Here, we present a comprehensive overview of the repertoire of miRNAs expressed in glioblastoma samples, and we identify several miRNAs that are dysregulated in glioblastoma. Additionally, we report a number of miRNA sequence variants that are expressed in the brain. MiRNA regulation of gene expression is an important aspect of tumorigenesis and cancer progression, and gaining a better understanding of the miRNAs that are dysregulated in brain cancers therefore has the potential to reveal mechanistic insights into the transformed state as well as open the door to new treatment modalities.
Our findings from deep sequencing of brain tissue and tumor samples significantly add to the list of miRNAs that are aberrantly expressed in brain tumors. We identified several new miRNAs that were not previously reported to be dysregulated in glioblastomas, including miR-95, miR-543, miR-598, and miR-873 (, Table S6
). Consistent with other glioma miRNA profiling studies 
, we also observed down-regulation of miR-124, miR-128, miR-132, and miR-7, and up-regulation of miR-10b, amongst others, demonstrating that high-throughput sequencing can be an effective method for profiling miRNA expression.
Compared to microarray and PCR-based profiling platforms, which detect a pre-determined set of miRNAs, high-throughput sequencing provides a more complete picture of the miRNAs expressed in a given sample and is not restricted in the number of individual miRNAs that can be detected. While these advantages of high-throughput sequencing allow for a greater dynamic profiling range, the method is not without limits. Using the standard FDR selection criterium of p<0.05, only nine miRNAs were identified here as significantly dysregulated in glioblastoma samples compared to non-tumor brain tissue (Table S5
). Based on the read counts for individual miRNAs in Tables S2
, and S4
, previous profiling studies (listed in Table S6
), and data shown here regarding miRNAs such as miR-21 and miR-128 (, ) that do not meet the standard FDR-adjusted p<0.05 criteria (Table S5
), clearly additional miRNAs identified here are dysregulated in glioblastoma (, Tables S4
). We therefore expanded our cut-off to p<0.1 to include 21 significantly dysregulated miRNAs, 17 of which were down-regulated in glioblastoma.
Importantly, high-throughput sequencing allowed us to analyze sequence variations in the detected miRNAs and investigate the possible presence of exogenous miRNAs such as viral miRNAs. In this study, we observed significant levels of A to I editing for seven cellular miRNAs expressed in the brain and 5′ end polymorphisms for at least 30 miRNAs, which could have dramatic consequences on mRNA targeting 
. In particular, 5′ end sequence heterogeneity may have functional implications as there are observable differences in the dominant isoforms for a few miRNAs (miR-335, miR-106b, miR-324) expressed in glioblastomas.
One question we were able to address by deep sequencing was whether any viral miRNAs might be expressed in the tumors and perhaps, might play a role in pathogenesis. Human cytomegalovirus (HCMV) and other neurotropic viruses, such as the polyomavirus JC virus (JCV), which replicates in glial cells, have recently been implicated in gliomas, tumors of the central nervous system, and other brain cancers 
. Although it is unclear at this point whether these viruses have any specific role in tumorigenesis, the incidence of HCMV infection in patients with glioblastoma multiforme is high; viral proteins and viral DNA have been detected in over 80% of glioblastomas 
. Both HCMV and JCV are known to express high levels of viral miRNAs during infection 
. Therefore, we aligned sequencing reads from all samples to known viral miRNAs present in miRBASE v 16.0 as well as the genomes of human α-, β- and γ-herpesviruses (HSV-1, HSV-2, HCMV, HHV-6A/B, HHV-7, EBV, KSHV) and neurotropic polyomaviruses (JCV and SV40). No viral small RNAs were detected in any of the nine samples analyzed (data not shown), indicating that viral miRNAs are likely not a contributing factor to the development or progression of glioblastoma.
It is hypothesized that glioblastomas, like other cancers such as leukemia 
, arise and are maintained by a population of progenitor cells with stem-like characteristics including the ability to self-renew for unlimited proliferation 
. Indeed, the glioblastoma miRNA signature supports this hypothesis. Brain-enriched miR-124 and miR-7, for example, show poor expression in neural progenitors, but are highly expressed in neurons and have been implicated in neural differentiation 
. Similarly, CREB-induced miR-132 and miR-212, which share seed sequence homology, promote dendritic outgrowth from newborn neurons 
and can target methyl CpG binding protein 2 (MeCP2), a regulator of neuroplasticity 
. Finally, miR-128, which is also abundantly expressed in neurons, has been shown to target the Bmi-1 stem cell renewal factor 
. miR-7, miR-124, miR-128, miR-132, and miR-212 are amongst the most highly down-regulated miRNAs found in glioblastomas compared to non-transformed cells (, Table S4
). Furthermore, miR-7, miR-124, and miR-128 have been reported to impair cell growth and proliferation when over-expressed in glioma-derived stem cells 
At least 20 cellular miRNAs were differentially expressed in the six glioblastomas assayed here compared to non-tumor brain tissue, many of which (miR-128, miR-124, miR-7, miR-132, miR-139) are consistently dysregulated in not only gliomas but also other brain cancers including medulloblastomas and neuroblastomas 
. It is conceivable that several of the down-regulated miRNAs with no defined functions at this point (i.e., miR-873, miR-95, miR-543) may exhibit tumor suppressor activity and normally target components of key signaling pathways that promote and maintain the growth and survival of glioma cells as has recently been reported for miR-10b 
While elucidating the roles of miRNAs in tumor formation and progression is certainly an important area of research, we took a different approach here and used our miRNA profiling data to explore the possible use of miRNAs in regulating cell suicide gene therapy. The use of tissue-specific miRNAs to regulate transgene expression for therapeutic applications has been investigated in multiple cell types, including lymphoid and myeloid cells, muscle cells, liver cells, and brain cells 
. One study done with brain developed an insect baculovirus-based vector expressing HSV-TK under the regulation of three miRNAs (miR-31, miR-127, and miR-143) reported to be under-expressed in glioblastoma cell lines 
. This vector showed the same therapeutic efficacy in glioblastoma U87 cells as a control HSV-TK vector lacking artificial miRNA target sites, but when introduced into human astrocytes, which express the three selected miRNAs, showed restricted HSV-TK expression, leading to an increase in cell viability. In our analysis of primary human glioblastoma tissue samples and brain tissues, miR-31 was not detectable, miR-143 was down-regulated in only half of the glioblastoma samples analyzed, and miR-127 exhibited only modest down-regulation in glioblastomas (Table S4
). Instead, we focused on miRNAs that are consistently down-regulated in glioblastomas 
(, Table S6
), and chose miR-128 as the best candidate to restrict cell suicide gene expression. While miRNA effects on target mRNAs are usually modest, using a miRNA such as miR-128, which is brain-enriched and highly expressed in non-glioma cells, potentially maximizes the efficacy of miRNA regulation. Furthermore, reduced expression of miR-128 is not specific to just glioblastomas as this miRNA is also down-regulated in other brain cancers 
. Using an in vitro model to recapitulate expression of brain-enriched miRNAs, we demonstrated that differentiated neuronal SH-SY5Y cells transduced with a miR-128-regulated HSV-TK lentiviral vector are indeed resistant to killing by GCV due to the expression of endogenous miR-128.
In conclusion, the data presented here provide a comprehensive analysis of the miRNA signature in glioblastoma and demonstrate the potential application of miRNA-regulated genes in the therapeutic treatment of brain cancers— an approach that could be readily extended to cancers of other organs.