Effect Of Pancreatic Cancer-Derived Exosomes On The Osteoclast Differentiation Of RAW264.7 Cells And The Underlying Mechanism

Background:Pancreatic cancer(PC)is one of the most malignant tumors with a very poor prognosis,with a 5-year survival rate of only about 7.2%.More than 70% of pancreatic cancer patients suffer from cachexia.The development of cachexia eventually leads to a decline in tolerance to radiotherapy and chemotherapy,and reduces overall survival.Therefore,improving cachexia is the key to improving the overall survival rate of pancreatic cancer patients.Cachexia,defined as weight loss and muscle wasting,is a complex metabolic syndrome,and more than 50% of cancer patients have cachexia manifestations.It is estimated that cachexia accounts for approximately 20% of all cancer patients’ direct causes of death.Studies have shown that the bone mineral content of lung cancer patients with 30% weight loss is significantly reduced,indicating that cachexia is related to bone loss.Similar results were shown in preclinical animal models of lung cancer,pancreatic cancer and colon cancer.Through intervention,the increased metabolic basis of pancreatic cancer patient can be reduced,and cachexia can be reduced through etiological treatment,thereby prolonging the time of chemotherapy.However,the mechanism of cancer-related bone loss is not clear.More and more evidences show that exosomes derived from pancreatic cells play an important role in tumour progression,metastasis,and chemoresistance,and cause para-cancer β-cell dysfunction,lipolysis of adipose tissue,and insulin resistance in skeletal muscle cells.Exosomes are secreted by a variety of cell types and cancer cells,and are loaded with various functional biomolecules(including DNA,m RNA,non-coding RNA(nc RNA),proteins and biologically active lipids).These biologically active components transferred from exosomes to recipient cells play a vital role in cell-to-cell communication.Micro RNA(mi RNA)is a 22-nucleotide single-stranded endogenous non-coding RNA that targets RNA and regulates post-transcriptional gene expression,and participates in many biological processes,such as cell proliferation,differentiation,and apoptosis.Exosomes can protect mi RNA from RNase degradation and transport mi RNA to recipient cells.Studies have shown that exosomal mi RNAs play an important role in metabolic diseases,and their dysregulation plays a vital role in the development and progression of cancer.They can suppress cancer or suppress cancer according to the targets regulated under specific circumstances.The role of oncogenes.However,little is known about the role of mi RNAs in pancreatic cancer-related bone loss.Therefore,this study aims to investigate whether exosomes derived from pancreatic cancer are involved in cachexia bone loss,and focus on the biological functions of exosomal-mi RNAs in this process to clarify its underlying mechanisms.At the same time,we will contribute to the discovery of potential targets for correcting pancreatic cancer cachexia and improve treatment in the context of pancreatic cancer.Methods:KPC-exosomes were extracted from the cell culture supernatant of pancreatic cancer cells(KPC cells)derived from Kras LSL-G12D/+,Trp53LSL-R172H/+,Pdx1-Cre mice by ultracentrifugation and the mouse pancreas Exosomes derived from ductal epithelial cells were used as a control(MPDC-exosomes)to process mouse macrophage cells(RAW264.7)to detect osteoclast differentiation and the expression of related key proteins to verify pancreatic cancer cell exocytosis Whether the body has the ability to induce osteoclast differentiation.We use mi RNA gene chip technology to detect differentially expressed mi RNAs in KPC-exosomes and MPDC-exosomes,and use KEGG(Kyoto Encyclopedia of Genes and Genomes)signal pathway enrichment to analyse Osteoclast differentiation-related signal pathways,and to screen differential mi RNAs.RAW264.7 cells were treated with mi RNA-mimics to detect the expression of key osteoclast-related proteins and m RNAs.The dual luciferase report experiments and reverting experiments were performed to analyse and determine the interaction of mi RNAs and targeted gene.Results:Our data showed that PC-derived exosomes significantly induced osteoclast differentiation and increased expression of NFAT2,TRAP,CTSK and MMP-9.The bone volume fraction and trabecular thickness of femor significantly reduced in osteoporotic model.Microarray analyses showed that the process is,at least partially,mediated by the mi R-125a-5p/TNFRSF1 B signaling pathways.We also verified the relationship between mi R-125a-5p and TNFRSF1 B gene through RT-PCR and dual luciferase report experiments.The results showed that mi R-125a-5p inhibited the expression of TNFRSF1 B by binding to the 3’-UTR of TNFRSF1 B.Reverting experiments verified that mi R-125a-5p promoted osteoclast differentiation by targeting TNFRSF1 B.Conclusion:We have shown that exosomes derived from pancreatic cancer can induce osteoclast differentiation,and this process is at least partially controlled by the mi R-125a-5p / TNFRSF1 B signaling pathway.The research provides new insights into the mechanism of pancreatic cell-derived exosomes on bone degeneration,and will contribute to a comprehensive understanding of the relationship between PC and systemic metabolism.Subsequent research will explore the potential of promising exosomal mi RNAs,including functional verification and mechanism elucidation.This research provides direction for potential targets for improving cachexia in pancreatic cancer.