Induction of epithelial mesenchymal transition, invasion, androgen independent Ngigem growth and sensitivity to inhibition of MUC1 C. MATERIAL SAND METHODS Cell culture Human LNCaP f prostate cancer in RPMI1640 medium with 10% heat-inactivated serum Fetal bovine serum, 100 units / ml penicillin, 100 mg / ml streptomycin and 2 mM glutamine were cultured L. LAPC4 AUY922 cells were cultured in Iscove, the updated Dulbecco, s medium containing 5% FBS HI, antibiotics and glutamine cultured L. LNCaP cells and were LAPC4 with lentiviruses expressing GFP or MUC1 C-infected and in select hygromycin. In some experiments, the cells in phenol red-free medium containing activated carbon were cultured removed. The cells were treated with bicalutamide. The cells were also measured using the inhibitor of MUC1 C GO 203 or controlled CP treated peptide 2 as described. Immunpr Zipitation and immunoblotting Subconfluent cell lysates were prepared as described. The L Soluble proteins Were coated with anti AR or anti-MUC1 C executed Filled exemplary immune system Filled and lysates not F Precipitation were subjected to immunoblotted anti-AR, anti-MUC1-C, anti-PSA and anti-b actin, anti-E- cadherin, and the fight against vimentin. The immune complexes were treated with horseradish peroxidase-conjugated secondary Rantik Body and reinforcing Detected markets chemiluminescence. To assess the effects of MUC1 C on AR gene transcription, we transfected LNCaP cells / GFP and C LNCaP/MUC1 with a vector, the promoter upstream Rts of the AR gene for luciferase. Analysis of luciferase activity t showed that the MUC1 C is so little influence on the AR promoter activation. Similar results were LAPC4 cells, suggesting that MUC1-C negatively regulates AR expression in post-transcriptional level. Previous work has found that some microRNAs down-regulated AR expression in prostate cancer cells. Of these, it was found that associated with the expression of MUC1 C in LNCaP cells with upregulation of miR 135b and miR 147th For the Best Confirmation that miR 135b down-regulated AR mRNA levels, we overexpressed miR 135b in LNCaP cells.
Analysis of mRNA levels AR 48 and 96 h after transfection, showed a decrease in AR transcription. An overexpression of miR 135b was also associated with decreased AR protein. These results show that the MUC1 C AR expression decreases at least partially by the upregulation of miR 135b. MUC1 CCytoplasmicDomainBindsDirectly toar earlier work showed that MUC1-C associated with the ERA and the cytoplasmic Cathedral Ne of MUC1-C-Dom Ne binds directly to DNA binding ERA. To determine whether MUC1-C associated with AR, co-Immunpr Zipitation of cells lysates carried out LNCaP/MUC1 C Detection of MUC1-C complex AR supports the assertion that MUC1-C interacts with AR. To partially define the base of the interaction ARMUC1 C LNCaP lysates with GST or GST MUC1 CD were incubated. The results show that AR binds to GST MUC1-CD and not GST, indicating that the AR with the cytoplasmic Dom assigned ne of MUC1 C AR consists of a domain AF1, DBD, hinge region, and the Bindungsdom Ne AF2/ligand. The incubation of MUC1 CD with GST AR, AR GST, GST or AR showed that MUC1-CD binds to the AR DBD region. To better assess the interaction between MUC1 CD and DBD AR, AR, we incubated with GST or GST-MUC1-CD. Analysis of the adsorbates with a file.