Alteration in Levels of Specific miRNAs and Their Potential Protein Targets between Human Pancreatic Cancer Samples, Adjacent Normal Tissue, and Xenografts Derived from These Tumors

Abstract

Herein, we describe the global comparison of miRNAs in human pancreatic cancer tumors, adjacent normal tissue, and matched patient-derived xenograft models using microarray screening. RNA was extracted from seven tumor, five adjacent normal, and eight FI PDX tumor samples and analyzed by Affymetrix GeneChip miRNA 4.0 array. A transcriptome analysis console (TAC) was used to generate comparative lists of up- and downregulated miRNAs for the comparisons, tumor vs. normal and F1 PDX vs. tumor. Particular attention was paid to miRNAs that were changed in the same direction in both comparisons. We identified the involvement in pancreatic tumor tissue of several miRNAs, including miR4534, miR3154, and miR4742, not previously highlighted as being involved in this type of cancer. Investigation in the parallel mRNA and protein lists from the same samples allowed the elimination of proteins where altered expression correlated with corresponding mRNA levels and was thus less likely to be miRNA regulated. Using the remaining differential expression protein lists for proteins predicted to be targeted for differentially expressed miRNA on our list, we were able to tentatively ascribe specific protein changes to individual miRNA. Particularly interesting target proteins for miRs 615-3p, 2467-3p, 4742-5p, 509-5p, and 605-3p were identified. Prominent among the protein targets are enzymes involved in aldehyde metabolism and membrane transport and trafficking. These results may help to uncover vulnerabilities that could enable novel approaches to treating pancreatic cancer.

miRNA- and Cell Line-Specific Constraints on Precursor miRNA Processing of Stably Transfected Pancreatic Cancer and Other Mammalian Cells

“…From our initial list of priority miRNAs that can be found in our previous publications [ 12 , 13 ], initial attempts to overexpress miR-29b, -1290, -2467-3p, and -6831-5p using overexpression vectors (described in methods) were unsuccessful, leading to no detectable expression of mature miRNAs by RT-qPCR in either MIA PaCa-2 or PANC-1 cells as shown in the figure in Section 2.5 . Compared to the four miRNAs listed above, miR-708-5p showed the highest levels of mature miRNA levels in MIA PaCa-2 (RQ = 4331) and PANC-1 (RQ = 419) and was chosen as the positive control miRNA for follow-up studies.…”

“…For primary and precursor miRNA detection, samples were prepared using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher, 4368814). The ddCt method was used to determine the relative quantification (RQ) of each miRNA with miR-16-5p and miR-24-3p used as endogenous controls [ 13 ]. Where parental levels of an miRNA were undetectable, the Ct value was set to 40 for completion of the RQ calculation as it is well accepted that a Ct greater than 35 is considered non-expressed.…”

“…In previous studies from our group, we carried out a global comparison of miRNAs in human pancreatic cancer tumours, adjacent normal tissue, and matched patient-derived xenograft models (PDX) using microarray screening [ 12 , 13 ]. From this screen, we compiled a list of priority miRNAs that were overexpressed in both comparisons between normal and tumour and tumour and PDX.…”