American Association for Cancer Research
Browse
00085472can150390-sup-145196_3_supp_3421818_34367m.doc (2.68 MB)

Supplementary Materials and Methods, Supplementary Figures 1 through 9, and Supplementary Tables 1 through 4 from Early Epigenetic Downregulation of microRNA-192 Expression Promotes Pancreatic Cancer Progression

Download (2.68 MB)
journal contribution
posted on 2023-03-30, 23:46 authored by Sandeep K. Botla, Soniya Savant, Pouria Jandaghi, Andrea S. Bauer, Oliver Mücke, Evgeny A. Moskalev, John P. Neoptolemos, Eithne Costello, William Greenhalf, Aldo Scarpa, Matthias M. Gaida, Markus W. Büchler, Oliver Strobel, Thilo Hackert, Nathalia A. Giese, Hellmut G. Augustin, Jörg D. Hoheisel

Supplementary Materials and Methods, Supplementary Figure 1. Representation of the DNA sequence upstream of the miR-192 gene and location of differentially methylated CpG dimers. Supplementary Figure 2. Assessment of in vitro DNA methylation of the CpG island sequence upstream the miR-192 transcription start site (TSS) by agarose gel electrophoresis after digesting with restriction endonuclease AciI. Supplementary Figure 3. Heatmap showing differentially expressed microRNAs in PDAC compared to normal pancreas tissue. Supplementary Figure 4. Heatmap displaying differentially expressed microRNAs in CP compared to normal pancreas tissue. Supplementary Figure 5. Heatmap showing a three-way comparison of microRNA expression in PDAC, CP and healthy pancreas tissues. Supplementary Figure 6. Result of bisulfite sequencing of the CpGI located 2.3 kb upstream of miR-181c. Supplementary Figure 7. Analysis of the effect of 5-azacytidine treatment on miR-192 expression in cell lines BXPC-3 and PANC-1. Supplementary Figure 8. Receiver Operating Characteristic (ROC) curve displaying the ability of miR-192 expression to discriminate between CP and PDAC samples. Supplementary Figure 9. Correlation of miR-192 expression and tumor stage. Supplementary Table 1. List of analyzed patient samples with information about relevant clinical annotations and the types of analysis that were performed with each sample. Supplementary Table 2. List of all DNA primer sequences used in this study. Supplementary Table 3. Summary of the methylation status of the analyzed CpG islands found upstream of the TSSs of the set of 13 miRNAs that exhibited differential expression of a similar nature in both CP and PDAC and had CpGIs within 10 kb upstream their TSSs. Supplementary Table 4. List of predicted miR-192 targets.

Funding

BMBF

Associazione Italiana Ricerca sul Cancro

History

ARTICLE ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) is characterized by very early metastasis, suggesting the hypothesis that metastasis-associated changes may occur prior to actual tumor formation. In this study, we identified miR-192 as an epigenetically regulated suppressor gene with predictive value in this disease. miR-192 was downregulated by promoter methylation in both PDAC and chronic pancreatitis, the latter of which is a major risk factor for the development of PDAC. Functional studies in vitro and in vivo in mouse models of PDAC showed that overexpression of miR-192 was sufficient to reduce cell proliferation and invasion. Mechanistic analyses correlated changes in miR-192 promoter methylation and expression with epithelial–mesenchymal transition. Cell proliferation and invasion were linked to altered expression of the miR-192 target gene SERPINE1 that is encoding the protein plasminogen activator inhibitor-1 (PAI-1), an established regulator of these properties in PDAC cells. Notably, our data suggested that invasive capacity was altered even before neoplastic transformation occurred, as triggered by miR-192 downregulation. Overall, our results highlighted a role for miR-192 in explaining the early metastatic behavior of PDAC and suggested its relevance as a target to develop for early diagnostics and therapy. Cancer Res; 76(14); 4149–59. ©2016 AACR.