Novel miRNAs Regulate mEAK-7 to Inhibit mTORC1 Signaling

Objectives: miRNAs are small non-coding RNAs that are capable of post-transcriptional regulation and represent a potential novel class of therapeutics. miRNAs regulate many cellular processes, such as cell proliferation, differentiation, cell death, and have important roles in cancer biology. mTOR (mammalian target of rapamycin) regulates major cellular functions and aberrant activation of this pathway may result in human cancer development. We identified novel miRNAs that target mEAK-7, which is a positive regulator of the mTORC1 pathway. The goal of this study was to determine the extent to which novel miRNAs regulate mEAK-7-mediated mTORC1 signaling in human cancer cells.
Methods: Non-small cell lung cancer H1975 and H1299 and breast cancer MDA-MB-231 cell lines were transfected with 100 nM miRNA mimics or control. For microarray analysis, RNA was extracted 48 hours after transfection using Qiazol and miRNeasy mini kit. Samples were then processed and hybridized to the Affymetrix Human Gene 2.1 ST Array. For protein analysis, samples were extracted with NP-40 lysis buffer 48 hours after transfection. mEAK-7 and its downstream targets were analyzed. Cell proliferation assays were performed 3 and 5 days after transfection using the Luna Cell Counter. For cell migration xCELLigence System was used to capture real-time cell motility for 48 hours.
Results: Microarray data showed that mEAK-7 was one of several genes down-regulated by 2-fold after miRNA transfection; pathway analysis showed that several biological processes were affected by the miRNA, including cell cycle and response to stress. Western blot analysis demonstrated a significant decrease for mEAK-7, phospho-S6, and phospho-4E-BP1 protein levels for all cell lines studied. Cancer cells treated with novel miRNAs resulted in a significant reduction of cell proliferation and migration after 24, 36 and 48 hours.
Conclusions: We have identified novel miRNAs that are required for mTORC1 signaling through mEAK-7, and may play a role in human tumorigenesis.