Hypoxia-Induced Exosomes Promote Pancreatic Cancer Cells Invasion And Migration Via Reciprocal Loop Of MiR-650 And HIF-1?

Purposes To explore the effect of exosomes from hypoxic pancreatic cancer cells on normoxic pancreatic cancer cells and further study the bioactive miRNAs of the exosomes in the process.This research may be conducive to understand the underlying mechanism of metastasis and invasion of pancreatic cancer and promising to supply a new therapeutic target for pancreatic cancer and improve clinical prognosis.Methods Exosomes were extracted from the culture of hypoxic pancreatic cancer cells and incubated with normoxic pancreatic cancer cells to observe the change of migration and invasion.Micro array assay and qRT-PCR was applied to analyze the expression of miRNAs in exosomes.Then,CHIP and Luciferase Reporter Assay of miR-650 promotor was applied to explore whether HIF-1? regulate the expression of miR-650 in transcriptional level.To testify the role of miR-650 of exosomes in the communication,we knocked down miR-650 in the hypoxic pancreatic cancer cells and then their exosomes were incubated with normoxic pancreatic cancer cells to explore the change of biological malignant progress.Next,normoxic cancer cells were overexpressed with miR-650 and their exosomes' capacity of facilitating migration and invasion was tested.Western blot and qRT-PCR were conducted to testify the hypoxia signal in the normoxic pancreatic cancer cells.The target of miR-650 was predicted by TargetScan and validated by western blot,qR T-PCR and Dual-Luciferase Reporter Assay.Besides,we further explore whether the target of miR-650 could weaken the effect of miR-650 on HIF-1?.ResultsExosomes of hypoxic pancreatic cancer cells could obviously promote the migration and invasion of normoxic pancreatic cancer cells.Micro array assay and qRT-PCR reveals miR-650 was up-regulated in the exosomes of hypoxic pancreatic cancer cells.CHIP and luciferase reporter showed that miR-650 was promoted in the transcriptional level via HIF-1?.Knockdown the level of miR-650 could dramatically inhibit the effect of exosomes on normoxic pancreatic cancer cells.While overexpression of miR-650 could enhance the capacity of migration and invasion of exosomes from normoxic cancer cells.Western blot and qRT-PCR revealed that the hypoxia signal of normoxic pancreatic cancer cells was activated after incubation with the exosomes of hypoxic pancreatic cancer cells or transfection with miR-650.TargetScan showed that VHL mRNA was a target of miR-650 and was validated by western blot,qRT-PCR and Luciferase Reporter Assay.Besides,Western blot revealed that VHL could obviously reverse the effect of miR-650 on the expression of HIF-1?.Conclusions Hypoxia promote the expression of miR-650 and their secretion to exosomes.The hypoxia signal pathway of normoxic pancreatic cancer cells can be activated after absorbing the exosomes from hypoxic pancreatic cancer cells and then the normoixc pancreatic cancer cells were facilitated to invade and migrate.There is a regenerative feedback loop between HIF-1? and miR-650.Inhibition miR-650 of exosomes may be a new therapeutic target for pancreatic cancer but this need further study.