Statistics Results are presented as mean standard deviation.

Materials and methods Cells and reagents MCF 7, MDA MB 157, and BT 549 human breast cancer cell lines were acquired from American Type Culture Col lection. Liquid nitrogen stocks were made upon receipt and maintained until the start of study. EREluciferase andor qPCR for ER and PgR enzyme 阻害剤 were used to con firm MCF 7 sustained estrogen responsiveness. Morphology and doubling times were also recorded regularly to ensure maintenance of phenotype for all cell lines. Cells were used for no more than 6 months after being thawed. Cells were maintained as previously described. RAD001 was pur chased from Selleck Chemicals LLC, and 17 beta Estradiol from Sigma. Animals 4 6 wks. old ovariectomized SCIDCB17 female mice were allowed a period of adaptation in a sterile and pathogen free environment with food and water ad libitum.<br><br> Cells were harvested in the exponential growth phase Lenalidomide 臨床試験 using a PBSEDTA solution and washed. Viable cells in 50 ul of sterile PBS suspension were mixed with 100 ul Reduced Growth Factor Matrigel. Injections were administered into the mammary fat pad using 27 ½ gauge sterile syringes. Animals were divided into treatment groups of five mice eachMCF 7 control vector, MCF 7 control vec tor plus E2, MCF 7 cells transduced to overexpress ma ture miR 155, MCF 7 cells transduced to overexpress mature miR 155 plus E2. Placebo or E2 pellets were implanted subcutaneously in the lateral area of the neck using a precision trochar. All procedures in animals were carried out under anesthesia using a mix of isofluorane and oxygen. RAD001 was administered as a micro emulsion dissolved in sugar water as 5 mgkgday.<br><br> Tumor size was measured every 2 3 days using digital calipers. The volume of the tumor was calculated using the formula43π LS2. Animals were euthanized by cervical dislocation after exposure to CO2. Tumors were removed and frozen in liquid nitro gen or fixed in 10% formalin for further LY2603618 911222-45-2 analysis. All procedures involving these animals were conducted in compliance with State and Federal laws, standards of the U. S. Department of Health and Human Services, and guidelines established by Tulane University Animal Care and Use Committee. The facilities and laboratory animals program of Tulane University are accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.<br><br> RNA Extraction and Quantitative Real Time RT PCR MCF 7 vector and MCF 7 miR 155 cells were harvested for total RNA extraction using Qiagen RNeasy RNA purification system or for microRNA miRNeasy purifica tion system per manufacturers protocol. Quantity and quality of the RNA and miRNA were determined by absorbance at 260 and 280 nm using the NanoDrop ND 1000. 2 ug of total RNA was reverse transcribed using the iScript kit and qPCR was performed using SYBR green. B Actin, PgR, ER, BCL 2, SDF 1, SERPIN3A, Rictor, TSC1, Raptor, Deptor, p70s6 kinase, and Rheb genes were amplified n 3. E2 stimulation experiments, cells were grown in 5% DMEM for 48 hours prior to treatment with 1 nM E2 or DMSO for 24 hours.