Exosomal let-7d-3p and miR-30d-5p as diagnostic biomarkers for non-invasive screening of cervical cancer and its precursors

Abstract

Cervical cancer screening through detection and treatment of high-grade cervical intraepithelial neoplasia (CIN) is most successful in cancer prevention. However, the accuracy of the current cervical cancer screening tests is still low. The aim of this study was to develop a more accurate method based on circulating exosomal miRNAs. The miRNA sequencing was performed to identify candidate exosomal miRNAs as diagnostic biomarkers in 121 plasma samples from healthy volunteers, cervical carcinoma patients, and CIN patients. A panel with eight differentially expressed exosomal miRNAs was identified to distinguish patients in the CIN II+ group (including advanced CIN II patients) from those in the CIN I- group (including CIN I patients and healthy volunteers). Let-7d-3p and miR-30d-5p showed significant difference between cervical tumors and adjacent normal tissues (P < 0.005), exhibited a consistent trend in plasma samples, and were further validated in 203 independent plasma samples. Integrating these two miRNAs yielded an AUC value of 0.828 to distinguish patients in CIN II+ group from those in CIN I- group. Further integrating them into a cytological test-based model resulted in a higher AUC of 0.887, while the AUC value based on the cytological test alone was 0.766. In summary, plasma exosomal miR-30d-5p and let-7d-3p are valuable diagnostic biomarkers for non-invasive screening of cervical cancer and its precursors. Further validation using large sample sizes is required for clinical diagnosis.

Keywords: Cervical cancer; Diagnosis; Early detection; Exosome; Liquid biopsy; Next-generation sequencing; miRNA.

Fig. 1

Identification of differentially expressed miRNAs in plasma exosomal sequencing samples. a Venn diagram of differentially expressed miRNAs between CIN I- and other groups (CIN II-III, CC, SCC, and ACC). b, c Principal component analysis (b) and clustering analysis (c) of all 61 significant exosomal miRNAs that were differentially expressed between CIN I- and other groups (CIN II-III, CC, SCC, and ACC). d ROC curves of the top eight significant miRNAs (let-7a-3p, let-7d-3p, miR-30d-5p, miR-144-5p, miR-182-5p, miR-183-5p, miR-215-5p, and miR-4443). ROC analysis was performed to evaluate the sensitivity and specificity of the eight-miRNA signature (i.e. a group of the top eight significant miRNAs) to discriminate CIN II+ from CIN I- subjects. e, f Principal component analysis (e) and clustering analysis (f) of the top eight significant miRNAs. g, h Expression levels and ROC curves of four down-regulated (g) and four up-regulated (h) miRNAs in CIN II+ group compared with those in CIN I- group. Exosomal miRNA expression levels were quantified as RPM in the sequencing data. i Biological pathways enriched for experimentally validated targets by at least five of the top eight miRNAs. Experimentally validated miRNA-target interactions were identified from the miRTarBase database. j miRNA-gene connection network. Circles represent miRNAs. Squares represent experimentally validated target genes by at least three of eight miRNAs. The pink, blue, and green squares represent target genes that were involved in < 5, 5–10, and > 10 significant pathways, respectively

Fig. 2

ddPCR validation of let-7d-3p and miR-30d-5p as diagnostic markers in 203 independent samples. a Expression and ROC analysis of let-7d-3p in validation samples (P = 1.4e-7 and AUC = 0.822). b Expression and ROC analysis of miR-30d-5p in validation samples (P = 5.4e-7 and AUC = 0.79). c ROC analysis of expression levels of two miRNAs (i.e., let-7d-5p and miR-30d-5p) from sequencing (AUC = 0.922, sequencing samples, n = 121) and ddPCR (AUC = 0.828, validation samples, n = 203). d ROC analysis of 166 validation samples that had at least one cytology test result. miRNA-AUC: ROC analysis based on two miRNAs (i.e., let-7d-5p and miR-30d-5p); cytology-AUC: ROC analysis based on cytology test results; combined-AUC: ROC analysis based on both miRNAs and cytology test results. All ROC analyses were performed to evaluate the sensitivity and specificity of exosomal miRNAs and/or cytology tests to discriminate CIN II+ from CIN I- subjects