miR 375 was among the Crenolanib GIST few miRNAs significantly downregulated in breast cancer cells treated with tras tuzumab. This miRNA was found to target IGF1R and was Inhibitors,Modulators,Libraries identified as the key regulator of trastuzumab re sponsiveness via targeting IGF1R. Ectopic expression of miR 375 inhibited IGF1R expression and restored sen sitivity of breast cancer cells to trastuzumab. These data suggest that miR 375 may be a novel therapeutic targets for trastuzumab resistant breast cancers. Methods Cell culture and generation of trastuzumab resistant cells The human breast cancer SKBr 3 and human embryonic kidney 293 cell Inhibitors,Modulators,Libraries lines were obtained from the Institute of Biochemistry and Cell Biology, Chinese Acad emy of Sciences. SKBr 3 cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum and HEK293 cells were cultured in D MEM high glucose medium containing 10% FBS.

Inhibitors,Modulators,Libraries Both cell lines were main tained at 37 C in a humidified atmosphere containing 5% CO2. Trastuzumab/Herceptin was dissolved in sterile water. Trastuzumab resistant cells were developed by continuous culture of SKBr 3 cells in the presence of 5 ug/ml trastuzumab for 6 months, as reported previously. Thereafter, trastuzumab resistant and parental SKBr 3 cells were cultured with or without trastuzumab, respectively. Plasmid construction and preparation of lentivirus Short hairpin RNAs were designed to target 21 nt sequences of IGF1R mRNA and GFP mRNA as a con trol. These sequences were subjected to BLAST query to confirm the lack of homology to other known genes. The shRNA targeted sequences were as follows.

Paired deoxyribonucle otide oligos encoding the shRNAs were synthesized, Inhibitors,Modulators,Libraries annealed, and cloned into the EcoRI and NcoI sites of the pLKO. 1 vector. Lentivirus pack aging and infection were performed according to stand ard protocols as recommended by the manufacturer. The sequences of the primers used for PCR ampli fication of the pre miR 375 coding sequence were as follows The resulting PCR fragment was cloned into the pMD 18 T vector and successful cloning was confirmed by DNA sequencing. The pre miR 375 coding sequence was then subcloned into the lentivirus based expression plasmid pLenti6/V5, and virus packaging and infection were performed accord ing to protocols as recommended by the manufacturer. The miR 375 mimics, miR 375 inhibitor, and negative controls were purchased from Shanghai Genechem Inc.

Transfection Inhibitors,Modulators,Libraries of cells with 50 nM of each miRNA was performed using Lipofectamine 2000 reagent, according to the manufacturers instructions. Colony formation assay Colony formation in soft agar was tested by plating 1 104 parental PXD101 and trastuzumab resistant SKBr 3 cells in 0. 4 ml of complete DMEM medium supplemented with 0. 3% low melting temperature agarose in 12 well plates coated with 0. 8 ml 0. 6% low melting temperature agarose.