The Analysis of Platelet-Derived circRNA Repertoire as Potential Diagnostic Biomarker for Non-Small Cell Lung Cancer

Silvia D'Ambrosi, Allerdien Visser, Mafalda Antunes-Ferreira, Ankie Poutsma, Stavros Giannoukakos, Nik Sol, Siamack Sabrkhany, Idris Bahce, Marijke J E Kuijpers, Mirjam G A Oude Egbrink, Arjan W Griffioen, Myron G Best, Danijela Koppers-Lalic, Cees Oudejans, Thomas Würdinger, Silvia D'Ambrosi, Allerdien Visser, Mafalda Antunes-Ferreira, Ankie Poutsma, Stavros Giannoukakos, Nik Sol, Siamack Sabrkhany, Idris Bahce, Marijke J E Kuijpers, Mirjam G A Oude Egbrink, Arjan W Griffioen, Myron G Best, Danijela Koppers-Lalic, Cees Oudejans, Thomas Würdinger

Abstract

Tumor-educated Platelets (TEPs) have emerged as rich biosources of cancer-related RNA profiles in liquid biopsies applicable for cancer detection. Although human blood platelets have been found to be enriched in circular RNA (circRNA), no studies have investigated the potential of circRNA as platelet-derived biomarkers for cancer. In this proof-of-concept study, we examine whether the circRNA signature of blood platelets can be used as a liquid biopsy biomarker for the detection of non-small cell lung cancer (NSCLC). We analyzed the total RNA, extracted from the platelet samples collected from NSCLC patients and asymptomatic individuals, using RNA sequencing (RNA-Seq). Identification and quantification of known and novel circRNAs were performed using the accurate CircRNA finder suite (ACFS), followed by the differential transcript expression analysis using a modified version of our thromboSeq software. Out of 4732 detected circRNAs, we identified 411 circRNAs that are significantly (p-value < 0.05) differentially expressed between asymptomatic individuals and NSCLC patients. Using the false discovery rate (FDR) of 0.05 as cutoff, we selected the nuclear receptor-interacting protein 1 (NRIP1) circRNA (circNRIP1) as a potential biomarker candidate for further validation by reverse transcription-quantitative PCR (RT-qPCR). This analysis was performed on an independent cohort of platelet samples. The RT-qPCR results confirmed the RNA-Seq data analysis, with significant downregulation of circNRIP1 in platelets derived from NSCLC patients. Our findings suggest that circRNAs found in blood platelets may hold diagnostic biomarkers potential for the detection of NSCLC using liquid biopsies.

Keywords: biomarkers; circular RNA; liquid biopsy; non-small cell lung cancer; platelets.

Conflict of interest statement

T.W. is a shareholder of GRAIL Inc. D.K.-L. is a shareholder of ExBiome BV.

Figures

Figure 1
Figure 1
Differential expression analysis of platelet-derived circRNAs distinguishes patients with NSCLC from asymptomatic individuals: (a) schematic representation of the workflow. Whole blood samples were subject to platelets isolation and total RNA extraction. Libraries were generated using SMARTer Stranded Total RNA-Seq Kit using N6-oligo primer lacking the oligo-dT primer. cDNA is sequenced using high-throughput paired-end sequencing on the Illumina HiSeq 4000 platform; (b) hierarchical clustering of differentially expressed circRNAs between NSCLC (n = 6; green) and asymptomatic individuals (control; n = 6; blue). Clustering was performed with PSO enhancement. Samples are indicated in the columns, circRNAs are indicated in the rows, and color intensity represents the Z score-transformed expression values; (c) volcano plot of differentially expressed circRNAs. The negative log of the p-value (base 10) is plotted on the Y-axis, and the log of the FC (base 2) is plotted on the X-axis. Red dots indicate significantly differentially expressed circRNAs (p-value < 0.05).
Figure 2
Figure 2
Validation of circNRIP1 expression in platelet-derived RNA. (a) Schematic illustration showing that circNRIP1 is formed by head-to-tail splicing of NRIP1 exon 2 and exon 3; (b) the expression of two circRNAs, NRIP1 and MAN1A2 in platelet samples derived from NSCLC patients (n = 23) and asymptomatic individuals (n = 24) was analyzed by RT-qPCR. The MAN1A2 circRNA was used as endogenous circRNA control for normalization as the “housekeeping gene”. Each sample was analyzed in triplicate. All data are presented as mean ± SEM. The 2−ΔΔCt method was used to calculate the relative expression of circNRIP1 between the different groups. For the statistical analysis, we used the unpaired nonparametric t-test with Welch correction with two-tailed p-value; (c) The expression level of circNRIP1 is significantly downregulated (p-value = 0.0302) in platelets of NSCLC patients, specifically in samples from patients diagnosed with late-stage disease (p-value = 0.0263, n = 12). Statistically non-significant (indicated as ns) downregulation is observed in patients with early stage NSCLC (p-value = 0.098, n = 11). * indicates p-value < 0.05.

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