MicroRNA-21 overexpression contributes to vestibular schwannoma cell proliferation and survival.

Vestibular schwannomas (VSs) are benign Schwann cell-derived tumors associated with the vestibular nerve and are a hallmark of the autosomal dominant genetic disorder neurofibromatosis type 2 (NF2). NF2 has been linked to mutations in the NF2 gene which encodes the tumor suppressor protein “merlin” (aka schwannomin) (1,2). Although the exact mechanisms whereby merlin prevents tumor formation are not completely understood, recent efforts to define the associated genes and molecular pathways involved in tumorigenesis and expansion have met with some success (3). VSs frequently go undiagnosed until clinical symptoms develop such as hearing loss, tinnitus, and balance impairment as the tumors grow larger. Left untreated, vestibular schwannomas can become life threatening. At present, the most common methods of treatment are observation with serial imaging studies, microsurgical removal and stereotactic radiosurgery. MicroRNAs are evolutionarily conserved, small (∼22 nt), non-coding RNA molecules that regulate gene expression post-transcriptionally. Mature microRNAs bind to specific mRNA targets in regions that are significantly complementary to the microRNA and, by a mechanism that is not completely understood, results in translational repression or mRNA degradation (4,5). The human genome encodes more than 1000 microRNAs with tissue- and cell-type specific expression (6). MicroRNAs have been shown to play important roles in such diverse cellular processes as differentiation, development, metabolism, apoptosis and cancer (7). Studies have shown that tumors generally exhibit aberrant microRNA expression profiles, and identified multiple microRNAs with reputed tumor suppressor or oncogenic properties (8-10). In preliminary studies investigating microRNA expression profiles in four human VSs using a microarray platform, we found that hsa-miR-21 (herein referred to as miR-21) was consistently over-expressed when compared to normal vestibular nerve. Over-expression of miR-21 has been observed in many cancers including breast, liver, and glioblastoma (11-13). Phosphatase and tensin homolog (PTEN) tumor suppressor gene has been identified as a target of miR-21 (12). PTEN acts as a tumor suppressor via its inhibitory effect on the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, which promotes cell survival, cell proliferation, and tumor formation (14). The PI3K/AKT pathway has recently been shown to be active in VSs (15). Therefore, elevated levels of miR-21 in VS may contribute to tumor growth by down-regulating PTEN and consequent hyperactivation of AKT signaling. The microRNA-21 gene has an upstream enhancer region containing two strictly conserved signal transducer and activator of transcription 3 (STAT3) binding sites and activation of STAT3 has been shown to induce the expression of miR-21 (16). STAT3 is a critical regulator of gene expression in response to many growth factors and cytokines (17). For example, the neuropoietic cytokines ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin 6 (IL-6) bind to specific ligand-binding receptor subunits and share the signal transduction subunit gp130 which signals through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway (18). Interestingly, merlin has been shown to play a role in suppressing STAT3 activation through its interaction with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) in a human schwannoma cell line (19). Furthermore, these authors showed that a naturally occurring NF2 missense mutation interferes with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) binding and abolishes the ability of merlin to inhibit STAT activation. This raises the possibility that over-expression of miR-21 in VSs may be a consequence of deregulated activation of STAT3 by an autocrine or paracrine mechanism involving neuropoietic cytokines or other growth factors. In the present study, we sought to confirm our earlier microarray results showing over-expression of miR-21 in the four original VSs using quantitative real-time polymerase chain reaction assays, measure the expression of PTEN mRNA and protein levels, and determine whether CNTF, LIF, IL-6, the receptor subunits neuropoietic cytokines ciliary neurotrophic factor receptor alpha (CNTFRα), leukemia inhibitory factor receptor (LIFR), interleukin 6 receptor alpha (IL-6Rα) and signal transduction subunit gp130, and STAT3 are expressed in these tumors. We also sought to examine miR-21 expression levels in additional VSs, greater auricular and normal vestibular nerve samples to increase the power of our statistical analyses. Finally, we sought to demonstrate that transfection of human VS cultures with anti-miR-21 oligonucleotides reduce their proliferative potential and promotes apoptosis which would suggest that over-expression of miR-21 contributes to VS growth.