MicroRNA Biomarkers in Glioblastoma

MicroRNA Biomarker
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MicroRNAs are excellent plasma biomarker candidates in cancer, as they are usually deregulated in glioblastomas. Compared to other blood biomarkers, such as mRNA, MicroRNAs have demonstrated to be the most reliable. The concentration of these blood biomarkers yields many factors that are associated with tumor activity. MicroRNAs regulate gene expression and can enhance or supress translational processes. Similar to oncogenes, “oncomirs” (MicroRNAs that have enhanced expression in cancer), can suppress the tumor suppressor genes in the surrounding genome[1]. Similarly, some Micro-RNAs can also function as tumor suppressors or effect methylation. Glioblastomas are the most common and malignant type of brain tumor found amongst adults. As a result, many Profiling techniques have been identified and implemented on distinct microRNA expression patterns in glioblastomas. Among these techniques are microarray, qRT-PCR, In situ hybridization, and RNA sequencing, each applying to a specific line of Micro-RNAs. Some have shown more advantageous in the quantity of samples, while others provide enhanced quality, encouraging further detection of biomarkers and tumors [2]. Currently, there are more than 1,400 microRNA sequences listed from studies, but much has yet to be clarified on their accuracy and precision, for their use as an early diagnostic method and perhaps a therapeutic approach.

MicroRNAs: An Introduction

MicroRNAs
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miRNA biogenesis and biological pathway of function.
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Biogenesis

MicroRNAs (miRNAs) are a class of, small single-stranded non-coding RNA molecules, varying from 18–25 nucleotides in length. The pathways involved in the biogenesis of miRNAs, play a significant role in their function, when regulating genes of their target mRNA strands. Mature miRNAs are first made from pri-miRNAs. These pri-miRNAs are transcribed, using RNA polymerase II, from miRNA coding genes or other genes residing in the genome, such as introns. [6]Whether the pri-miRNAs are made from the miRNA genes or from introns, effects on the function of miRNA have not been demonstrated, but instead have been observed in the type of mRNA gene targeted, therefore affecting their role in tumor metastasis and other functions beyond that point. Once the pri-miRNA is created they undergo another pathway, using Drosha, Pasha and Dicer, RNA polymerase III enzymes. [7] Each enzyme has different functions. Drosha and Pasha cleave the pri-miRNAs producing pre-miRNAs, while Dicer cleaves it further to create miRNA but after it has been exported into the cytoplasm. The product formed by Dicer is then considered the mature miRNA, where it is sent to the miRNA-induced silencing complex.[8] Argonaute and GW182 proteins are involved in the assembly of this complex. This miRISC, is where the miRNAs can base-pair with the specific mRNAs chosen, then creating RNA interference. RNAi performs the translational repression or gene silencing of the target gene by the miRNA.

Function: Roles in Gene Expression

MicroRNAs are short non-coding RNA molecules that contribute to the physiological and developmental pathways by regulating and modifying the gene expression of target mRNAs. MicroRNAs regulate gene expression in a variety of manners, including post-transcriptional regulation, translational repression and deadenylation. [9] They play important roles in the majority of cancers. Each type of miRNA regulates processes involving biochemical and extracellular mechanisms during tumorigenesis such as metastasis, apoptosis, proliferation, or angiogenesis, giving oncogene or tumor suppressor genes like qualities/functions.

MicroRNAs in Diagnosis

Micro-RNA-21

MicroRNA-21, is an miRNA that is coded by the miRNA21 gene and specifically targets tumor suppressor mRNAs. It is one of the most studied microRNAs and has been reported to be highly upregulated in glioblastoma tissue. In the study by Mutlu et al., they characterized the presence of the microRNA-21 in the plasma of 10 glioblastoma patients. They found that microRNA-21 plasma levels are higher in glioblastoma patients than in healthy normal brain controls, leading to the ability to confer tumor presence and growth by the presence of the miRNA in blood.[1]

