Article | Published:

miRNA profiling of small intestinal neuroendocrine tumors defines novel molecular subtypes and identifies miR-375 as a biomarker of patient survival

Modern Pathologyvolume 31pages13021317 (2018) | Download Citation


The aim of this study was to define the miRNA profile of small intestinal neuroendocrine tumors and to search for novel molecular subgroups and prognostic biomarkers. miRNA profiling was conducted on 42 tumors from 37 patients who underwent surgery for small intestinal neuroendocrine tumors. Unsupervised hierarchical clustering analysis of miRNA profiles identified two groups of tumor metastases, denoted cluster M1 and M2. The smaller cluster M1 was associated with shorter overall survival and contained tumors with higher grade (WHO grade G2/3) and multiple chromosomal gains including gain of chromosome 14. Tumors of cluster M1 had elevated expression of miR-1246 and miR-663a, and reduced levels of miR-488-3p. Pathway analysis predicted Wnt signaling to be the most significantly altered signaling pathway between clusters M1 and M2. Analysis of miRNA expression in relation to tumor proliferation rate showed significant alterations including downregulation of miR-137 and miR-204-5p in tumors with Ki67 index above 3%. Similarly, tumor progression was associated with significant alterations in miRNA expression, e.g. higher expression of miR-95 and miR-210, and lower expression of miR-378a-3p in metastases. Pathway analysis predicted Wnt signaling to be altered during tumor progression, which was supported by decreased nuclear translocation of β-catenin in metastases. Survival analysis revealed that downregulation of miR-375 was associated with shorter overall survival. We performed in situ hybridization on biopsies from an independent cohort of small intestinal neuroendocrine tumors using tissue microarrays. Expression of miR-375 was found in 578/635 (91%) biopsies and survival analysis confirmed that there was a correlation between downregulation of miR-375 in tumor metastases and shorter patient survival. We conclude that miRNA profiling defines novel molecular subgroups of metastatic small intestinal neuroendocrine tumors and identifies miRNAs associated with tumor proliferation rate and progression. miR-375 is highly expressed in small intestinal neuroendocrine tumors and may be used as a prognostic biomarker.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Niederle B, Pape UF, Costa F, et al. ENETS Consensus Guidelines update for neuroendocrine neoplasms of the jejunum and ileum. Neuroendocrinology. 2016;103:125–38.

  2. 2.

    Pavel M, O’Toole D, Costa F, et al. ENETS Consensus Guidelines update for the management of distant metastatic disease of intestinal, pancreatic, bronchial neuroendocrine neoplasms (NEN) and NEN of unknown primary site. Neuroendocrinology. 2016;103:172–85.

  3. 3.

    Ahmed A, Turner G, King B, et al. Midgut neuroendocrine tumours with liver metastases: results of the UKINETS study. Endocr Relat Cancer. 2009;16:885–94.

  4. 4.

    Modlin IM, Moss SF, Chung DC, Jensen RT, Snyderwine E. Priorities for improving the management of gastroenteropancreatic neuroendocrine tumors. J Natl Cancer Inst. 2008;100:1282–9.

  5. 5.

    Andersson E, Arvidsson Y, Swärd C, et al. Expression profiling of small intestinal neuroendocrine tumors identifies subgroups with clinical relevance, prognostic markers and therapeutic targets. Mod Pathol. 2016;29:616–29.

  6. 6.

    Andersson E, Swärd C, Stenman G, Ahlman H, Nilsson O. High-resolution genomic profiling reveals gain of chromosome 14 as a predictor of poor outcome in ileal carcinoids. Endocr Relat Cancer. 2009;16:953–66.

  7. 7.

    Banck MS, Kanwar R, Kulkarni AA, et al. The genomic landscape of small intestine neuroendocrine tumors. J Clin Invest. 2013;123:2502–8.

  8. 8.

    Francis JM, Kiezun A, Ramos AH, et al. Somatic mutation of CDKN1B in small intestine neuroendocrine tumors. Nat Genet. 2013;45:1483–6.

  9. 9.

    Karpathakis A, Dibra H, Pipinikas C, et al. Prognostic impact of novel molecular subtypes of small intestinal neuroendocrine tumor. Clin Cancer Res. 2016;22:250–8.

  10. 10.

    Hashemi J, Fotouhi O, Sulaiman L, et al. Copy number alterations in small intestinal neuroendocrine tumors determined by array comparative genomic hybridization. BMC Cancer. 2013;13:505.

