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Messenger RNA capture sequencing of extracellular RNA from human biofluids using a comprehensive set of spike-in controls.

ABSTRACT: Comprehensive transcriptome analysis of extracellular RNA (exRNA) purified from human biofluids is challenging because of the low RNA concentration and compromised RNA integrity. Here, we describe an optimized workflow to (1) isolate exRNA from different types of biofluids and (2) to prepare messenger RNA (mRNA)-enriched sequencing libraries using complementary hybridization probes. Importantly, the workflow includes 2 sets of synthetic spike-in RNA molecules as processing controls for RNA purification and sequencing library preparation and as an alternative data normalization strategy. For complete details on the use and execution of this protocol, please refer to Hulstaert et al. (2020).

SUBMITTER: Hulstaert E

PROVIDER: S-EPMC8076706 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAccumulating evidence has indicated that methylation status is closely related to tumourigenesis and a few aggressive features of diverse cancers. However, as an important methylation regulation modification, the distribution of 5-methylcytosine (m5C) in high-grade serous ovarian cancer (HGSOC) remains unclear.Materials and methodsWe collected three pairs of human HGSOC tissues and adjacent non-tumour tissues to analyse the transcriptome-wide m5C methylation of messenger RNAs (mRNAs) by methylated RNA immunoprecipitation sequencing. Gene ontology (GO) enrichment analysis and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis were performed to eExplore the potential biological functions of these genes and important cancer pathways. We used immunohistochemistry to analyse the expression of the m5C modification regulatory gene MAP2K3 in 80 HGSOC tissue samples, and their associations with clinical parameters were analyzed using the Spearman-rho test. Univariate and multivariate Cox regression analyses were performed to identify potential prognostic factors. Kaplan-Meier analysis was performed to analyze overall survival.ResultsWe identified 2050 dysregulated m5C peaks, 1767 of which were significantly upregulated, while 283 were significantly downregulated. GO enrichment analysis showed that genes altered by the m5C peak played a key role in system development, transporter complex, and transporter activity. KEGG pathway analysis revealed that these genes were enriched in some important pathways in cancer regulation, such as inflammatory mediator regulation of the TRP channels pathway, Wnt signalling pathway, and focal adhesion pathways. In addition, through joint analysis of MeRIP-seq and RNA-seq data, we identified 125 differentially methylated m5C peaks and synchronous differentially expressed genes. These genes play key roles in cell growth, maintenance, plasma membranes, and cell adhesion molecule activity. Immunohistochemical staining results showed that high expression of MAP2K3 was significantly correlated with CA125 level (p < 0.001), tumour size (p = 0.001), lymph node metastasis (p = 0.008), depth of myometrial invasion (p < 0.001), and FIGO stage (p < 0.001), indicating a poor prognosis.ConclusionOur results reveal the different distribution patterns of m5C in HGSOC and adjacent tissues and the possible involvement of m5C in HGSOC cell functions. Our study provides new insights into the epi-transcriptomic dysregulation of m5C in the tumourigenesis of HGSOC.

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Messenger RNA capture sequencing of extracellular RNA from human biofluids using a comprehensive set of spike-in controls.

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