The Role And Mechanism Of Circ＿0024107 Regulating GC-MSC Fatty Acid Oxidative Metabolism Reprogramming To Promote Gastric Cancer Metastasis

Objective: The purpose of the study is to clarify the role and mechanism of circ＿0024107 regulating gastric cancer tissue derived mesenchymal stem cells(GCMSCs)fatty acid oxidative metabolism reprogramming to promote gastric cancer metastasis.To provide new molecular mechanism for explaining GC-MSCs promoting gastric cancer metastasis and a potential molecular target for gastric cancer treatment though targeted intervention of GC-MSCs.Methods: Microarray screened the aberrant expression profiles of circRNAs between BM-MSCs and GC-MSCs.q PCR was used to verify the upregulated expression of circ＿0024107 in GC-MSCs.The characteristics of circ＿0024107 were identified by convergent and divergent primer amplification,sequencing analysis,RNase R test and nuclear-cytoplasmic separation experiment.Design and synthesis of circ＿0024107 si RNA fragments were based on the sequences of the back-splicing site and the effective si RNA fragment was then screened.The supernatants of GC-MSCs with knockdown of circ＿0024107 were collected to treat AGS and HGC-27.Transwell migration and invasion assay,and the lymphatic metastasis model were used to evaluate the role of circ＿0024107 regulating GC-MSCs promoting gastric cancer metastasis in vitro and in vivo.Microarray screened the differentially expressed genes between BMMSCs and GC-MSCs and examined the FAO metabolic pathway activation by bioinformatics analysis.To verify the difference in fatty acid oxidative metabolism between BM-MSCs and GC-MSCs,CPT1 A expression detected by q PCR and western blot and CPT1 activity and fatty acid β oxidation rate tested by colorimetry were used to analyze FAO metabolism capacity.GC-MSCs was treated with CPT1 A inhibitor Etomoxir and their supernatants were then collected to further treat gastric cancer cells.To verify the role of CPT1 A in GC-MSCs promoting metastasis,transwell migration and invasion assay were used to detect the changes of migration and invasion ability of gastric cancer cells.The changes of CPT1 A expression,CPT1 activity and fatty acid βoxidation rate were detected after knocking down circ＿0024107 in GC-MSCs.The effect of circ＿0024107 on CPT1 A expression and fatty acid oxidative metabolism of GC-MSCs was observed.Bioinformatic analyses was conducted to predict the potential binding miRNAs of circ＿0024107 and CPT1 A.The direct interaction between miR-5572 and circ＿0024107 or CPT1 A were verified using luciferase reporter vector construction and activity assay.The negative correlation between miR-5572 and circ＿0024107 or CPT1 A was tested by q PCR and western blot.circ＿0024107 and miR-5572 mimics co-transfection was used to elucidate the molecular mechanism of circ＿0024107 as ce RNA regulating CPT1 A.Results: Successfully screened,verified and identified circ＿0024107 as a novel circRNA,which was abnormally increased in GC-MSCs.Knocking down circ＿0024107 significantly inhibited the migration and invasion of gastric cancer cells in vitro and lymph node metastasis in vivo promoted by GC-MSCs.Compared with BM-MSCs,the CPT1 A expression and CPT1 activity and fatty acid β oxidation rate were enhanced significantly in GC-MSCs.Etomoxir treatment significantly suppressed the role of GC-MSCs in promoting migration and invasion of gastric cancer cells compared with DMSO,indicating that CPT1 A played an important role in GC-MSCs promoting gastric cancer metastasis.The fatty acid β oxidation rate and CPT1 A expression and CPT1 activity were decreased significantly in GC-MSCs after knockdown of circ＿0024107,suggesting that circ＿0024107 regulated CPT1 A expression and fatty acid oxidative metabolism reprogramming in GC-MSCs.Bioinformatic analysis predicted that circ＿0024107 could act as miR-5572 sponge to regulate CPT1 A expression indirectly.Luciferase reporter gene assay confirmed the direct interaction between circ＿0024107 and miR-5572.The expression of miR-5572 was significantly increased in GC-MSCs with circ＿0024107 knockdown while was significantly decreased in BM-MSCs with circ＿0024107 overexpression.circ＿0024107negatively regulated miR-5572.miR-5572 negatively regulated CPT1 A through the predicted 3’UTR site,which were analyzed and verified by luciferase reporter gene assay,q PCR and western blot.In co-transfection model of circ＿0024107 and miR-5572 mimics,miR-5572 reversed the role of circ＿0024107 upregulating CPT1 A and the function of promoting gastric cancer metastasis in MSCs.Conclusion: GC-MSCs derived circ＿0024107 may serve as miR-5572 sponge to upregulate CPT1 A and fatty acid oxidative metabolism reprogramming to mediate GCMSCs promoting gastric cancer metastasis,which provides a new molecular mechanism for explaining GC-MSCs promoting gastric cancer metastasis and a potential molecular target to interfere GC-MSCs for gastric cancer treatment.