MiR-592 activates the mTOR kinase, ERK1/ERK2 kinase signaling and imparts neuronal differentiation signature characteristic of Group 4 medulloblastoma

The expression of miR-592 was found to reduce the malignant potential of Group 3 medulloblastoma cell lines, D283, D425 and HD-MB03. RNA-seq analysis was carried out to identify genes differentially expressed upon miR-592 expression in the medulloblastoma cell lines. MiR-592 expression was found to upregulate mTOR and ERK1/ERK2 signaling and bring about neuronal differentiation in D283 and D425 cells. Transcriptome sequencing was also carried out on the RNA extracted from the polyclonal population of D283 and D425 expressing miR-592 as well as the vector control population cells treated with rapamycin or U0126, a MAPK inhibitor to study if the mTOR signaling or MAPK signaling is instrumental in bringing about the neuronal differentiation. MiR-592 was expressed in a doxycycline-inducible manner in the medulloblastoma cell line using the lentiviral vector pTRIPZ. Total RNA was prepared from doxycycline-treated cells expressing miR-592 and those expressing the vector alone. Total RNA was also extracted from the doxycycline induced vector control and polyclonal population expressing miR-592, following treatment with mTOR signaling inhibitor or MAPK signaling inhibitor. Libraries were prepared from the polyA-enriched RNA fraction using the Illumina Truseq RNA library preparation kit and sequenced on the Illumina Hiseq platform