Pancreatic Cancer-Derived Exosomal MiR-Let-7b-5p Stimulates Insulin Resistance in Skeletal Muscle Cells Through RNF20/STAT3/FOXO1 Axis Regulation

Background: Cancers trigger systemic metabolic disorders usually associated with glucose intolerance, which is an initially apparent phenomenon. One of the features of pancreatic cancer (PC) metabolic reprogramming is the crosstalk between PC and peripheral tissues (skeletal muscle and adipose tissues), emphasized by insulin resistance (IR). Our previous study reported that mice pancreatic cancer-derived exosomes could induce skeletal muscle cells (C2C12) IR, and exosomal microRNAs (miRNAs) may exert an important effect. However, the underlying mechanism remains to be further elucidated.

Methods: qPCR was used to determine the expression of let-7b-5p in normal pancreatic islet cells and PC cells. Exosomes were purified from PC cell culture medium by ultracentrifugation. The role let-7b-5p on IR-mediated by PC cells-derived exosomes was asses by Oil Red O staining using miRNA inhibitor. Western blot assay was performed to examine the expression of IR-related genes and the activation of signaling pathways. A Luciferase experiment was applied to confirm how let-7b-5p regulated the expression of RNF20. IP/WB analysis further determined whether RNF20 promoted STAT3 ubiquitination. Rescue experiment using RNF20 overexpression plasmid was performed to confirm the role of RNF20 on IR-mediated using PC cell-derived exosomes in C2C12 myotube cells.

Results: miRNA-let-7b-5p was identified as the key exosomal miRNA, which could promote the IR in C2C12 myotube cells supported the lipid accumulation, the activation of STAT3/FOXO1 axis, and the decreased expression of IRS-1 and GLUT4. RNF20, an E3 ubiquitin ligase, was confirmed as the target gene of let-7b-5p and was found to improve IR by downregulating STAT3 protein expression via ubiquitination-mediated protein degradation. The ectopic expression of RNF20 could effectively attenuate the IR mediated by the pancreatic cancer-derived exosomes in C2C12 myotube cells.

Conclusion: Our data suggest that exosomal miRNA-let-7b-5p may promote IR in C2C12 myotube cells by targeting RNF20 to activate the STAT3/FOXO1 axis.