Ann Surg Treat Res.  2021 Sep;101(3):140-150. 10.4174/astr.2021.101.3.140.

Circulating microRNAs as biomarkers in bile-derived exosomes of cholangiocarcinoma

• Han JY
• Ahn KS
• Kim YH
• Kim TS
• Baek WK
• Suh SI
• Kang KJ

• KMID: 2519844
• DOI: http://doi.org/10.4174/astr.2021.101.3.140

Abstract

Purpose
In this pilot study, using next-generation sequencing and integrated messenger RNA (mRNA) sequencing, we investigated circulating microRNA (miRNA) expression profiling from bile-derived exosomes to identify dysregulated miRNA signatures and oncogenic pathways and determine their effects on targeted mRNAs in cholangiocarcinoma (CCA). Moreover, we explored the possibility that genetic analysis using bile-derived exosomes may replace gene analysis using tissue.
Methods
Bile was collected from a patient with perihilar CCA before curative resection. As a control, bile was collected from a patient with a common bile duct stone. Exosomes were isolated from the bile, and we performed next-generation miRNA sequencing using isolated exosomes. To evaluate miRNA-mRNA interactions, mRNA sequencing was performed using bile fluid in both patients.
Results
We identified 22 differentially expressed miRNAs. More than 65% of the predicted mRNA targets of those miRNAs were actually differentially expressed between control and CCA bile samples. In functional pathway analysis, targets of 22 miRNAs were primarily enriched in mitogen-activated protein kinase, platelet derived growth factor, vascular endothelial growth factor, epidermal growth factor receptor, and p53 signaling. In particular, in the functional assessment of miRNAmRNA interactions, RAS pathways, including downstream pathways (PI3K-AKT-mTOR and RAS-RAF-MEK-ERK), were determined to be enriched.
Conclusion
Circulating miRNAs in bile-derived exosomes provide new information for the development of miRNA analysis in CCA. These miRNAs may represent the oncogenic characteristics of CCA tissue, enabling them to be used instead of tissue samples for the diagnosis of CCA. Further research investigating circulating miRNAs in bile exosomes may lead to more rational, targeted approaches to treatment.

Keyword

Bile; Exosomes; MicroRNAs; Tumor microenvironment

Figure

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  Fig. 1 Heat map of the 1-way hierarchical clustering using Z-score for normalized value (log2 based) based on 52 mature microRNAs satisfying |fold change| ≥ 2 and raw. P < 0.05.

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  Fig. 2 MicroRNA (miRNA)-messenger RNA (mRNA) regulatory network. Among 22 differentially expressed miRNAs (DEmiRNAs), 14 netDEmiRNA had a network of miRNAs–mRNAs that has a threshold for the minimum number of miRNA-target interactions of 4 and false discovery rate < 0.01. Bold blue boxes indicate differentially expressed mRNAs between normal and cancer bile samples. CCNT2, cyclin T2; ALDH5A1, aldehyde dehydrogenase 5 family member A1; DICER1, dicer 1, ribonuclease III; CNOT6, CCR4-NOT transcription complex subunit 6; ITGB1, integrin subunit beta 1; AKT3, AKT serine/threonine kinase 3; CTNNBIP1, catenin beta interacting protein 1; DNMT1, DNA methyltransferase 1; ZEB2, zinc finger E-box binding homeobox 2; WDR37, WD repeat domain 37; MYB, MYB proto-oncogene, transcription factor; DCBLD2, discoidin, CUB and LCCL domain containing 2; RPS6KA3, ribosomal protein S6 kinase A3; EGR1, early growth response protein 1; NLK, nemo like kinase; PLAG1, PLAG1 zinc finger; BCL2L11, BCL2 like 11; CASP8, caspase 8; BMPR2, bone morphogenetic protein receptor type 2; VHL, Von Hippel-Lindau syndrome; IRS1, insulin receptor substrate 1; CUL5, cullin 5; FOS, Fos protooncogene; PIK3CG, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma; NCOA3, nuclear receptor coactivator 3; SIRT1, sirtuin 1; PTEN, Phosphatase and tensin homolog; BCL2, B-cell lymphoma 2; VEGFA, vascular endothelial growth factor A; CDK6, cyclin dependent kinase 6; MYC, MYC proto-oncogene; MMP2, matrix metallopeptidase 2; DNMT3B, DNA methyltransferase 3 beta; LOX, lysyl oxidase; ADAM12, ADAM metallopeptidase domain 12; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; SERPINH1, serpin family H member 1; COL4A2, collagen type IV alpha 2 chain; FBN1, fibrillin 1; LAMC2, laminin subunit gamma 2; COL5A2, collagen type V alpha 2 chain; TDG, thymine-DNA glycosylase; SPARC, secreted protein acidic and rich in cysteine; TET2, Tet methylcytosine dioxygenase 2; IRS1, insulin receptor substrate 1; BMPR2, bone morphogenetic protein receptor type 2; PIK3R1, phosphoinositide-3-kinase regulatory subunit 1; COL4A1, collagen type IV alpha 1 chain; AKT2, AKT serine/threonine kinase 2; LRP6, LDL receptor related protein 6.

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  Fig. 3 RAS pathway in functional assessment of microRNA (miRNA)-messenger RNA (mRNA) interaction. Highlighted red-colored and yellow-colored boxes indicate differentially expressed miRNA and mRNA signaling in the present study, respectively. This image was obtained by Kyoto Encyclopedia of Genes and Genomes with copyright permission (Kanehisa [14]). RAS, rat sarcoma virus; IKK, inhibitor of nuclear factor-κB (IκB) kinase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PI3K, phosphoinositide 3-kinase; PIP3, phosphoinositide 3-kinase; AKT, protein kinase B; BAD, BCL2 associated agonist of cell death; BCL-X, B-cell lymphoma-extra large; AFX, FOXO4; FasL, Fas ligand; AF6, ALL1-fused gene from chromosome 6 protein; Raf-1, rapidly accelerated fibrosarcoma-1; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; ERK, extracellular-signal-regulated kinase; KSR, kinase suppressor of Ras; PLA, poly-lactic acid; ELK, ETS transcription factor; ETS, electron transport chain; Repac, another Epac subfamily member called.

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