Inhibition of metastamiR -335 and -373, playing different functions in cancer stem cell rich MDA-MB231 cell line

Introduction: MicroRNAs which were known as a master regulator of gene expression, are small non coding RNAs that suppress gene expression post-transcriptionally by sequence-specific interactions with the miRNA response elements. MiR-335 is suppressing metastamiR but miR-373 is promoting. The existence of microRNA in metastasis pathways (metastamiRs) is hoping to discover a way to suppress miRs for reducing in metastasis. And also for cancer therapy by decreasing the number of stem cells, Inhibition of metastamiRs can possibly reduce the risk of breast cancer. Materials and methods: MDA-MB231 (Metastatic), MCF-7 (Tumourigenic) and MCF-10A (Non-tumourigenic) cell lines have been cultured. The proportions of CD44+/CD24-/low cells to detect breast cancer stem cell were evaluated by flow-cytometery. The double strand oligo of mature miR and inhibitor were designed and cloned to pcDNA 6.2gw/EmGFP according to the manufacturer's instruction of the BLOCK-iT™ Pol II miR RNAi Expression Kit. All cells have been transfected by lipofectamin 2000. Isolation of miRNA was performed by High Pure miRNA Isolation kit and expression of miR-335 and miR-373 were quantified by LNA enhanced RT microRNA PCR primer (real time PCR). Invasion and migration assay have been carried out. Results: The result of our evaluation shows that MDA-MB231 has a high proportion of CD44+/CD24-/low cells in comparison to the other cells. The relative expression of miR-335 and miR-373 in metastatic MDA-MB231 was 0.03% and 1.19%. After transfection miR-335 increase to 2.58% and miR-373 decrease to 0.71%. Matrigel-coated Transwell assay showed extreme reduction in invasiveness. Conclusion: Since cancer stem cells are resistant against common treatments and their isolation are arduous, MDA-MB231 cell line could be used as amenable BCSCs models to study innovative methods of cancer therapy. In this regard, miR-therapy could open new horizons in the field of cancer treatment and introduction of novel and promising drug candidates

Keywords:

Breast cancer stem cells , metastamiR