Function And Mechanism Of PDTC Alleviating Cancer Cachexia And Mechanism Of Exosome Regulating Cancer Cachexia

Cancer cachexia is a systemic metabolic disorder syndrome with an irreversible weight loss.It is caused by cytokines(inflammatory factors,exosomes,etc.)secreted by cancer cell and the body.The most important feature of cancer cachexia is skeletal muscle atrophy and fat degradation.Cancer cachexia is the main cause of death in many cancer patients,so its pathogenesis remains unclear up to now.The clinical treatment has limited effectiveness.Therefore,it is urgent to find new therapeutic drugs and to further explore the mechanism of cancer cachexia.In this thesis,TNF?and C26 medium were used to establish cancer cachexia model in vitro,C2C12 muscle atrophy model and 3T3-L1 lipolysis model.C26 tumor-bearing mice were used to establish a cancer cachexia model in vivo.Based on the studies on cancer cachexia models in vivo and in vitro,the mechanism of PDTC in relieving cancer cachexia and the mechanism of exosomes regulate cancer cachexia were explored.The first part of the paper explored the mechanism of action of PDTC in relieving tumor cachexia.NF-?B signaling plays an important role during skeletal muscle atrophy and fat lipolysis.As an inhibitor of NF-?B signaling,PDTC was reported to relieve cancer cachexia.In our study,we showed that PDTC inhibited muscle atrophy and lipolysis in cells models in vitro induced by TNFa and C26 tumor medium.What's more,PDTC attenuated cancer cachexia symptom in C26 tumor bearing mice models in vivo without influencing tumor volume.Meaningfully,it was found that PDTC has little effect on NF-?B signaling pathways while attenuating skeletal muscle atrophy and fat degradation.We observed that PDTC also inhibited p38 MAPK signaling besides the NF-?B signaling in cancer cachexia in vitro models.In addition,PDTC also influenced the protein synthesis of skeletal muscle by activating AKT signaling and regulated fat energy metabolism by inhibiting AMPK signaling.Therefore,PDTC primarily influenced different pathways in different tissues.The second part of the paper explores the mechanism of exosomes regulating the development of cancer cachexia.Exosomes are nanometer-sized vesicles secreted by cells into the extracellular environment,and they are involved in the cells distant regulation through proteins,lipids and nucleic acids exosomes carried.Based on the previous PDTC study,we systematically studied the function of C26 exosomes regulating cancer cachexia muscle atrophy.Cell culture medium and exosomes of C26 and MC38 cells were acted on C2C12 myotubes,and only C26 medium and C26 exosomes were able to induce C2C12 myotube atrophy.And the diameter of C2C12 myotubes gradually decreased with the increase of C26 exosomes concentration.High doses of C26 exosomes intramuscular injection can affect muscle strength,and reduce the weight of the tibialis anterior muscle.Experiments in vitro and in vivo showed that C26 exosomes are involved in the regulation of cancer cachexia muscle atrophy.In order to clarify the mechanism,miRNA-seq was used to analyze the contents of C26 exosomes and MC38 exosomes.Combined with miRNA-seq analysis,literature investigation and C2C12 myocyte functional experiment,it was found that miR-195a-5p and miR-125b-1-3p were able to induce atrophy of C2C12 myotubes.The contents of miR-195a-5p and miR-125b-1-3p in serum and gastrocnemius muscle of C26 tumor-bearing mice were significantly higher than those in healthy controls,indicating that miR-195a-5p and miR-125b-1-3p were involved in the progress of cancer cachexia muscle atrophy.The apoptosis inhibitory protein Bcl-2 may be a downstream target gene of miR-195a-5p and miR-125b-1-3p based on bioinformatics analysis and literature investigation.We successfully demonstrated the targeting of Bcl-2 to miR-195a-5p/miR-125b-1-3p using a dual luciferase assay.Levels of m RNA and protein of Bcl-2 were down-regulated and the apoptotic signaling pathway was activated and the ubiquitination pathway was not affected in C2C12 myotubes transfected with miR-195a-5p mimic and miR-125b-1-3p mimic.The TUNEL assay also found that the apoptosis rate of gastrocnemius muscle of C26 tumor-bearing mice was significantly higher than that of the healthy mice.WB assays showed that the expression of BCL-2 was down-regulated in the myocytes,and the apoptotic signaling pathway was activated.The apoptotic rate of tibialis anterior muscle injected with high dose C26 exosomes was significantly higher than that of the control group.WB assays found that the expression of BCL-2 was down-regulated in the high dose group.The apoptotic signaling pathway is activated and and the ubiquitination pathway was not affected.It was demonstrated that C26 exosome-derived miRNAs(miR-195a-5p and miR-125b-1-3p)induce skeletal muscle atrophy by regulating the apoptotic signaling pathway.In conclusion,this thesis first proves that PDTC does not function solely as an NF-?B inhibitor in the treatment of cancer cachexia,but attenuates skeletal muscle atrophy by inhibiting the p38 MAPK signaling pathway and activating AKT signaling pathway and by inhibiting p38 MAPK signal pathway and AMPK signaling pathway to alleviate fat degradation.The mechanism of PDTC on relieving cancer cachexia will provide a theoretical basis for subsequent research and treatment.PDTC is expected to become a new class of effective compounds for the treatment of cancer cachexia.Furthermore,this thesis firstly explored the mechanism of C26 exosomes regulating cancer cachexia muscle atrophy.C26 exosome-derived miRNAs(miR-195a-5p and miR-125b-1-3p)participate in skeletal muscle atrophy by activating the apoptotic signaling pathway not the ubiquitination pathway,which can serve as a new “target” for cancer cachexia treatment and will be contribute to provide new research strategies and therapeutic targets for subsequent treatment of cancer cachexia.