Exosome-Derived MiR-130a Activates Angiogenesis In Gastric Cancer By Targeting C-MYB In Vascular Endothelial Cells

Background: The epidemiological status of gastric cancer is still serious.The invasion and metastasis of cancer cells result in disease progression and ultimately poor prognosis.Angiogenesis is involved in every stage of tumor development,especially in the process of tumor invasion and metastasis.While the current anti-vascular therapy for gastric cancer is not effective enough.Exosomes are small membrane vesicles and can conduct various bioactive substances to mediate the communication between tumor cells and tumor stromal cells.Mi RNAs play a key role at the posttranscriptional level by binding complementary sequences in 3'UTRs of target m RNA to negatively regulate gene expression.Previous studies and preliminary experimental results of our team have confirmed that c-MYB,which acts as a transcription factor,is closely related to tumor angiogenesis.Bioinformatics has demonstrated that mi R-130 a has specific binding sites in the 3'UTR of c-MYB m RNA.Objective: The purpose of this study was to further explore the mechanism of tumor angiogenesis,and to verify that exosome-derived mi R-130 a from GC cells transferred into vascular endothelial cells to target c-MYB,thereby promoting angiogenesis.Our study aimed to provide a new therapeutic target for anti-vascular therapy of gastric cancer and to reveal a potential novel biological maker for tumor progression.Methods: 1.Clinical level:? The effect of c-MYB on the prognosis of GC was predicted and analyzed by Kaplan-Meier plotter.? GC tissues and paired noncancerous tissues were collected to analyze the expression of c-MYB and mi R-130 a.2.In vitro:? Exosomes secreted from SGC7901 cells were isolated by sequential differential centrifugation,and the morphology was testified by electron microscopy and exosome markers were assessed by western blotting? Mi R-130 a can directly target the 3'UTR of c-MYB m RNA,which was validated by bioinformatics analysis and luciferase assay.? Donor cells(SGC7901)were transfected to obtain mi R-130a-knockdown exosomes(transfected with mi R-130 a inhibitors)and NC exosomes(transfected with NC inhibitors).After coculturing HUVECs with these exosomes,we detected biological behaviors related to angiogenesis.Meanwhile,we explored the expression of mi R-130 a and c-MYB of HUVEC after coculturing with exosomes.? Mi R-130 a mimics,miR-130 a inhibitors,NC mimics,and NC inhibitors were transfected directly into HUVECs by lipo-2000,so as to analyze the biological behaviors related to angiogenesis.In addition,we detected the expression of mi R-130 a and c-MYB of HUVEC after transfection.3.In vivo:? Establishment of transplanted tumor model in mice: SGC7901 cells were pretreated withmi R-130 a lentivirus,mi R-130a-inhibitors lentivirus,or control lentivirus.Then,the cells mentioned above were subcutaneously injected into BALB/c-nude mice.? Detection during tumorigenesis: tumor size was recorded to demonstrated that mi R-130 a overexpression can promote tumor growth.? Tumors were harvested by surgical removal: We validated the level of mi R-130 a and c-MYB expression in tumor tissues from each group.The level of mi R-130 a in exosomes isolated from the serum of mice was analyzed by RT-q PCR.And tumor angiogenesis was evaluated by immunohistochemistry(IHC)using CD31.Results: 1.Clinical level: By Kaplan-Meier Plotter,we found that during the follow-up,the survival rate of the high c-MYB expression group was consistently higher than that of the group with low expression.And the expression of c-MYB protein was dramatically downregulated in the GC tissues compared with that in the normal adjacent tissues.Meanwhile,the expression of mi R-130 a in cancer tissue is upregulated.2.In vitro: Combined with previous research and our preliminary conclusion,we demonstrated that c-MYB is closely associated with angiogenesis.Both bioinformatics analysis and luciferase assay result revealed that mi R-130 a can directly target the 3'UTR of c-MYB m RNA.HUVEC cocultured with different exosomes validated that exosome-derived mi R-130 a can promote angiogenesis by targeting c-MYB.Meanwhile,the direct transfection of HUVEC further certified the angiogenesis activation by mi R-130 a.3.In vivo: During a tumor-burdened period,we found that tumors of the miR-130 apositive group presented with a high rate of tumor growth.However,the growth of tumors in the mi R-130a-negative group was shown to be significantly slower than that in the control group.The tumor tissue of mi R-130a-positive group showed a higher expression of mi R-130 a than that of the control group,and the expression of c-MYB protein was downregulated,accordingly.The density of the positive CD31 expression was used to assess the angiogenesis rate.We confirmed that,compared with the mi R-130a-negative group,the angiogenesis rate in the mi R-130a-positive group was enhanced,as indicated by the increased expression of CD31.Conclusion: 1.Mi R-130 a is upregulated in gastric cancer cells,and can be secreted within exosomes.2.Gastric cancer cells are rich in mi R-130 a,and can be transferred into vascular endothelial cell to promote angiogenesis by targeting c-MYB.3.In vivo results confirm that suppressing the expression of mi R-130 a or blocking the transmission of these exosomes that contain overexpressed mi R-130 a might be a novel strategy for antiangiogenic therapy of gastric cancer.