensitive of cells to5Fu, IC50was calculated. (3) The cells transfected with vectors were treated with100mg/L5-Fu, FACS was applied to exam the apoptosis changes, Caspase-9and Caspase-7activity were evaluate by enzymes labelling instrument.(4) Flow cytometry was used to examine the ratio of breast cancer cells after overexpress or knockdown Bmil.(5) microRNAs who target Bmil were predict through bioinformatics, conserved microRNAs in all species were choosed to study. The microRNAs lever was quantified by RT-qPCR. Transfect MCF-7/5Fu, MDA-MB-231, MDA-MB-453cells.3’-UTR of Bmil mRNA was cloned into pMIR-Report Vector Dual-luciferases assay was used to detect the relation ship of Bmil and microRNAs.(6) miRNAs RT-qPCR was used to check if the transfection was successful or not. Wild-type p53was transfect into all cells, then exam the miRNAs also. Western Blot assay was used to check the p53lever after transfect with the pEGFP-N1-p53, Co-immunoprecipitaion assay was used to test the interaction between Bmil and p53Result(1) There is no difference in mRNA lever between each cell lines, but the protein lever is different. Bmil protein lever in MCF-7cell is lower than in MCF-7/5Fu, MDA-MB-231and MDA-MB-453cells.(2)Overexpress Bmil enhance the resistance to5-Fu of MCF-7cell, Knockdown Bmil will enhance the sensitivity to5Fu in MCF-7/5Fu, MDA-MB-231and MDA-MB-453cells, and inhibit cells to apoptosis.(3)Bmil up-regulate the protein Bcl-2but inhibit Bax, knockdown Bmil will enhance the induction of5-Fu to cells through mitochondrial pathway, showed as the more cytochrome C release, and activate caspase-9and caspase-7.(4)Bmil will up-regulate CD44+/CD24-stem cell ratio in breast cancer,(5)Bioinformatics assay suggest that miRNA200c and miRNA203was conserved target Bmil between all kinds of species,Dual-Luciferase result proved that miRNA200c and miRNA203can bind to the3’-UTR of Bmi1mRNA. Leading the protein Bmi1was down-regulated.(6)Bmi1can negative feedback regulated miRNA200c but not miRNA203. Overexpress wild-type p53will reverse the inhibition of Bmi1to miRNA200c.(7)Bmi1can bind to p53, then decrease the protein lever of p53.Conclusion(1)Bmi1mediate breast cancer cell resistance to5-Fu through inhibit the mitochondrial apoptosis pathways,(2) Bmi1can up-regulate the CD44+/CD24-subpopulation cells,(3) miRNA200c repress Bmi1in breast cancer cells,(4) Identified miRNA203target Bmi1in breast cancer cells at the first time,(5) Bmi1negative feedback repress miRNA200c mediate by p53...