The Mechanism Study On Secreted MiR-9 Deliveried By Exosome Suppresses Nasopharyngeal Carcinoma Metastasis

Background and ObjectNasopharyngeal carcinoma is prevalent in South religion of China and Southeast Asia,which has clinical characteristics of low differentiation and high metastasis.75%?90%newly diagnosed nasopharyngeal carcinoma patients had locally advanced disease combined with metastatic of lymph nodes.Although the local control rate of nasopharyngeal carcinoma has been significantly improved along with the chemoradiotherapy based on IMRT,it has been still impossible to early predict and effectively intervent metastasis of nasopharyngeal carcinoma in clinical.The metastasis rate and overall survival rate have not been improved significantly,the median survival time is only 7.3-10 months once the recurrence or metastasis emerged,and distant metastasis is still the main cause of death in patients with nasopharyngeal carcinoma.Therefore,a comprehensive analysis of the molecular mechanism of NPC metastasis,and actively seeking new therapeutic method which is effective,safe and reliable,is the key to effectively control the metastasis of nasopharyngeal carcinoma and improve the survival rate of patients with nasopharyngeal carcinoma.Exosomes have been proved to mediate signal and contents such as protein and nucleic acid transduction between tumor cells and peripheral components,which may induce pre-metastatic niche to promote tumor metastasis.Therefore,exosomes play a very important role in tumor metastasis,which could be a new biomarker and therapeutic target to predict and prevent tumor metastasis.Tumor angiogenesis and increased permeability of vascular is also one of the characteristics of pre-metastatic niche,which is necessary for promoting metastasis and is considered as a target for the treatment of cancer and the prevention of tumor recurrence and metastasis.Although drugs which target angiogenesis have already been used in clinic and achieved encouraging clinical efficacy,the angiogenesis of tumor is extremely complex,and the majority of tumor vascular targeted therapy resulted in side effects such as high blood pressure and drug resistance because of non specific blocking VEGF signaling pathway.It is necessary to search for a more specific target and to enhance the effect of tumor vascular targeting therapy.MicroRNA has been proved to play an important role in the mechanism of tumor angiogenesis.MiRNA that affects angiogenesis is termed angiomiR,which will provide new target for tumor vascular targeting therapy.Compared with a single target medicine which regulate signaling pathway of angiogenesis,it may avoid drug side-effects and drug resistance to suppress tumor angiogenesis through correcting the abnormality of angiomiR.Tumor cells have been shown to secrete microRNA into the vascular endothelial cells via exosomes to mediate angiogenesis.In our previous studies,miR-9 was confirmed to be involved in metastasis of nasopharyngeal carcinoma,but there is no report whether its abnormal expression would have relationship with tumor angiogenesis which results in metastasis of nasopharyngeal carcinoma,and whether nasopharyngeal carcinoma cells could secreted miR-9 to vascular endothelial cells via exosomes.Thus,we carried out corresponding research in order to determine the effect of abnormal miR-9 expression on nasopharyngeal carcinoma angiogenesis,confirm nasopharyngeal carcinoma cells secrete miR-9 into endothelial cell via exosome,and illuminate the molecular mechanism and signal pathway of secretory miR-9 effects on vascular endothelial cells.Materials and Methods1.Immunohistochemical stain and in situ hybridization:Immunohistochemical stain of CD31 and in situ hybridization of miR-9 in 50 nasopharyngeal carcinoma tissue specimens;Immunohistochemical stain of MDK in 100 nasopharyngeal carcinoma and 80 nasopharyngitis tissue specimens2.Lentivirus packaging and transfection,exosome extraction and identification,trace experiment:to build stable expression miR-9 and contrast 5-8F and CNE1 cell lines;Collect supernatant fluid of Lv-miR-9 NPC cells or Lv-control NPC cells to extract exosomes;Nanosight,western blot(CD63?TSG101),transmission electron microscopy;Extract RNA and detection the expression of miR-9 by RT-qPCR;CY3 labeled miR-9 and PKH67 labeled exosomes,tracing miR-9 and exosomes by laser confocal microscope3.Functional experiment in vitro and vivo:CCK8,invasion assay,tube formation assay,matrigel plug assay4.Prediction and verification of target gene:Prediction of target gene by bioinformatics databases Targetscan?Pictar?miRBase;RT-qPCR screen candidate target gene;Dual luciferase reporter gene experiment and western blot were used to verify candidate target gene MDK;Phospho-Kinase Array and western blot were used to verify PI3K/AKT and MAPK signaling pathway5.Statistical analysis:SPSS 20.0 software was used for statistical analysis.Independent-samples t test was used for two groups of measurement data.Spearman method was used for correlation analysis of immunohistochemistry and in situ hybridization semi quantitative data.CCK8 assay were analyzed by Factorial design analysis of variance.Non-parametric tests were used to compare independent groups of numerical data(Kruskal-Wallis test)and categorical data(x2-test).Survival curves were estimated using the Kaplan-Meier method