| ObjectiveOvarian cancer is the leading cause of death in gynecological malignancies.Although the early diagnosis has a good prognosis,most cases are diagnosed in advanced.Five-year survival rate of patients with the ovarian cancer is only 30%-40%.The low survival rate might be due to the lack of overt symptoms and about 75%patients in advanced stages are diagnosed with peritoneal metastasis and chemotherapy resistance.Therefore,it's very important to understand the mechanism of infiltration and metastasis of ovarian cancer and to discover new tumor markers and therapeutic treatment.Methods1.Isolation and Characterization of exosomesFour ovarian cancer cell lines(A2780,UWB,HEY,Anglne)were cultured in hypoxia(1%O2)and normoxia(5%CO2)condition,independently.Exosomes were isolated from conditioned medium supernatant using Exo Isolation Kit.Transmission electron microscopy(TEM)and Nano particle tracking analysis(NTA)revealed the morphology and particle size of exosomes.Western Blot detected the expression of exosome-specific protein CD81,CD63.2.Screening and validating differentially expressed miRNAsA High-Throughput sequencing(lllumina PE150)was performed to detect miRNAs expression profile of exosomes under normoxia and hypoxia condition.Four miRNAs were verified by quantitative-polymerase chain reaction(Q-PCR).miR-199a-5p was been chosen,and verified in four ovarian cancer cell lines(UWB,HEY,A2780 and Anglne)and exosome derived from them through Q-PCR.Ovarian cancer(n=20)and normal fallopian tube tissues(n=20)was recruited from Shan Dong provincial Qian Fo Shan hospital.Both of them are the same age.Then Q-PCR was used to detect the expression of miR-199a-5p in tissues.Ovarian cancer OC-1601 tissue microarray was purchased.Fluorescence in situ hybridization(FISH)is used to analyze the expression level of miR-199a-5p and the clinicopathological characteristics of ovarian cancer patients(age,tumor infiltration,lymph node metastasis,and tumor stage).3.Bioinformatics analysis of miR-199a-5pThe potential target gene of miR-199a-5p was predicted by miRwalk which contains twelve databases.The function of miR-199a-5p was enriched by DAVID(https://david.ncifcrf.gov/)database,which contained GO and KEGG,to analyze the biological functions and signaling pathways of miR-199a-5p target gene.4.The migration and invasion effect of miR-199a-5p on ovarian cancer cell and molecular mechanism.Wound-Healing,Transwell and Matrigel Transwell assay were performed to explore the role of miR-199a-5p on ovarian cancer cell invasion and migration,we transfected with miR-199a-5p plasmid or inhibitor to A2780 cells under normoxia and hypoxia condition in vitro.Luciferase Reporter Assay confirmed the correlation between HIF-2? and miR-199a-5p.The expression of HIF-2?,Wnt3a and ?-catenin were detected after treated with overexpression or inhibition of miR-199a-5p in ovarian cancer A2780 cell by Western Blot.The expression of HIF-2?,wnt3a and?-catenin protein in ovarian cancer tissues was also detected by Western Blot.Immunohistochemistry(IHC)detection of HIF-2? in ovarian cancer tissue chip,and explore the potential correlation between HIF-2? and miR-199a-5p.5.Effect of miR-199a-5p on the junction of endothelial cells by exosomal transfermiR-199a-5p was labeled with FITC,and ovarian cancer cell-secreted exosome was tagged with PKH26,then co-cultured with HUVECs.The images were photographed by confocal microscope.miR-199a-5p plasmid and miR-199a-5p inhibitor were transfected into endothelial cells,and the effects of miR-199a-5p on endothelial cell adhesion were observed by Western blot.The expression of(junctional adhesion molecule(JAM-A)and VE-Cadherin in tissue chips was detected by immunohistochemistry.Result1.Extraction and identification of exosomesTEM revealed there were typical cup-shaped round vesicles.NTA exhibited the size of those vesicles were mainly 30?170nm,and Western Blot showed that exosome-specific protein CD81,CD63 were positive.2.Screening and validation of miR-199a-5pHigh-Throughput sequencing showed a total of 359 differentially