Another study conducted by Chan et al., confirmed miRNA-21’s antiapoptotic activity. In their experiment, they performed a Knockdown of microRNA-21 in cultured glioblastoma cells. They found an increase in the caspase activity, involved with apoptotic cell death, which provided evidence for the antiapoptotic functions of microRNA-21 in GBM tissue. Although the paper was very informative on the procedures, presenting thorough evidence that the overexpression of the miR-21 functions as a micro-oncogene; it lacked the support and evidence found through biological pathways that would be affected and lacked the association of the genes and proteins probably suppressed as a result of the miRNA, therefore lacking in support to their conclusion on its ability to produce an antiapoptotic effect. [4]Another study by Gabriely et Al., shed some light on the matter. They had been able to demonstrate specific proteins involved in the pathway, altered by miRNA-21. In the paper, they demonstrated that miRNA-21 regulates genes involved in glioma apoptosis, migration, and invasiveness, including the RECK and TIMP3 genes, which are suppressors of inhibitors of matrix metalloproteinases (MMPs). [5]They presented that the downregulation of miR-21 impacts the migratory and invasive characteristics found in MMPs. They had done this by inhibiting the miR-21 with antisense oligonucleotides, which in turn produced overexpression of RECK and TIMP3 proteins which reduced MMP activities in both in vitro assays and in vivo through gliomaspresent in mice. In conclusion it is evident that plasma microRNA-21 could be used as a predictive clinical biomarker for the glioblastomas in patients.

MicroRNA Functions
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Different functions of MicroRNAs based on their selectivity of mRNA genes and the translational repression of those genes.
Picture from: Emmanuel Barillot, Laurence Calzone, Philippe Hupé, Jean-Philippe Vert, Andrei Zinovyev,
Computational Systems Biology of Cancer Chapman & Hall/CRC Mathematical & Computational Biology , 2012

Micro-RNA-181d

MicroRNA-181d is a microRNA that is known to predict the drug treatment response of Temozolomide (TMZ). Temozolomide is a common chemotherapy drug used in treating brain cancers, in particular, glioblastoma multiforme. This alkylating agent is used in GBMs because it can breach the brain-blood barrier, unlike many other drug treatments. TMZ functionally should halt or decrease the pace at which the tumor or cancerous cell tissue grows, ultimately leading to their death.[11] Mechanistically it delivers a methyl group to purine bases of DNA. The base O6-methylguanine (O6-MeG) can be removed by methylguanine methyltransferase (MGMT) in tumours. MGMT is a type of resistance to TMZ. This resistance to TMZ can destroy many paths to successful treatment of tumors. There has been an association between miR-181d expression and survival in patients treated with Temozolomide (TMZ). [11]

In the study by Zhang et al., the experiment conducted was to find the link between miRNAs and their predictive value. They profiled the miRNAs from 82 glioblastoma tissue samples collected from patients that previously undergone radiation therapy, and a portion having undergone treatment with TMZ as well. [10]They used a dataset to make connections between the miRNA-181d and the survival. This had supported their hypothesis, as they found higher levels of miRNA-181d associated with overall survival rate. To demonstrate the biomarker’s relevance to MGMT, they hypothesized that miR-181d would downregulate the MGMT therefore increasing the sensitivity of the tumors to TMZ. The results provided evidence that miR-181d did decrease the MGMT activity and that it also did this directly, ie, not through another pathway, thus increasing the TMZ responsiveness in the patients. [11]

Micro-RNA-124-5p

MicroRNA-124-5p is a microRNA that is known to have tumor suppressor-like functions. The recent primary paper by Chen Q. et al demonstrates the pathway affected by the Glioblastomas, linking the Micro-RNA function to the down-regulation of genes linked with tumor cell proliferation. In cell biology it has been known that laminin-8 expression can act a predictor of tumor recurrence and patient survival and so this study has demonstrated that LAMB1 protein, a protein involved in the production of b1 chain of laminin-8 which is altered in tumors, is a mediator of GBM progression. In mouse models, the miRNA-124-5p is involved with genes that regulate the LAMB1 pathway.[3] It was demonstrated that the overexpression of miRNA-124-5p could halt the cell turnover and inhibit the tumor cell migration and invasion. They first observed the effects of LAMB1 regulation by miRNA-124-5p when it was left at normal levels. The effects on the proliferation, growth, and invasiveness showed a negative correlation between the microRNA and LAMB1.[3] The upregulation of LAMB1 would inversely affect the miRNA-124-5p level. Also laminin-9 changed to laminin-8 expression, due to the lack of b1 subunits, leaving b2 as the only type filling in for the other subunit leaving the gliomas more susceptible to microvessel destruction or deformation, enhancing the tumor’s strength.[3] The profiling has demonstrated that with the increased expression of miR-124-5p, the posttranscriptional reduction of microvessels is shown and as a result, altering the pathway and expression of LAMB1 early on in the tumor. A LAMB knockdown was conducted to confirm the finding and it had completely restored the microRNA levels, improving the state. Thus, miR-124-5p not only assumes a strong therapeutic method but can be useful for early detection of the proteins associated with the tumor.

