Cancer-associated Fibroblasts Activation By Extracellular Vesicles Packaged MiR-4488 Promotes The Metastasis Of Nasopharyngeal Carcinoma Through MIF

Background and Purposes: NPC(nasopharyngeal carcinoma)is the most common malignant tumor of the head and neck,and metastasis is the main cause of clinical treatment failure of NPC.Studies have shown that distant metastasis is a multi-stage,multi-factor reaction.TDSFs(Tumor-Derived Secreted Factors)derived from the primary tumor were conducive to from a suitable micro-environment for colonization of tumor cells at the metastatic site,known as a "premetastatic niche"(PMN),which happened before tumor cells reached the distant organs.PMN is characterized by interstitial cell remodeling,extracellular matrix remodeling,angiogenesis and immunosuppression.Cancer associated fibroblasts(CAFs),as the most important stromal cell components in tumor microenvironment,mediated PMN formation by the secretion of oncogenic cytokines such as IL-6,VEGFA,thereby promoted distant metastasis of tumors.CAFs originate from a variety of cells including epithelial cells,endothelial cells,adipose cells and normal fibroblasts(NFs)which are the main sources of CAFs.In addition to cytokines,tumor derived extracellular vesicles(EVs)are also involved in NFs activation.However,the involvement of CAFs in tumor PMN formation and metastasis in NPC has not been reported.Therefore,it is of great significance to elucidate the roles of CAFs activated by EVs in PMN formation and the mechanisms in metastasis of NPC.Methods:(1)EVs mi RNA sequencing was used to detect the expression of plasma EVs mi RNA in patients with metastatic and nonmetastatic NPC;(2)The expression of mi RNA in cells and EVs was detected by q RT-PCR;(3)The isolated EVs were identified by Transmission Electron Microscopy,Particle Size Analysis and Western blotting(WB);(4)6-10B-EVs and 5-8F-EVs,6-10B/Vec-EVs and 6-10B/mi R-4488-EVs were treated with NFs(hepatic stellate cell LX-2 and human embryonic lung cell MRC-5)to detected CAFs marker moleculesα-SMA and FAP by WB;(5)mi RNA target gene online prediction,m RNA sequencing,luciferase reporter gene and Akt and NF-κ B inhibitor,to analysis the expression of MEN1,α-SMA,FAP,AKT/NF-κ B;(6)EVs packaged with mi R-4488 were injected into tail vein,PMN was detected by immunofluorescence and immunohistochemistry;(6)Cytokine chip and ELISA were used to screen the key cytokines in the activated CAFs-CM;(7)Activated CAFs-CM co-culture with vascular endothelial cells(HUVEC)and macrophages(THP-1)respectively and combined with target MIF inhibitors to detect angiogenesis and macrophage M2polarization;(8)EVs packaged with mi R-4488 were injected into the tail vein of mice for 21 days,NPC cells were injected into the tail vein to detect the distant metastasis of NPC;(9)After EVs loaded with mir-4488 were injected into the tail vein of mice,NPC cells were injected.In addition,cpsi-1306 was injected intraperitoneally to detect the distant metastasis of NPC;(10)q RT-PCR was used to detect mir-4488 loaded by EVs in clinical serum samples,and the relationship between mir-4488 loaded by EVs and NPC metastasis was analyzed in combination with clinical data.Results: 1 mi R-4488 was highly expressed in plasma EVs of patients with NPC metastasis;2.mi R-4488 loaded by EVs can promote the activation of CAFs;3.mir-4488 targets MEN1 through Akt/NF-κ B upregulated of α-SMA and FAP and activated of CAFs;4.in mice,EVs loaded with mi R-4488 promoted the formation of CAFs and PMN in liver and lung;5.Activated CAFs-CM can promote angiogenesis and increase vascular permeability;6.Activated CAFs-CM promotes macrophage polarization to M2 type;7.MIF was highly expressed in activated CAFs-CM;8.Activated CAFs-CM can promote angiogenesis and increase vascular permeability through MIF;6.Activated CAFs-CM can promote macrophage polarization to M2 type through MIF;7.EVs loaded with mi R-4488 can promote the distant metastasis of NPC;8.mi R-4488 was highly expressed in EVs of serum samples from patients with nasopharyngeal carcinoma metastasis.In conclusion,this study for the first time elucidated the molecular mechanisms of extracellular vesicles packaged with mi R-4488 activation of normal fibroblasts by targeting MEN1.In addition,activated CAFs promoted angiogenesis and M2 polarization of macrophages through MIF,which was conductive to the formation of pre-metastasis niches and occurrence of NPC metastasis in lung.This study provides a new theoretical basis for further understandings of the molecular mechanism of distant metastasis of NPC,and also a new diagnostic index and intervention target for anti-early metastasis of NPC.