expressed miRNAs,among which 153 were up-regulated and 206 were down-regulated(FDR<0.05).Four miRNAs were selected,miR-199a-5p and miR-214-5P were downregulated in hypoxia condition compared with normoxia.miR-199a-5p(Z=-4.438,P=0.0030),miR-214-5P(Z=-4.1828,P=0.0240),and miR-216a-5p and miR-216b-5p were upregulated in hypoxia condition compared with normoxia.miR-216a-5P(Z=2.5317,P=0.0064),miR-216b-5p(Z=2.9195,P=0.0053).miR-199a-5p was the most significant difference.The verification of Q-PCR was consistent with the result of high-throughput sequence.Q-PCR showed the expression of miR-199a-5p in cells,exosomes and fresh tissues,compared with normal fallopian tube tissue,miR-199a-5p was lower expressed in ovarian cancer.Compared with normoxic condition,miR-199a-5p was lower expressed in hypoxic A2780 cells and exosome.FISH showed that miR-199a-5p expression in normal fallopian tube tissue showed stronger fluorescence than in the ovarian cancer tissue.Correlation of exosomal miR-199a-5p with clinicopathologic characteristics in ovarian cancer patients indicated that miR-199a-5p was associated with tumor infiltration(P<0.0010),lymph node metastasis(P<0.0010),tumor TNM stage(P<0.0010),not associated with age(P=0.5740).3.Bioinformatics analysis of miR-199a-5pThrough at least three databases in the miRwalk database to predict the potential target gene of miR-199a-5p.We enriched the intersection of these database and,obtained 70 candidate target genes which used to analyze the miR-199a-5p biology function.GO annotation enriched miR-199a-5p molecular function,biological process and cellular component.KEGG signaling pathway enriched the cancer related multiple signaling pathways:Wnt/?-catenin pathway,P13K pathway,NF-?B pathway,etc.4.Through miR-199a-5p-HIF-2?-Wnt/?-catenin axis,suppress ovarian cancer cell migration and invasion.Transfection of miR-199a-5p plasmid under normoxia and hypoxia condition can inhibit the invasion and metastasis of ovarian cancer A2780 cells,while treated with miR-199a-5p inhibitor,the result was reversed.Through biological analysis HIF-2?was a potential target gene of miR-199a-5p.Double luciferase report showed that after transfected with HIF-2? wild-type,miR-199a-5p could significantly inhibit the luciferase activity,but had no effect on the HIF-2? mutant type,which directly proved that HIF-2? was a direct target of miR-199a-5p.Immunoblotting showed that the overexpression of miR-199a-5p significantly inhibited the expression of HIF-2?,Wnt3a and ?-catenin,while miR-199a-5p inhibitor reverse the results in A2780 cells.HIF-2?,Wnt3a and ?-catenin were highly expressed in ovarian cancer tissues compared with normal fallopian tube tissues by immunoblotting.Besides,immunohistochemical showed that HIF-2? was strong positive in ovarian cancer tissues,and negative or weak positive in normal fallopian tissues.It was speculated that the expression of HIF-2? may be negatively correlated with miR-199a-5p in ovarian cancer tissues.5.Exosome miR-199a-5p can enhance the tight connection of endothelial cellsConfocal microscopy showed that endothelial cells could phagocytose exosomes which are secreted by A2780 cell and contain miR-199a-5p.miR-199a-5p can increased endothelial cell tight junction protein VE-cadherin and JAM-A and IHC results showed that VE-Cadherin and JAM-A were negative or weakly positive in ovarian cancer compared to normal fallopian tube tissue.ConclusionThe expression of ovarian cancer cell secreted exosomal miR-199a-5p was downregulated in ovarian cancer tissues,which is correlated with the clinical TNM stage,lymphatic metastasis and tumor infiltration.Under hypoxia microenvironment,the downregulated exosomal miR-199a-5p promoted the migration and invasion of ovarian cancer cells.The target gene of miR-199a-5p is HIF-2?,and miR-199a-5p may play a role through Wnt/?-catenin signaling pathway.Exosomal miR-199a-5p acts on endothelial cells via paracrine,the downregulated of miR-199a-5p may inhibit the tight connection of endothelial cells. |
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