Sequence of qRT-PCR products amplified from GBM serum maps to human HOTAIR locus
To validate our biomarker assay, we sought to determine whether the HOTAIR products we are amplifying contain the known HOTAIR sequence. To this end, we amplified HOTAIR from GBM patient serum using qRT-PCR, subcloned the corresponding qRT-PCR products into the pCR™4-TOPO®TA vector, and subsequently performed DNA sequencing of the cloned products. 7
Taq polymerase-amplified qRT-PCR products were picked randomly among all the samples and transformed into competent
E. coli. Colonies were isolated the next day and plasmid DNA was isolated for subsequent sequencing. DNA sequencing demonstrated that the HOTAIR amplified from serum has the same sequence as the known sequence of HOTAIR. As shown in Fig.
2e,
f and
g, the sequence of all 7 HOTAIR inserts aligned to the HOTAIR locus exon at the USC browser. Furthermore, the amplified HOTAIR mapped to chromosome 12q13, which is the known location of HOTAIR in the human genome (Fig.
2g). As the HOTAIR gene has several splice variants, the PCR products amplified in this study detect HOTAIR splice variant HOTAIR-205. Collectively, these studies suggest that we are indeed measuring HOTAIR levels in our biomarker assay.
In this study we demonstrate that HOTAIR can be utilized as a peripheral biomarker for detecting GBM. HOTAIR, or HOX transcript antisense intergenic RNA, has been shown to be an important regulator of tumor growth and a possible serum biomarker in several cancers [
5,
13]. First identified in 2007, HOTAIR is a polyadenylated RNA with 2158 nucleotides and 6 exons [
7]. HOTAIR is transcribed from the Hox gene cluster [
13]. The Hox genes, a subgroup of the homeobox superfamily, are 39 transcription factors classified in 4 clusters (HOXA, HOXB, HOXC, HOXD) that guide correct spatial organization during development. Aberrant expression of Hox genes has oncogenic effects, and many noncoding transcripts (miRNA and lncRNAs) have been detected in all Hox clusters [
14]. HOTAIR is initially transcribed from HOXC locus, but later represses the expression of distal HOXD locus and other genes on chromosomes [
7]. Importantly, HOTAIR has been found to
trans act on multiple regions in the genome to regulate genes involved in metastasis and proliferation. Our own studies have demonstrated that HOTAIR controls GBM cell growth and we found that HOTAIR is highly expressed in GBM tumors yet completely undetectable in normal brain through single molecular sequencing (SMS) analysis [
5]. This is in line with similar reports from other groups where HOTAIR was detected in the serum of patients suffering from melanoma, esophageal cancer, breast cancer, bladder cancer, and pancreatic cancer [
7‐
11]. Hence, we propose that detection of the lncRNA termed HOTAIR in the serum of GBM patients can be utilized as a serum biomarker to detect GBM growth or recurrence.
Several proteins and microRNAs such as GFAP, lactate, miR-504, and miR-137 have been previously described as possible biomarkers for GBM [
15‐
17]. However, a recent report by Vietheer et al. demonstrated that serum GFAP levels do not correlate with tumor recurrence in GBM in 33 samples in their studies [
17]. Similar results were also observed for another protein, lactate, in serum [
15]. Very few studies have been done on lncRNAs as biomarker in GBM. In the current study, serum lncRNA can be easily detected by qRT-PCR. Many groups are looking at lncRNAs now as they show highly promosing results in other cancers including melanoma, esophageal cancer, and breast cancer [
7,
10]. Our study is one of the first studies to show that the levels of lncRNAs can be used as a biomarker in GBM. While most studies focus only on the use of biomarkers for predicting the survival of the GBM patients or prediagnostic alone, our studies focus on both diagnostic and prognostic value of HOTAIR.
Exosomes are small membrane-bound vesicles that are secreted into biological fluids such as blood, urine, saliva and milk from tumor cells. Recent studies have shown that RNAs, including lncRNA, microRNAs, and mRNAs are concentrated in these exosomes as they play a role in distance cell-to-cell communication. For instance, microRNAs in GBM cell derived exosomes have been postulated to control PTEN, SOX2, PI3K/AKT, STAT3 and ERK pathways [
18]. We detected HOTAIR mainly in GBM serum exosomes, suggesting that HOTAIR could be possibly released from tumor tissue into serum in extracellular vesicles. Our initial studies suggest that freeze-thaw cycles affect the integrity of exosomes. Therefore, the levels of HOTAIR within exosomes were reduced after repeated freeze-thawing. However, the levels of HOTAIR within total serum were not affected. We postulate that the circulating HOTAIR we are detecting in serum is RNA and not DNA since amplifying HOTAIR from serum without reverse transcription yielded less PCR products relative to when reverse transcriptase was included (Additional file
2: Figure S1). In addition, we suggest that what we are amplifying from serum is indeed HOTAIR as DNA-sequencing of the qPCR products corresponded to the known HOTAIR sequence (Fig.
2e–g).
A longitudinal study of one recurrent GBM patient that was treated at our institution was performed to monitor HOTAIR expression in serum before and after tumor resection. We observed a reduction of serum HOTAIR levels after surgery and a further reduction at the 2 week post-surgery follow-up (Additional file
3: Figure S2). Although this is only one patient, this promising result suggests that further experiments are warranted to determine whether HOTAIR levels do indeed decrease after GBM tumor removal. Furthermore, prior studies have demonstrated that HOTAIR expression is greatest in GBM tumors with a mesenchymal classification [
19]. However, we were unable to detect correlation of HOTAIR serum levels with any particular subgroup of GBM tumors (unpublished studies). Further studies are required to determine whether HOTAIR expression correlates with any particular tumor type in GBM. Similarly, more work is needed to determine whether HOTAIR serum levels can be used as a diagnostic peripheral marker for other brain tumor patients.
GBM, a WHO Grade IV tumor, is the most malignant primer tumor of the human central nervous system with an extremely poor prognosis. An efficient biomarker that can monitor GBM progression and treatment response will improve the clinical management of this lethal disease. Our studies suggest that we have developed a novel peripheral biomarker assay for GBM. To our knowledge this is the first report of a peripheral biomarker for GBM using a long noncoding RNA. This is also the first report that HOTAIR is a peripheral biomarker for GBM that is expressed in exosomes from serum of GBM patients.