Melatonin Regulates The Exosomes Produced By Gastric Cancer Cells To Regulate The Immune Function Of Macrophages

Objectives: To explore whether melatonin can affect the function of macrophages in the tumor microenvironment by regulating the production of exosomes in gastric cancer cells,regulate the human body’s anti-tumor immunity,and ultimately produce anti-tumor effects.Methods:1.Obtain exosomes-free serum by ultracentrifugation,culture gastric cancer cell lines,obtain gastric cancer cell exosomes from the gastric cancer cell culture medium by differential-ultracentrifugation,and detect the purity of exosomes.The exosomes of gastric cancer cells were stained with PKH67 and co-cultured with the macrophage cell line.The phagocytosis of exosomes was observed under a fluorescent microscope.2.Co-culture the exosomes of gastric cancer cells with the macrophage cell line,and use ELISA to detect the levels of TNF-α,IL-6,IL-10,CXCL10,MCP-1,Western-blot and RT-q PCR in the culture medium The method detects the level of PDL1 in macrophages.3.Melatonin interferes with gastric cancer cell lines,extracts exosomes,and detects the level of PDL1 in gastric cancer cells and exosomes.4.Melatonin intervenes in gastric cancer cell lines,extracts exosomes,and co-cultures them with macrophages,and uses ELISA to detect TNF-α,IL-6,IL-10,CXCL10 in the culture medium of each group of co-culture systems,The level of MCP-1.Western-blot and RT-q PCR methods were used to detect the level of PDL1 in co-cultured macrophages.5.Use the database to screen the microRNA against PDL1,synthesize microRNA in vitro and transfect the macrophage cell line,and detect the level of PDL1 in each group of macrophages by Western-blot and RT-q PCR methods.RT-q PCR method is used to detect the level of microRNA in gastric cancer cells and their exosomes intervened by melatonin.6.The obtained gastric cancer cell exosomes were injected under the skin of the right axillary of C57BL/6 mice,and gastric cancer cells were planted under the skin of the right axillary of the mouse to monitor the tumor formation cycle and tumor mass growth.Results:1.Successfully obtain high purity gastric cancer cell exosomes.RAW 264.7macrophages can swallow the exosomes produced by gastric cancer cells.Gastric cancer cell exosomes can inhibit the secretion of TNF-α,CXCL10 and CXCL10 by RAW 264.7 macrophages.The secretion of IL-10 and the exosomes of gastric cancer cells can promote the expression of PDL1 protein in macrophages.2.Compared with the Control group,the exosomes produced by gastric cancer cells treated with melatonin can promote the secretion of TNF-α and CXCL10,and inhibit the expression of MCP-1,IL-6 and PDL1.3.The database predicts and undergoes in vitro synthesis of microRNA to interfere with macrophage experiments,which verifies that microRNA-20b-5p,microRNA-17-5p and microRNA-93-5p can inhibit the expression of RAW 264.7macrophage cell line PDL1.4.Melatonin can inhibit the expression of PDL1 in gastric cancer cells,regulate the level of PDL1 protein in gastric cancer cells,and promote the expression of microRNA-20a-5p,microRNA-20b-5p,microRNA-106b-5p and microRNA-93-5p in gastric cancer cells.Promote the expression of microRNA-20a-5p and microRNA-106b-5p in the exosomes of gastric cancer cells in a dose-dependent manner.5.Exosomes from gastric cancer cells can promote the development of tumors.The exosomes produced by melatonin treatment of gastric cancer cells can reduce the immune suppression effect of the body.Conclusions:1.Exosomes secreted by gastric cancer cells can inhibit the secretion of macrophages TNF-α,IL-10,CXCL10,promote the expression of PDL1,and inhibit the immune function of macrophages;2.Exosomes produced by gastric cancer cells after melatonin treatment can promote the secretion of TNF-α and CXCL10,inhibit the expression of MCP-1,IL-6 and PDL1,and reverse the inhibition of gastric cancer cells exosomes on the immune function of macrophages;3.microRNA-20b-5p,microRNA-17-5pand microRNA-93-5p can inhibit the expression of PDL1 in RAW 264.7 macrophages;4.Melatonin can promote the expression of microRNA-20a-5p,microRNA-20b-5p,microRNA-106a-5p,microRNA-106b-5p,microRNA-17-5p and microRNA-93-5p in gastric cancer cells.Increase the expression levels of microRNA-20b-5p,microRNA-17-5p and microRNA-93-5p in the exosomes of gastric cancer cells and inhibit the expression of PDL1 in macrophages.