Hypoxic Tumor-derived Exosomal MiR-301a Mediates M2 Macrophage Polarization Via PTEN/PI3K? Pathway To Promote Pancreatic Cancer Metastasis

Background: Exosomes are emerging as important mediators of the crosstalk between tumor cells and the microenvironment.However,the mechanisms by which exosomes modulate tumor development under hypoxia in pancreatic cancer(PC)remain largely unknown.Here,our study aims to explore the role and mechanisms by which hypoxic pancreatic cancer cells-derived exosomes mediate macrophage polarization and tumor metastasis.Methods: Pancreatic cancer(PC)cells-derived exosomes during normoxic or hypoxic conditions were isolated by ultracentrifugation and kit assay.And the exosomes were identified by TEM,NTA and WB.The effect of hypoxic exosomes in macrophages polarization was detected by q RT-PCR and WB.The invasion and metastatic ability,and the EMT markers expression levels of PC cells co-cultured with M2 macrophages were performed by using transwell and WB respectively.The expression of exosomal miR-301 a derived from PC patients' serum was examined by q RT-PCR and the correlation between exosomal miR-301 a expression and clinicopathological features as well as prognosis was analyzed.Double luciferase reporter assay was used to prove the binding effects of miR-301 a on PTEN promoter regions.WB was used to detect the effect of exosomal miR-301 a under hypoxia in the PTEN/PI3K? signaling pathway in macrophages.Finally,lung metastasis model in nude mice was used to demonstrate the effect of macrophages induced by exosomal miR-301 a under hypoxia in PC cells metastasis.Results: Electron microscopy revealed typical rounded particles ranging from 30 to150 nm in diameter,and NTA exhibited a similar size distribution of exosomes.Increased levels of CD9 and CD81 were observed in exosomes under the hypoxia.The expression of M2 markers(CD206 and Arg-1 et.al)increased in macrophages incubated with hypoxic exosomes.M2 macrophages induced by hypoxic tumor-derived exosomes significantly increased the migration and invasion of PC cells compared with normoxic exosomes.And the the expression levels of mesenchymal cell markers(N-cadherin et.al)increased.The expression of miR-301 a in exosomes derived from the serum of PC patients was markedly up-regulated.And circulating exosomal miR-301 a expression was significantly associated with depth of tumor invasion,lymph node metastasis,late TNM stage and prognosis.The expression of exosomal miR-301 a reduced in HIF-1a or HIF-2a-downregulated PC cells.Macrophages treated with hypoxic exosomes derived from PC cells or transfected with miR-301 a minics significantly inhibited the expression of PTEN and promoted the expression of PI3K?,p-AKT and p-m TOR,while inhibition of miR-301 a exhibited the opposite effects.MiR-301 a bound to the 3 'UTR region of its target gene PTEN and inhibited PTEN protein expression.In addition,PC cells mixed with macrophages treated with hypoxic exosomes or transfected with miR-301a/mimics generated more and larger nodules of metastatic lung tumors.Conclusions: Circulating exosomal miR-301 a levels positively associated with depth of invasion,lymph node metastasis,late TNM stage,and poor prognosis of PC.MiR-301a-rich exosomes generated from PC cells in a hypoxic microenvironment induced the M2 polarization of macrophages via activation of the PTEN/PI3K?signaling pathway in a HIF-1a or HIF-2a-dependent manner,which then facilitates the migration,invasion,and EMT of PC cells.