  11. 11.

    Crona J, Gustavsson T, Norlén O, et al. Somatic mutations and genetic heterogeneity at the CDKN1B locus in small intestinal neuroendocrine tumors. Ann Surg Oncol. 2015;22:1428–35.

  12. 12.

    Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–97.

  13. 13.

    Mendell JT, Olson EN. MicroRNAs in stress signaling and human disease. Cell. 2012;148:1172–87.

  14. 14.

    Calin GA, Ferracin M, Cimmino A, et al. A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med. 2005;353:1793–801.

  15. 15.

    He H, Jazdzewski K, Li W, et al. The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci USA. 2005;102:19075–80.

  16. 16.

    Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature. 2005;435:834–8.

  17. 17.

    Volinia S, Calin GA, Liu CG, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA. 2006;103:2257–61.

  18. 18.

    Svoronos AA, Engelman DM, Slack FJ. OncomiR or tumor suppressor? The Duplicity of MicroRNAs in Cancer. Cancer Res. 2016;76:3666–70.

  19. 19.

    Bosman TF CF, Rhuban RH (eds). WHO Classification of Tumors of the Digestive Systems Vol 4th edn, pp 102–107 (IARC: Lyon, France, 2010).

  20. 20.

    Sobin L, Gospodarowicz M, Wittekind C (eds). TNM Classification of Malignant Tumors. Vol 7th edn, pp 310 (Wiley-Blackwell: Oxford, UK, 2010)

  21. 21.

    Tang LH, Gonen M, Hedvat C, Modlin IM, Klimstra DS. Objective quantification of the Ki67 proliferative index in neuroendocrine tumors of the gastroenteropancreatic system: a comparison of digital image analysis with manual methods. Am J Surg Pathol. 2012;36:1761–70.

  22. 22.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402–8.

  23. 23.

    Li SC, Khan M, Caplin M, et al. Somatostatin analogs treated small intestinal neuroendocrine tumor patients circulating microRNAs. PLoS ONE. 2015;10:e0125553.

  24. 24.

    Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004;3:Article3.

  25. 25.

    Ritchie ME, Silver J, Oshlack A, et al. A comparison of background correction methods for two-colour microarrays. Bioinformatics. 2007;23:2700–7.

  26. 26.

    Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003;19:185–93.

  27. 27.

    Huang W, Li H, Luo R. The microRNA-1246 promotes metastasis in non-small cell lung cancer by targeting cytoplasmic polyadenylation element-binding protein 4. Diagn Pathol. 2015;10:127.

  28. 28.

    Kim G, An HJ, Lee MJ, et al. Hsa-miR-1246 and hsa-miR-1290 are associated with stemness and invasiveness of non-small cell lung cancer. Lung Cancer. 2016;91:15–22.

  29. 29.

    Liu ZY, Zhang GL, Wang MM, Xiong YN, Cui HQ. MicroRNA-663 targets TGFB1 and regulates lung cancer proliferation. Asian Pac J Cancer Prev. 2011;12:2819–23.

  30. 30.

    Neerincx M, Sie DL, van de Wiel MA, et al. MiR expression profiles of paired primary colorectal cancer and metastases by next-generation sequencing. Oncogenesis. 2015;4:e170.

  31. 31.

    Yi C, Wang Q, Wang L, et al. MiR-663, a microRNA targetingp21(WAF1/CIP1), promotes the proliferation and tumorigenesis of nasopharyngeal carcinoma. Oncogene. 2012;31:4421–33.

  32. 32.

    Zhao Y, Lu G, Ke X, et al. miR-488 acts as a tumor suppressor gene in gastric cancer. Tumour Biol. 2016;37:8691–8.

  33. 33.

    Chai S, Ng KY, Tong M, et al. Octamer 4/microRNA-1246 signaling axis drives Wnt/beta-catenin activation in liver cancer stem cells. Hepatology. 2016;64:2062–76.

  34. 34.

    Miao CG, Shi WJ, Xiong YY, et al. MicroRNA-663 activates the canonical Wnt signaling through the adenomatous polyposis coli suppression. Immunol Lett. 2015;166:45–54.

  35. 35.

    Song Q, Xu Y, Yang C, et al. miR-483-5p promotes invasion and metastasis of lung adenocarcinoma by targeting RhoGDI1 and ALCAM. Cancer Res. 2014;74:3031–42.

