Copy number-based quantification assay for non-invasive detection of PVT1-derived transcripts

Abstract

BackgroundOne of the most important susceptibility loci for cancer is the 8q24 human chromosomal region. The non-protein coding gene locus plasmacytoma variant translocation 1 (PVT1) is located at 8q24 and is dysregulated in prostate cancer. PVT1 gives rise to multiple transcripts which may have different functions. Here, we describe a real-time quantitative polymerase chain reaction (qPCR)-based assay for copy number-based quantitation of PVT1 exons 4A, 4B, and 9 to enable accurate, reproducible, and quantifiable detection.MethodsPVT1 exons 4A, 4B, and 9 were cloned into a plasmid vector to create standards for subsequent creation of linear standard curves representing a broad range of concentrations. PCR was carried out using SYBR-Green signal detection to quantify PVT1 exons 4A, 4B, and 9. The efficacy of this assay was evaluated by using it to detect these transcripts in prostate epithelial and prostate cancer cell lines, normal and cancerous human prostate tissues, human serum, mouse plasma, and urine samples.ResultsThe results indicate that the assay can be used to quantify both low and high copy numbers of PVT1-derived transcripts. This is the first report of a copy number-based quantification assay for non-invasive detection of PVT1 derived transcripts.ConclusionsThis novel assay holds promise for routine non-invasive testing in diseases where PVT1 is dysregulated.

Population Differentiation at the PVT1 Gene Locus: Implications for Prostate Cancer

“…PVT1 promotes proliferation, invasion, and metastasis, and promotes epithelial to mesenchymal transition in PCa ( Chang et al 2018 ). Though the role of PVT1 is well-established in different cancers and other diseases, very few reports are available on the alternatively spliced transcripts of PVT1 ( Guo et al 2017 , Pal and Ogunwobi 2019 ). We previously demonstrated that PVT1 exon 9 is associated with aggressive PCa in men of African ancestry ( Ilboudo et al 2015 ).…”

Biomarkers in Colorectal Cancer: Current Research and Future Prospects

“…The PVT1 gene is differentially expressed among populations [ 127 ]. Furthermore, quantification of PVT1 expression pattern reveals variations between tissue types with maximal expression in ovaries, lymph nodes and bone marrow and moderate levels of expression in the colon [ 128 ]. Of note, the PVT1-217 transcript is the most abundant in the gastrointestinal tract mucosa [ 115 ].…”

“…High expression of specific splice variants like PVT1 -214 is associated with poor overall survival and acquisition of stem-cell like properties including invasion and cell migration [ 137 ]. Furthermore, downstream targets of PVT1 such as miR-26b could provide both a mechanism as well as more specific biomarker readouts of PVT1 activity in CRC [ 128 ]. Poor overall survival with elevated PVT1 expression as well as increased cell proliferation, invasion and metastasis has been shown in further studies [ 138 ].…”

Prostac: A New Composite Score With Potential Predictive Value in Prostate Cancer

“…Primers for PVT1 exons 4A, 4B, and 9 were designed using Primer3Plus. The assay was performed as previously described (17). Concentration of PCR products were measured spectrophotometrically using NanoDrop ® ND100 (Thermo Scientific NanoDrop Products, Wilmington, Delaware) in nanograms per microliter and converted to copies per microliter using the formula y copies ml = ½x ng ml  10 −9 ½pcDNA vector and 273 301 j j130 DNA bps  660 !…”

“…In particular, PVT1 exon 9 was shown to be significantly overexpressed in moAA with aggressive PCa ( 14 ). The promise of these potential biomarkers to improve the clinical diagnosis of PCa, among this most vulnerable population group, was subsequently elevated by the development of a non-invasive copy number-based quantification assay for detecting PVT1-derived transcripts from prostate tissues, serum, and urine samples ( 17 ). Whereas expression levels of PVT1 exons 4A, 4B, and 9 are promising biomarkers in their own right, it is reasonable to expect that a single numeric score that combines information from all two or more transcripts would be a more robust and easier to use diagnostic tool for clinicians.…”