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Profiling of downregulated blood-circulating miR-150-5p as a novel tumor marker for cholangiocarcinoma

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Tumor Biology

Abstract

Altered microRNA (miRNA) expression plays a role in cholangiocarcinoma (CCA) development; thus, detection of blood-circulating miRNAs could be useful as CCA markers. This study profiled serum miRNA levels in patients with primary sclerosing cholangitis (PSC) and CCA and then assessed the role of miR-150-5p in CCA progression in vitro. Three samples were randomly selected from each of 50 sera of healthy controls, 30 PSC sera, and 28 CCA sera with matched bile samples for miRNA microarray profiling. The dysregulated miRNAs were confirmed using qRT-PCR, and miR-150-5p was selected for further in vitro and ex vivo studies. The miRNA microarray identified three dysregulated miRNAs in both CCA and PSC samples, while miR-150-5p level was consistently lower in CCA sera, bile, and tissues than in normal control and PSC sera (P < 0.05). Furthermore, levels of miR-150-5p were associated with serum carbohydrate antigen 19-9 (CA19-9) levels and CCA pathological grade. Bioinformatic Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses showed that miR-150-5p could regulate hand-full gene pathways, including cancer pathway (P < 0.01). However, overexpression of miR-150-5p inhibited proliferation, migration, and invasion capability of CCA cells (P < 0.05). Luciferase reporter assay showed that miR-150-5p bound to an oncogene Ets including gene-1 (ELK1), and Western blot data confirmed that miR-150-5p suppressed ELK1 expression in CCA cell lines. These results suggest that reduced miR-150-5p expression could contribute to CCA development and progression due to uncontrolled ELK1 expression. Thus, further study could evaluate miR-150-5p as a novel target and predictor for CCA prevention and treatment.

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Acknowledgments

The authors would like to thank our staff in both Departments for their contributions to this study and Genminix Company for their technical assistance. This study was supported in part by grants from the National Natural Science Foundation of China (no. 81502038 to FA, no. 81302382 to YJ, and no. 31400720 to WW), the Natural Science Foundation of Jiangsu Province (no. BK2012098 to FA), and the Wuxi Municipal Science and Technology Development Planning Funds (no. CSZ00N1304 to FA).

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Authors and Affiliations

  1. Department of Gastroenterology, Wuxi People’s Hospital Affiliated to Nanjing Medical University, 299 Qingyang Road, Wuxi, Jiangsu, 214023, China

    Xiongbo Wu, Min Xia, Dayang Chen, Fang Wu, Zhifa Lv, Qiang Zhan & Fangmei An

  2. Department of Gastroenterology, Xuzhou NO.1 People’s Hospital, Xuzhou, Jiangsu, 221002, China

    Guangxia Chen

  3. School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Medical School of Soochow University, Suzhou, 215123, China

    Yang Jiao & Wenjie Wang

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  1. Xiongbo Wu
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  2. Min Xia
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  4. Fang Wu
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Correspondence to Guangxia Chen or Fangmei An.

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This study was approved by the Institutional Review Board (IRB) of Nanjing Medical University. All patients provided written informed consent before participation of this study.

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Xiongbo Wu and Min Xia are authors contributed equally to this work.

Electronic supplementary material

Table S1

(Clinical characteristics of CCA patients. The table shows the relevant clinical information of CCA patients. The gender, age, tumor location, CA-19-9 and pathological grade of 13 CCA patients with tumor tissues and matched adjacent-tumor tissues were listed. DOCX 13 kb)

Fig. S1

(The Gene Ontology (GO) analyze of 3 differentially expressed miRNAs-regulated genes in body fluids of CCAs. The GO term predicted functions of genes regulated by these 3 miRNAs (P < 0.001). The vertical axis represented GOs and the horizontal axis is enrichment degree. Enrichment degree means the contribution of one miRNA to the surrounding GOs or the contribution one GO to the surrounding miRNAs. The key functions always have the higher enrichment degrees. The top 50 enrichment of GOs were showed. JPEG 434 kb)

Fig. S2

(The Kyoto Encyclopedia of Genes and Genomes (KEGG) analyze of 3 differentially expressed miRNAs-regulated pathways in body fluids of CCAs. KEGG predicted 29 pathways related to tumors regulated by these 3 miRNAs, (P < 0.001). The vertical axis is the pathway category and the horizontal axis is the P value of each pathway. The lower P value was, the more miRNAs regulated the pathways and the pathways had more important roles in CCA. JPEG 195 kb)

Fig. S3

(miRNA-mRNA network. The miRNAs target genes were predicted by TargetScan and Sanger and then miRNA–mRNA-network was generated. The blue circles represent genes, and the blue squares represent miRNAs. The lines indicate the relationship between genes and miRNA. The degree (size) of the blue square indicates the regulatory functionality of miRNA, i.e., the greater the degree, the more functions of the miRNA possess. The figure shows the relationship among these 3 overlapping miRNAs and their targeted genes, including tumor development and miR-150-5p targeted ELK1, Notch3, and MMP14. PNG 1689 kb)

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Fig. S4

(Assay for the transfection efficiency. 150-5pM, NSM, 150-5pI, NSI were transiently transfected into HCCC-9810 and RBE cells respectively for 24 h and then subjected to qRT-PCR analysis of miR-150-5p expression. qRT-PCR was in triplicate and repeated three times. JPEG 590 kb)

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Wu, X., Xia, M., Chen, D. et al. Profiling of downregulated blood-circulating miR-150-5p as a novel tumor marker for cholangiocarcinoma. Tumor Biol. 37, 15019–15029 (2016). https://doi.org/10.1007/s13277-016-5313-6

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  • DOI: https://doi.org/10.1007/s13277-016-5313-6

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