and differences among groups were analysed using the log-rank test.The Cox regression model was used to perform multivariate analysis.Results1.The expression of miR-9 was significantly decreased(Z=-3.635,P<0.001)and MVD was significantly increased(t=-3.507,P=0.001)in III-IV nasopharyngeal carcinoma biopsy specimens.Expression of miR-9 has negative correlation with MVD in nasopharyngeal carcinoma biopsy specimens(r=-0.3075,P=0.0298).2.Establishment of stable overexpression of miR-9 nasopharyngeal carcinoma cell lines,identification and extraction of exosome:Compared with 5-8F Lv-control cells,expression of miR-9 was upregulated by 61.487-fold in 5-8F Lv-miR-9 cells(P=0.0016);expression of miR-9 was upregulated by 48.05-fold in CNE1 Lv-miR-9 cells when compared with CNE1 Lv-control cells(P=0.0048).Lv-miR-9 cells and Lv-control cells could secrete exosomes,which have similar quantity and morphology.Expression of miR-9 in 5-8F Lv-miR-9 exosomes was upregulated by 36.56-fold when compared with 5-8F Lv-control exosomes(P=0.0016);Expression of miR-9 in CNE1 Lv-miR-9 exosomes was upregulated by 24.77-fold when compared with CNE1 Lv-control exosomes(P=0.0015).Confocal laser scanning showed that CY3-labeled-miR-9 secreted by nasopharyngeal carcinoma cells was transported into endothelial cells by PKH67-labeled-exosomes.3.Functional experiment in vitro and vivo:The result of CCK8 showed that HUVECs cultured by 5-8F Lv-miR-9 exosomes grew slower significantly when compared with 5-8F Lv-control(P<0.001),HUVECs cultured by CNE1 Lv-miR-9 exosomes grew slower significantly when compared with CNE1 Lv-control(P<0.001);The result of transwell invasion assay showed that the number of HUVECs cultured by 5-8F Lv-miR-9 exosomes decreased by 78.11%(P<0.001),the number of HUVECs cultured by CNE1 Lv-miR-9 exosomes decreased by 63.51%(P<0.001);The result of tube formation assay showed that the relative tube length of HUVECs cultured by 5-8F Lv-miR-9 exosomes decreased by 65%(P<0.001),the relative length of the relative tube length of HUVECs cultured by CNE1 Lv-miR-9 exosomes decreased by 56.86%(P<0.001);The result of matrigel plug assay showed that new blood vessels in matrigel plug markedly reduced in Lv-miR-9 exosome group when compared with Lv-control exosome group,the relative tube length of HUVECs cultured by Lv-miR-9 exosomes decreased by 68.18%(P<0.001).4.Prediction and verification of target gene:Bioinformatics software predicted 17 target genes,in which the result of target gene screening showed 9 expressed differently induced by exosomal miR-9,and the most obvious difference was between the multiple of MDK(t=37.500,P=0.001);Dual luciferase assay showed exosomal miR-9 combined with MDK 3'UTR,which resulted in decrease of fluorescence activity(t=33.924,P=0.000),whereas not combined with the MDK MT 3'UTR(t=0.975,P=0.385).The combination of exosomal miR-9 and MDK 3'UTR could be inhibited by transient transfection of miR-9 inhibitor,which resulted in increase of fluorescence activity(t=26.077,P=0.001),whereas there was no effect on MDK MT 3'UTR group(t=1.823,P=0.142);Western blot showed that expression of MDK in HUVECs cultured by Lv-miR-9 exosomes decreased significantly when compared with HUVECs cultured by 1640 complete medium or Lv-control exosomes;The result of antibody microarray showed that phosphorylation levels of seven proteins in PI3K/AKT signaling pathway decreased in HUVECs cultured by Lv-miR-9 exosomes when compared with Lv-control exosomes,and the difference of phosphorylation of P70S6k and PDK1 which is most obviously has been verified in western blot5.MDK was barely expressed in nasopharyngeal epithelial tissues of nasopharyngitis whereas predominantly expressed in cytoplasm of nasopharyngeal carcinoma cells;The expression of MDK manifested differently among different clinical stage of NPC(P<0.001);Primary tumors staging(P = 0.001),lymph node staging(P = 0.005)and clinical stage(P = 0.001)were significantly higher in patients with high expression of MDK when compared with low expression of MDK group in 100 cases of nasopharyngeal carcinoma specimens;Kaplan-Meier survival analysis showed that the prognosis of NPC patients with high expression of MDK(OS:53.33 months,95%confidence interval:45.56-61.11;PFS:50.02 months,95%confidence interval:41.51-58.52 months)was worse than the patients with low MDK expression(OS:78.13 months,95%confidence interval:72.95-83.31 months;PFS:69.25 months,95%confidence interval:62.81-75.69 months).Differences between groups were analyzed by Log-rank test which showed that OS:Log-rank= 14.923,P = 0.0001,PFS:Log-rank= 10.205,P = 0.0014;In multivariate Cox proportional hazard models analysis,MDK expression showed to be negative independent prognostic factor for OS(RR=0.351,P=0.030)Conclusion1.Expression of miR-9 is negatively correlated with MVD in nasopharyngeal carcinoma tissue.2.Nasopharyngeal carcinoma cells secrete miR-9 to vascular endothelial cells via exosomes.3.Nasopharyngeal carcinoma cells-secreted miR-9 inhibit proliferation,migration and tube formation of endothelial cells.4.Nasopharyngeal carcinoma cells-secreted exosomal miR-9 inhibit angiogenesis through combining with target gene MDK and inhibiting PI3K/AKT signaling pathway in HUVECs.5.The expression of MDK in nasopharyngeal carcinoma tissues can be used to predict prognosis in patients with nasopharyngeal carcinoma.