| Purpose:Circular RNA(circRNAs),as one of the hot molecules in the field of cancer,circular RNA has shown its unique advantages in tumor occurrence,development and diagnosis and treatment application,and has been favored by researchers in recent years.In the previous work focusing on the abnormal expression of circRNAs in GC-MSCs,we found and identified a new circRNA,circ＿0024107.It has been shown that the abnormal upregulation of circ＿0024107 is crucial for GC-MSCs to play a role in promoting gastric cancer lymph node metastasis.However,it is unclear whether this circRNA is abnormally expressed in gastric cancer cells and has direct regulation of the biological behavior of gastric cancer cells.This study focused on investigating the expression of circ＿0024107 in gastric cancer cells and whether it mediates GC-MSCs exerting pro-metastatic effect of gastric cancer and the possible molecular mechanism.Methods:Using qPCR to compare circ＿0024107 in different gastric cancer cells,gastric cancer tissues and adjacent tissues,we selected MGC-803 with RNA interference fragment and overexpression vector for circRNA expression,and analyzed the clinical significance of circ＿0024107 expression with clinical pathological data.Transwell test analyzed the effects of knockdown and overexpression of circ＿0024107 on the migration and invasion ability of gastric cancer cells.BM-MSCs and GC-MSCs were isolated by adherent culture,and MSCs were obtained by morphology,osteogenic adipogenic induced differentiation assay,and flow cytometry.Culture supernatant of MSCs were collected and treated with GC cell and circ＿0024107 in pre-knockdown gastric cancer cells followed by supernatant treatment of GC-MSCs.Changes in gastric cancer cell migration,invasion ability and circ＿0024107 expression were analyzed by Transwell test and qPCR,respectively,to clarify whether GC-MSCs enhance the metastatic ability of gastric cancer cells by upregulating circ＿0024107 in cancer cells.Analysis of CPT1 A m RNA and protein expression by qPCR and western blot,CPT1 activity and the rate of fatty acid β-oxidation were determined by a colorimetric assay to analyze and compare changes in the metabolic capacity of the FAO after gastric cancer cells were treated with the supernatant of BM-MSCs and that of GC-MSCs,Gastric cancer cells before and after circ＿0024107 knockdown,GC-MSCs supernatant treated GC cells with pre-knockdown circ＿0024107.Transwell test tested the effect of blocking FAO metabolism of gastric cancer cells on GC-MSCs supernatant and mi R-5572 overexpression on the migration and invasion ability of gastric cancer cells,respectively.The qPCR test compared CPT1 A and mi R-5572 content in gastric cancer and adjacent tissues,and analyzed the correlation between both and pathological features combined with clinical data.The Spearman-test was used to analyze the correlation of circ＿0024107 / CPT1A/mi R-5572 expression.The Kaplan Meier Plotter database analysis was used to evaluate the correlation of CPT1 A,mi R-5572 and gastric cancer prognosis based on the abundance of MSCs in gastric cancer tissues.RBPDP and RBPsuite databases were used to predict RBP which regulating formation of circ＿0024107,combined with qPCR screening to regulate the expression of KHDRBS3 in GC cancer cells and GC-MSCs by qPCR to knock down the effect of KHDRBS3 on circ＿0024107 expression in GC cancer cells and GC-MSCs,respectively.Results:The expression of circ＿0024107 was significantly higher in gastric cancer cells and gastric cancer tissues than in gastric mucosal epithelial cells and the corresponding adjacent tissues.The expression of circ＿002417 in gastric cancer tissues was associated with lymph node metastasis and TNM stage.Knockdown of circ＿0024107 significantly inhibited AGS migration and invasion in gastric cancer cells,while overexpression enhanced the migration and invasion ability of MGC-803 in gastric cancer cells.BM-MSCs and GC-MSCs were successfully isolated and obtained,and the supernatant of GC-MSCs significantly promoted gastric cancer cell migration and invasion and circ＿0024107 expression.Prior knockdown of circ＿0024107 in gastric cancer cells significantly blocked the regulation of the supernatant of GC-MSCs.The supernatant of GC-MSCs significantly enhanced the FAO metabolic activity of GC cells compared with that of BM-MSCs,and Etoxomir pretreatment blocked the migration and invasion ability of GC-MSCs.Knockdown of circ＿0024107 inhibited FAO metabolism,CPT1 A expression and upregulated mi R-5572 expression in gastric cancer cells,while mi R-5572 over-expression showed a similar effect as circ＿0024107knockdown.Pre-knockdown of pick＿0024107 significantly blocked the regulatory effects of GC-MSCs to enhance FAO metabolism,promoting CPT1 A expression and inhibiting mi R-5572 in gastric cancer cells.CPT1 A was aberrantly elevated and only associated with lymph node metastasis compared to adjacent tissues,whereas mi R-5572 expression was significantly reduced in cancerous tissues with no significant association with clinical features.The expression level of circ＿0024107 was positively correlated with CPT1 A level,and mi R-5572 was longer with CPT1 A expression level,circ＿0024107 and mi R-5572 in MSC deficiency group,3-year overall survival(OS);patients with MSC expression in GC had longer 3-year recurrence-free survival(RFS),while mi R-5572 served as a good prognostic factor in patients with MSC deficiency group in GC tissue.The database prediction,detection screen selected KHDRBS3 as an important RBP that might regulate the formation of circ＿0024107,and knockdown of KHDRBS3 significantly downregulated circ＿0024107 expression in gastric cancer cells and GC-MSCs.Conclusion:The circ＿0024107 as an oncogene directly regulates the migration and invasion function of gastric cancer cells and mediated the pro-gastric cancer effect of GC-MSCs.GC-MSCs promote FAO metabolism and enhance GC cell migration and invasion ability by regulating circ＿0024107 / mi R-5572 / CPT1 A axis in cancer cells.Abnormal expression of circ＿0024107 / mi R-5572 / CPT1 A signaling axis molecules in gastric cancer tissues and correlation with lymph node metastasis and prognosis.KHDRBS3 may be an important regulatory RBP regulating the cyclic formation of circ＿0024107.This paper will provide a new molecular mechanism for explaining GC-MSCs,and provide potential molecular targets and experimental basis for effectively co-targeting microenvironment cells and gastric cancer cells. |
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