  36. 36.

    Wu J, Ji X, Zhu L, et al. Up-regulation of microRNA-1290 impairs cytokinesis and affects the reprogramming of colon cancer cells. Cancer Lett. 2013;329:155–63.

  37. 37.

    Sorby H, Welin S, Langer SW, et al. Predictive and prognostic factors for treatment and survival in 305 patients with advanced gastrointestinal neuroendocrine carcinoma (WHO G3): the NORDIC NEC study. Ann Oncol. 2013;24:152–60.

  38. 38.

    Hu Y, Xie H, Liu Y, et al. miR-484 suppresses proliferation and epithelial-mesenchymal transition by targeting ZEB1 and SMAD2 in cervical cancer cells. Cancer Cell Int. 2017;17:36.

  39. 39.

    Lin M, Shi C, Lin X, et al. sMicroRNA-1290 inhibits cells proliferation and migration by targeting FOXA1 in gastric cancer cells. Gene. 2016;582:137–42.

  40. 40.

    Shen H, Wang L, Ge X, et al. MicroRNA-137 inhibits tumor growth and sensitizes chemosensitivity to paclitaxel and cisplatin in lung cancer. Oncotarget. 2016;7:20728–42.

  41. 41.

    Wang Z, Yang H, Ren L. MiR-21 promoted proliferation and migration in hepatocellular carcinoma through negative regulation of Navigator-3. Biochem Biophys Res Commun. 2015;464:1228–34.

  42. 42.

    Yin Y, Zhang B, Wang W, et al. miR-204-5p inhibits proliferation and invasion and enhances chemotherapeutic sensitivity of colorectal cancer cells by downregulating RAB22A. Clin Cancer Res. 2014;20:6187–99.

  43. 43.

    Song G, Sharma AD, Roll GR, et al. MicroRNAs control hepatocyte proliferation during liver regeneration. Hepatology. 2010;51:1735–43.

  44. 44.

    Wan LY, Deng J, Xiang XJ, et al. miR-320 enhances the sensitivity of human colon cancer cells to chemoradiotherapy in vitro by targeting FOXM1. Biochem Biophys Res Commun. 2015;457:125–32.

  45. 45.

    Vishnubalaji R, Hamam R, Yue S, et al. MicroRNA-320 suppresses colorectal cancer by targeting SOX4, FOXM1, and FOXQ1. Oncotarget. 2016;7:35789–802.

  46. 46.

    Fan D, Ren B, Yang X, Liu J, Zhang Z. Upregulation of miR-501-5p activates the wnt/beta-catenin signaling pathway and enhances stem cell-like phenotype in gastric cancer. J Exp Clin Cancer Res. 2016;35:177.

  47. 47.

    Vinas JL, Ventayol M, Brune B, et al. miRNA let-7e modulates the Wnt pathway and early nephrogenic markers in mouse embryonic stem cell differentiation. PLoS ONE. 2013;8:e60937.

  48. 48.

    Ullmann P, Qureshi-Baig K, Rodriguez F, et al. Hypoxia-responsive miR-210 promotes self-renewal capacity of colon tumor-initiating cells by repressing ISCU and by inducing lactate production. Oncotarget. 2016;7:65454–70.

  49. 49.

    Anton R, Chatterjee SS, Simundza J, Cowin P, Dasgupta R. A systematic screen for micro-RNAs regulating the canonical Wnt pathway. PLoS ONE. 2011;6:e26257.

  50. 50.

    Dai J, Wu H, Zhang Y, et al. Negative feedback between TAp63 and Mir-133b mediates colorectal cancer suppression. Oncotarget. 2016;7:87147–60.

  51. 51.

    Zhang GJ, Zhou H, Xiao HX, Li Y, Zhou T. MiR-378 is an independent prognostic factor and inhibits cell growth and invasion in colorectal cancer. BMC Cancer. 2014;14:109.

  52. 52.

    Li SC, Essaghir A, Martijn C, et al. Global microRNA profiling of well-differentiated small intestinal neuroendocrine tumors. Mod Pathol. 2013;26:685–96.

  53. 53.

    Miller HC, Frampton AE, Malczewska A, et al. MicroRNAs associated with small bowel neuroendocrine tumours and their metastases. Endocr Relat Cancer. 2016;23:711–26.

  54. 54.

    Ruebel K, Leontovich AA, Stilling GA, et al. MicroRNA expression in ileal carcinoid tumors: downregulation of microRNA-133a with tumor progression. Mod Pathol. 2010;23:367–75.

