The Role And Molecular Mechanism Of Lymph Node Metastatic Gastric Cancer Cell-derived Exosome Educating Bm-MSCs By Modulating Fatty Acid Oxidation Metabolism Reprogramming

Purpose: Lymph node metastasis is a common way of gastric cancer metastasis,which seriously affects the prognosis and outcome of gastric cancer.Bone marrow derived mesenchymal stem cells(BM-MSCs)can be chemotactic by gastric cancer cells and participate in the formation of gastric cancer microenvironment which are closely related to lymph node metastasis.Fatty acid oxidation(FAO)metabolic reprogramming plays an important regulatory role in tumor metastasis.The purpose of this study is to investigate whether the exosomes of gastric cancer cells with lymph node metastasis can educate BM-MSCs by regulating FAO’s metabolic reprogramming and its molecular mechanisms.Method: The highly lymph node metastatic gastric cancer cells were isolated and obtained using the continuous passage of the lymph node metastasis model in vivo and the adherence method.The transwell migration and invasion assay in vitro and the metastasis model in vivo were performed to indentify the highly lymph node metastatic gastric cancer cell line,HGC-27 L.Ultracentrifugation was used to isolate exosomes from HGC-27 L,parental cell line HGC-27 and primary gastric cancer cell line AGS.Exosomes were identified by Nanoparticle Analyzer(NTA)and Western blot(WB).The above-mentioned three kinds of gastric cancer cell exosomes were used to treat BM-MSCs.In vitro,the immunofluorescence staining was used to detect the expression of α-SMA in BM-MSCs,the effect of the supernatant from the treated BM-MSCs on the migration and invasion ability of AGS and HGC-27 L were detected by transwell,and the effect of the supernatant of BM-MSCs after treatment on the tubule formation ability of lymphatic endothelial cells(HLEC)was detected by tubule formation assay.In vivo,the effect of the treated BM-MSCs supernatant on the metastatic ability of HGC-27 L was analyzed using the lymphatic metastasis model.Both of the in vitro and in vivo assay were used to evaluated the effect of gastric cancer cell exosomes on BMMSCs education.Western blot and colorimetric methods were separately used to detect CPT1 A expression,CPT1 activity and β-oxidation rate of BM-MSCs after treatment,which was used to analyze the effect of gastric cancer cell exosomes on the metabolic capacity of FAO in BM-MSCs.The FAO metabolic inhibitor Etomoxir was used to pretreat BM-MSCs to analyze whether lymph node metastasis-derived gastric cancer cell exosomes educated BM-MSCs through FAO metabolic reprogramming.Western blot,lentiviral infection and RNA interfering techniques were used to analyze whether exosomal CD44 mediated lymph node metastasis-derived gastric cancer cell regulating the FAO metabolic reprogramming of BM-MSCs.Results: The gastric cancer cells with highly lymph node metastasis were successfully isolated,identified and was named as HGC-27 L.HGC-27 L,HGC-27 and AGS exosomes were isolated and obtained.Compared with AGS exosomes,HGC-27 L and HGC-27 exosomes significantly induced BM-MSCs to express α-SMA and enhanced their ability to promote the migration and invasion of gastric cancer cells,tubule formation of HLEC and lymph node metastasis of gastric cancer cells in vivo.The CPT1 A protein expression,CPT1 activity and β-oxidation rate of BM-MSCs in the HGC-27 L and HGC-27 exosome treatment groups were increased compared with the AGS exosome treatment group.Among them,the HGC-27 L exosomes treatment group has the most obvious effect.Etomoxir treatment can block the expression of α-SMA of BM-MSCs induced by HGC-27 L exosomes and their ability to promote the metastasis of gastric cancer cells in vivo and in vitro.The content of CD44 in HGC-27 L and HGC-27 cells and their exosomes were higher than that in AGS,and the content of CD44 in HGC-27 L cells and exosomes was the highest.Knockdown of CD44 in HGC-27 L exosomes significantly inhibited their effect on BM-MSCs education and inducing FAO metabolic reprogramming of BM-MSCs.On the contrary,CD44-enriched AGS exosomes obtained by CD44 overexpression presented the similar ability as lymph node metastatic gastric cancer exosomes educating BM-MSCs and inducing their metabolic reprogramming.Conclusion: Lymph node metastatic gastric cancer cells-exosomes significantly educate BM-MSCs compared with those of primary gastric cancer cells.Lymph node metastatic gastric cancer cells-exosomes educate BM-MSCs by regulating FAO metabolism reprogramming.CD44 is indentified as an important protein mediated the regulatory role of lymph node metastatic gastric cancer cells-exosomes,and it is expected to provide a potential molecular target for the treatment of gastric cancer with lymph node metastasis.