الفهرس 
MATERIALS AND METHODSOnly 14 pages are availabe for public view
 |  | from | 120 |  |  |
| | | from | 120 | | |
Abstract
Triple negative breast cancer (TNBC) is the most aggressive subtype and represents up to 20% of breast malignancy. TNBC is uniquely characterized by lack of expression of surface receptors for estrogen, progesterone and Her2/neu, therefore it doesn’t respond to ordinary treatments such as tamoxifen or aromatase inhibitors or therapies that target and inhibit HER2 receptors, such as trastuzumab.
MiRNAs are class of promising emerging regulatory molecules. They are short, non-coding form of RNAs that are differentially expressed in cancer tissues. miRNAs can either act as tumor suppressors or promotors by regulating their target proteins through suppression of gene translation.
MiR-206 has been reported to be down-regulated in several types of cancer including gastric, liver, lung and breast cancer. Latest reports have linked miR-206 in inhibition of cell proliferation, migration, stemness and metastasis in breast cancer
Vascular endothelial growth factor (VEGF) and the Kirsten rat sarcoma viral oncogene homolog (KRAS) are two of the deriving genes in cancer progression . In the current study we aimed to investigate the possible differential expression of miR-206, VEGF and KRAS in TNBC compared to non-TNBC and normal tissues. We also investigated the regulatory role of miR-206 on genetic expression of VEGF and KRAS in TNBC.
Aim of the Work: The aim of this study was to examine miR-206 expression in triple negative breast cancer cells and its correlation with the expression of vascular endothelial growth factor (VEGF) and Kirsten rat sarcoma viral oncogene homolog (KRAS) genes.
Materials and Methods: Human Breast Cancer Tissue Samples: Forty-two formalin-fixed, paraffin-embedded (FFPE) archived breast cancer tissues and corresponding apparently normal adjacent breast tissues were collected from BC patients who underwent surgical resection. Samples were then subdivided according to their receptor status into triple negative (n=15) and non-triple negative (n=37) groups. The age of patients ranged from 32-69 years and their clinicopathological characteristics were recorded.
Summary
80
Cell Lines and Culture Conditions :The human breast adenocarcinoma triple negative cell line MDA-MB-231 (ATCC(R) HTB-26) and the ER expressing MCF-7 cell line (ATCC(R) HTB-22TM) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 mg/ml of streptomycin sulfate. Cells were allowed to grow in humidified atmosphere at 37°C under 5% CO2 in 6 and 96–well tissue culture plates.
The miR-206 miScript miRNA mimic (As a drug) was transfected into MDA-MB-231 and MCF-7 cells at using stock concentration of 100 nM. Reverse transfection started with adding miR-206 mimic to 96-well plate followed by the addition of HiPerFect transfection reagent diluted in culture medium.
The viability of Cells MDA-MB-231 and MCF-7 cells were evaluated using the MTT assay. Cells were seeded into 96-well plates. 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was added 48h following the treatment. Cells were then incubated for 3 hours. Viable cells are detected by its mitochondrial conversion of the tetrazolium salt MTT into formazan crystals, which can be solubilized for homogenous measurement at OD 540. The control cell viability was defined as 100%.
:Total RNA was extracted from formalin-fixed, paraffin-embedded tissues using RNeasy FFPE Kit and from cell lines using RNeasy Mini Kit according to manufacturer instructions. The purity and concentration of extracted RNA were evaluated by NanoDrop(R) ND-1000 UV-Visible Spectrophotometer. Total RNA was reverse transcribed using TaqMan™ advanced miRNA cDNA synthesis Kit for miR-206 and high-capacity cDNA reverse transcription Kit with RNase Inhibitor for VEGF and KRAS, according to the manufacturer’s instructions. Real-time PCR was performed by TaqMan kits. U6 small nuclear RNA and GAPDH were used as internal controls to normalize the expression of miR-206, VEGF and KRAS respectively.
Results: Downregulation of miR-206 and upregulation of VEGF and KRAS in TNBC tissues: Quantitative real time PCR results showed that miR-206 expression in TNBC tissue is significantly lower than non-TNBC and normal breast tissues (p=0.002 and
Summary
81
<0.001 respectively The levels of both VEGF and KRAS were inversely correlated to miR-206 expression