  55. 55.

    Hua HW, Jiang F, Huang Q, Liao Z, Ding G. MicroRNA-153 promotes Wnt/beta-catenin activation in hepatocellular carcinoma through suppression of WWOX. Oncotarget. 2015;6:3840–7.

  56. 56.

    Jiang J, Yu C, Chen M, et al. Reduction of miR-29c enhances pancreatic cancer cell migration and stem cell-like phenotype. Oncotarget. 2015;6:2767–78.

  57. 57.

    Xu L, Wen T, Liu Z, et al. MicroRNA-375 suppresses human colorectal cancer metastasis by targeting Frizzled 8. Oncotarget. 2016;7:40644–56.

  58. 58.

    Fotouhi O, Adel Fahmideh M, Kjellman M, et al. Global hypomethylation and promoter methylation in small intestinal neuroendocrine tumors: an in vivo and in vitro study. Epigenetics. 2014;9:987–97.

  59. 59.

    Karpathakis A, Dibra H, Pipinikas C, et al. Progressive epigenetic dysregulation in neuroendocrine tumour liver metastases. Endocr Relat Cancer. 2017;24:L21–L5.

  60. 60.

    Jeon MK, Klaus C, Kaemmerer E, Gassler N. Intestinal barrier: molecular pathways and modifiers. World J Gastrointest Pathophysiol. 2013;4:94–9.

  61. 61.

    Kim JT, Li J, Jang ER, et al. Deregulation of Wnt/beta-catenin signaling through genetic or epigenetic alterations in human neuroendocrine tumors. Carcinogenesis. 2013;34:953–61.

  62. 62.

    Knudsen LA, Petersen N, Schwartz TW, Egerod KL. The microRNA repertoire in enteroendocrine cells: identification of miR-375 as a potential regulator of the enteroendocrine lineage. Endocrinology. 2015;156:3971–83.

  63. 63.

    Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 2007;129:1401–14.

  64. 64.

    Ludwig N, Leidinger P, Becker K, et al. Distribution of miRNA expression across human tissues. Nucleic Acids Res. 2016;44:3865–77.

  65. 65.

    He J, Cao Y, Su T, et al. Downregulation of miR-375 in aldosterone-producing adenomas promotes tumour cell growth via MTDH. Clin Endocrinol (Oxf). 2015;83:581–9.

  66. 66.

    Yan JW, Lin JS, He XX. The emerging role of miR-375 in cancer. Int J Cancer. 2014;135:1011–8.

  67. 67.

    Avnit-Sagi T, Kantorovich L, Kredo-Russo S, Hornstein E, Walker MD. The promoter of the pri-miR-375 gene directs expression selectively to the endocrine pancreas. PLoS ONE. 2009;4:e5033.

  68. 68.

    Keller DM, McWeeney S, Arsenlis A, et al. Characterization of pancreatic transcription factor Pdx-1 binding sites using promoter microarray and serial analysis of chromatin occupancy. J Biol Chem. 2007;282:32084–92.

Download references


The expert technical assistance of Pauline Brattberg and Linda Inge is greatly appreciated. This study was supported by the Swedish Cancer Society, Sahlgrenska Academy (the government ALF agreement), BioCARE- a National Strategic Research program at University of Gothenburg, Chalmers University of Technology, The Sahlgrenska University Funds, and the Assar Gabrielsson Research Foundation.

Author information


  1. Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden

    • Yvonne Arvidsson
    • , Anders Bergström
    • , Ellinor Andersson
    • , Gülay Altiparmak
    •  & Ola Nilsson
  2. Department of Mathematical Sciences, Chalmers University of Technology, Gothenburg, Sweden

    • Anna Rehammar
    •  & Erik Kristiansson
  3. Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden

    • Christina Swärd
    •  & Bo Wängberg


  1. Search for Yvonne Arvidsson in:

  2. Search for Anna Rehammar in:

  3. Search for Anders Bergström in:

  4. Search for Ellinor Andersson in:

  5. Search for Gülay Altiparmak in:

  6. Search for Christina Swärd in:

  7. Search for Bo Wängberg in:

  8. Search for Erik Kristiansson in:

  9. Search for Ola Nilsson in:

Conflict of interest:

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Yvonne Arvidsson.

Electronic supplementary material

About this article

Publication history





Issue Date


Further reading