Repeat RNA infection
Description
Aberrant expression of repeat RNAs in pancreatic ductal adenocarcinoma (PDAC) mimic viral-like responses with implications on tumor cell state and the response of the surrounding microenvironment. To better understand the relationship of repeat RNAs in human PDAC, we performed spatial molecular imaging at single cell resolution in 46 primary tumors revealing correlations of high repeat RNA expression with alterations in epithelial state in PDAC cells and myofibroblast phenotype in cancer-associated fibroblasts (CAFs). This loss of cellular identity is observed with dosing of extracellular vesicles (EVs) and individual repeat RNAs of PDAC and CAF cell culture models pointing to cell-cell intercommunication of these viral-like elements. Differences in PDAC and CAF response are driven through distinct innate immune signaling through interferon regulatory transcription factor 3 (IRF3). The cell context specific viral-like responses to repeat RNAs provides a mechanism for modulation of cellular plasticity in diverse cell types in the PDAC microenvironment.
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For Western blotting, Cells were collected, washed, and lysed with lysis buffer (50 mM Tri-Cl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate, 1 mM 1,4-dithiothreitol and Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific, USA, cat# 78440). Total protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, cat# 23227). The cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride membrane (MilliporeSigma, MA, USA, cat# IPVH00010). The membranes were blocked with 3% of bovine serum albumin (BSA, Sigma-Aldrich, cat# A2058) for 1 hr and applied with primary antibodies for overnight at 4◦C. Membranes were washed with 1X phosphate-buffered saline (PBS) containing 0.1% Tween-20 (Sigma-Aldrich, cat# P1379) (PBST) for 10 minutes 3 times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Signal was detected with enhanced chemiluminescence (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific, cat# 34577, SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, cat# 34094), and images were developed using G:BOX (SYNGENE).