27 We found that mTOR and p70S6K,28 which are key downstream ABT-263 mouse signals of PI3K, also contained miR-7 target sites in their 3′UTR. Using PIK3CD siRNAs (Fig. 2A; Supporting Figs. 2, 3B, 7, and and mTOR siRNAs (Supporting Figs. 14 and 15) as positive controls, we concluded that miR-7 regulates the PI3K/Akt/mTOR-signaling pathway. The repression of

both mTOR and p70S6K by miR-7 and the upstream regulator, PIK3CD, interfered with the transcription of various proteins, including cell-cycle–associated proteins,27 providing a possible basis for the observed cell-cycle delay. It was recently revealed that miR-7 is induced by a dysregulation of EGFR signaling and that it acts as an important modulator of EGFR-mediated oncogenesis in lung cancer cells.25 EGFR is a known target of miR-7.7 These

findings suggest that a negative feedback loop might exist between miR-7 and its targets (Fig. 8, left). We hypothesized that the transcription factors associated with miR-7, such as c-myc25 and HoxD10,10 might be activated by the PI3K/Akt/mTOR pathway through an unknown mechanism and induce miR-7 expression, resulting in the suppression of miR-7′s molecular targets. This homeostasis could be dysregulated in cancer cells by a failure to activate the transcriptional factors, inhibition of the transcription of miR-7, or aberrant action of miR-7 without altering its expression (Fig. 8, right). It is established that polymorphisms within miRNA target sites can impact the

interaction between miRNA and target mRNAs, a process that is associated with neoplasia,29 disease,30 and organismal development.31 In addition to miRNA target-site mutations, BAY 73-4506 chromosomal translocations, which separate the oncogene open reading frame (ORF) from its 3′UTR, containing related miRNA target site(s),32 and alternative splicing events33 may also lead to the loss of miRNA function in the post-transcriptional regulation. We explored miR-7 function in the context of HCC. We previously compared the endogenous expression of Carnitine palmitoyltransferase II miR-7, PIK3CD mRNA, and p110δ proteins in QGY-7703 with L-02, a normal liver cell line (Supporting Fig. 16A). We found that both PIK3CD mRNA and p110δ protein were overexpressed in QGY-7703, although they had a similar level of miR-7 expression. We then aligned PIK3CD 3′UTRs cloned from QGY-7703 and L-02, but no mutations in the miR-7 target regions were found (Supporting Fig. 16B). It has not been reported that chromosomal translocation or alternative splicing events occur at the PIK3CD gene locus in HCC. We demonstrated that overexpression of miR-7 markedly down-regulated the reporter luciferase activity, indicating that luciferase expression was suppressed when PIK3CD 3′UTR was cloned into the 3′ terminal region of the luciferase ORF. When miR-7 was transiently transfected into cells, both PIK3CD mRNA and p110δ expression was repressed, compared to the controls (Supporting Fig. 1A).