Effect Of Pancreatic Cancer Derived Exosomes On Insulin Resistance Of Skeletal Muscle Cells And The Underlying Mechanism

Background:Biochemical examinations,such as with tumour markers,and imaging examinations,which are commonly employed in the clinic,are not accurate for the diagnosis of pancreatic cancer(PC)at the early stage.Therefore,the main goals of pancreatic cancer research lie in looking for markers of the early stage,understanding the biological behaviours of the tumour and developing novel therapeutic methods and targets.One of the features of metabolic reprogramming is the crosstalk between PC and peripheral tissues(skeletal muscle and adipose tissues),emphasized by insulin resistance(IR).This cancer hallmark,besides IR,triggers systemic metabolic disorder which has associated glucose intolerance as initial apparent phenomenon.This systemic metabolic disorder is closely related to tumor proliferation,metastasis and cancer cachexia.Exosomes play an important role for cell-to-cell communication.Mature microRNAs(miRNAs)identify target mRNAs and then regulate post-transcriptional gene expression.Exosomes protect miRNAs from degradation induced by RNA enzymes in body fluid.The exosomes transport miRNAs to recipient cells,participating in genes expression,signal transduction and playing a key role in the process of various kinds of diseases.Plenty of researches suggest that exosomal miRNA could be molecular markers for metabolic diseases and potential therapeutic targets for correcting metabolic disorders.Hence,this study aims to explore whether PC-derived exosomes induce IR of skeletal muscle cells,and focus on the biological functions of exosomal miRNAs in this process,so as to preliminary elucidate the mechanism underlying.The results would contribute to discover potential targets for correcting metabolic disorders and improving diagnosis/treatment in the context of PC.Methods:KrasLSL-G12D/+,Trp53LSL-R172H/+,pdxl-Cre(KPC)mouse is a kind of classical pancreatic ductal carcinoma model.KPC cells are developed from tumor tissue of KPC mice.By employing ultracentrifugation,KPC-exosomes were extracted from the supernatant,and then used to treat the differentiated C2C12 cells.Glucose uptake capacity,lipid deposition and the expression of key proteins in Insulin&PI3K/Akt&FoxO signal pathways were detected to demonstrate whether insulin resistance of skeletal muscle cells occurred.MPDC-exosomes secreted from murine pancreatic ductal epithelial cell line(MPDC)were used as a negative control.Through miRNA microarray analysis,the differently expressed miRNAs in KPC-exosomes are screened out and subsequent Kyoto Encyclopedia of Genes and Genomes(KEGG)signal pathway analysis was used to detect the signal pathways we emphasized whether been enriched by the predicted genes of miRNAs.Other signal pathway information can also provide ideas for further study.MiRNA-mimics and Immunoblotting were used to detect the effect of some miRNAs on the signal pathways.The results would provide an alternative for our future research based on human cell line and mice models.Results:After 20μg/ml of PKH67-dyed KPC-exosomes treated differentiated C2C12 myotubes for 24 hours,we observed that exosomes can readily enter myotube structure.KPC-exosomes(5μg/ml)exhibited minor impact on the survival behavior of C2C12 cells in 24 hours,but triglyceride level of C2C12 cells treated by KPC-exosomes(0.2-0.8μg/ml)was increased significantly in a dose-dependent manner.Besides,the glucose uptake capacity of C2C12 cells was significantly decreased(the concentration of exosomes 1μg/ml),and the key proteins on IRS/PI3K/Akt signal axis was down-regulated(the concentration of exosomes 0.2-0.8μg/ml).FoxO1 and Glut4 are the essential downstream targets of PI3K/Akt signaling.The former exerts transcription activity in cell nucleus and is closely related with lipidosis and skeletal muscle insulin resistance,while the latter is directly correlated with glucose uptake of skeletal muscle.20μg PKH67-dyed exosomes in 100μL PBS were injected through caudal vein into the body of each nude mouse.The results suggested that FoxO1 protein accumulated in muscle cell nuclei(red overlapped with blue)of the nude mice was increased while Glut4 protein distributed on plasma membrane was decreased apparently.We conducted further analysis on miRNAs in KPC-exosomes by employing MPDC-exosomes as the control in MicroRNA microarray analysis,through which the differently expressed miRNAs in KPC-exosomes were screened out.Subsequent KEGG pathway analysis proved that the predicted genes of these miRNAs mainly targets insulin signal pathway,PI3K/Akt signal pathway,FOXO pathway and ubiquitin proteasome system(UPS).After RT-qPCR verification on miRNA microarray results,immunoblotting technique was used to test the influence of the first nine highly expressed KPC-exosomal miRNAs on Insulin and PI3K/Akt signal pathways.The results showed that miR-450b-3p and miR-151-3p could significantly down regulate the expression of IRS1 and P110α.Conclusion:These results suggest that pancreatic cancer derived exosomes could induce the increase of lipidosis in skeletal muscle cells and the decrease of glucose uptake capacity and finally result in the occurrence of insulin resistance.This pathological process is closely related with PI3K/Akt/FoxO1 signal pathway.MiRNAs in exosomes may play an essential role,which is expected to be intervention targets for improving the diagnosis and treatment of pancreatic cancer.