Comparison to mRNA Markers

MicroRNA biomarkers are more advantageous than mRNA markers. mRNA biomarkers are used to identify the expression and presence of proteins in bodily fluids and other mediums. This is very similar to microRNAs but mRNA hinder the reasoning behind why the protein or gene is not expressed, just simply predicting their lack of. Also mRNA markers can vary with different hormonal fluctuations, broadening the confounds that could arise in the experiment. This also limits their use in some fluids, such as vaginal or sperm secretions or blood plasma during a woman’s menstrual cycle. MicroRNAs in general can predict the presence of tumors and factors associated with any mechanistic feature related to the mRNA they target, not limiting themselves to the actual protein or gene expression data.

Techniques & Implementation

- Microarrays
- qRT-PCR
- In situ hybridization
- RNA sequencing

The Use of Microarray
Microarray procedure used to detect MicroRNA transcripts

Techniques for MicroRNA
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4 Techniques currently used in MicroRNA analysis.
Picture from: Hermansen SK, Kristensen BW. (2013, 3 January )
MicroRNA biomarkers in glioblastoma. Neuro-Oncology 2013; 114:13–23

Microarrays are a type of analysis that combines miRNAs and their complementary transcripts, which are planted on a microarray chip. [13] These miRNAs are labeled before being added to the chip and the fluorescent intensity leads to the analysis of a value presented in each spot, representing the presence of the miRNA being tested. Microarrays have advantages and disadvantages in their use in the lab.[13] They allow the profiling of many samples at once, which is quite efficient in wet labs requiring many samples to be run but don’t provide numerical data, that can be plotted on charts, only images of fluorescent intensities. Another disadvantage is that the technology is quite pricey.[14] qRTPCR has the highest sensitivity, whether using TaqMan probes or SYBR green.[17] qRTPCR is based miRNA profiling that depends on reverse transcription with miRNA-specific primers. This is then undergoes a real time PCR reaction with MGB fluorescent probes. The splicing of the transcript during PCR results in the emission of fluorescence, which analyzes the presence of the target; it has high specificity. The technology has low cost but limits the quantity making samples Microarrays are better option, regarding quantity.[18] If the microRNA biomarker relies on morphology or segregation of tissue compartments then ISH is the only method. This is usually the case with microRNA 21. [15] This technique is better in understanding the cellular interactions going on cells regarding their miRNA expression levels. Another method is RNA sequencing which provides a far more precise measurement of levels of transcripts and their isoforms than other methods deeming it another advantageous option.[16]

Current Research and Therapeutic Approaches

Although the miRNA were first discovered more than a decade ago, it is only in the last set of years that researchers have begun to discover the presence and regulatory functions of miRNAs. With more research, scientists have been able to present miRNAs as vital regulators in gene expression and cell growth, all linking to cancers. More than Several miRNAs have been linked to malignant incurable cancers such as glioblastoma multiforme. [2] Therefore, miRNAs more research has been done to extrapolate the most information on their functions and variability as to serve the purpose of biomarkers, especially for future cancer diagnostics. Currently scientists think that further studies are necessary to clarify not only the boundaries but the abilities of plasma microRNA levels as blood biomarkers. An example could be the analysis of plasma microRNA concentrations as indicator of tumor recurrence in larger patient studies, leading to more precise and accurate results to yield better and faster healthcare for Glioma patients.[2] There are many MicroRNA transcripts known and more to be discovered. So far, science has been able to discover these short RNAs and uncover their vital role in gene related biological pathways. These pathways in our body affect the immune response, genetic turnover and replication, energy metabolism, cell differentiation, and most importantly cancers. It has been discovered that antisense oligonucleotide inhibitors of miRNAs, can disturb these diseases by affecting their regulation on gene expression. As a result we can alter cell activities through the alterations of these miRNAs. An example would be the inhibition of a single oncomir, like miR-21, with antisense oligonucleotides, like those mentioned above. This cannot only be a form of diagnostics for tumors but as a therapeutic technique in biological of pathways and expression of proteins involved in glioblastomas. [1] MicroRNAs as drugs are an entirely new, and efficient, creating opportunities to cure cancer naturally, without using radiation or other harmful treatments.

Bibliography
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2. Hermansen SK, Kristensen BW. (2013, 3 January ) MicroRNA biomarkers in glioblastoma. Neuro-Oncology 2013; 114:13–23
3. Chen Q, Lu G, Cai Y, Li Y, Xu R, Ke Y, Zhang S.( 2014 Feb 3). MiR-124-5p inhibits the growth of high-grade gliomas through posttranscriptional regulation of LAMB1. Neuro-Oncology 2014; 0, 1–15
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