【作者】 张莹；

【导师】 谢莹；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2021， 硕士

【摘要】 目的:本研究从肿瘤相关内皮细胞的诱导入手,构建鼻咽癌相关内皮细胞(Nasopharyngeal Carcinoma-Associated Endothelial Cell,NPCEC)模型。明确miR-92a-3p在NPCECs中的表达对内皮细胞屏障功能的影响。预测并验证miR-92a-3p的靶基因及调控机制,了解miR-92a-3p与鼻咽癌(Nasopharyngeal Carcinoma,NPC)经循环系统转移之间的关系。方法:1.培养人淋巴管内皮细胞(Human Lymphatic Endothelial Cell,HLEC)、人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cell,HUVEC)和人鼻咽癌高转移细胞株(5-8F)。2.构建NPCEC模型:建立两种共培养体系,即嵌入式细胞共培养体系和条件培养基(CM)共培养体系,将5-8F分别与HLEC/HUVEC共培养。48h后,通过倒置相差显微镜观察共培养后两种内皮细胞的形态学变化;实时荧光定量聚合酶链反应(q RT-PCR)检测共培养后两种内皮细胞中肿瘤内皮标志物8(TEM8)和miR-92a-3p的表达;划痕实验和小管形成实验检测共培养后两种内皮细胞的迁移和成管能力。3.分别将miR-92a-3p mimic、miR-92a-3p inhibitor及其相应的阴性对照转染至HLEC/HUVEC中,上调或下调内皮细胞中miR-92a-3p的表达水平。48h后,采用CCK-8和Ed U实验检测miR-92a-3p对内皮细胞增殖的影响;Transwell实验检测miR-92a-3p对内皮细胞通透性的影响。4.应用q RT-PCR实验验证NPCECs(C-HLEC/C-HUVEC)中miR-92a-3p与PECAM1的表达相关性;Western Blot和免疫荧光实验验证miR-92a-3p对PECAM1的反向调控作用。应用生物信息学软件分析预测miR-92a-3p与PECAM1的靶向结合关系,并通过双荧光素酶报告基因实验进行验证。5.构建稳定过表达PECAM1的HLEC和HUVEC细胞株,并通过绿色荧光蛋白(GFP)法和q RT-PCR实验验证慢病毒感染效率。6.挽救实验:分别将miR-92a-3p mimic或miRNA mimic Negative Control(即:miR-92a-3p mimics或mi-NC)与PECAM1过表达重组慢病毒颗粒或阴性对照慢病毒颗粒(即:PECAM1 OE或Vector)共转染至HLEC/HUVEC中。48h后,采用q RT-PCR和免疫荧光实验检测共转染后PECAM1基因的表达情况;Transwell实验检测共转染后对HLEC/HUVEC通透性的影响。结果:1.5-8F分别与HLEC/HUVEC间接接触共培养48h后,两种内皮细胞形态伸长、触角增多,变成多角形,细胞间连接疏松,细胞结构紊乱,TEM8和miR-92a-3p的m RNA表达水平均显著升高,且迁移能力及成管能力均明显增强。2.与相应的阴性对照组比较,过表达miR-92a-3p后能显著增加内皮细胞通透性;反之,下调miR-92a-3p的表达量后,则会显著降低内皮细胞通透性。但上调或下调内皮细胞中miR-92a-3p的表达水平,对HLEC/HUVEC的增殖能力无明显影响。3.间接接触共培养后,与对照组相比,两种共培养方式诱导的NPCECs中PECAM1的表达量均显著减少,而miR-92a-3p的表达量却显著增加,两者间存在负相关性。4.Western Blot和免疫荧光实验结果显示,下调miR-92a-3p后,PECAM1蛋白表达量显著增加,表现为细胞膜上的荧光信号明显增强。而过表达miR-92a-3p后,PECAM1蛋白表达量显著减少,表现为内皮细胞间连接被破坏,荧光信号减弱、出现断裂,呈锯齿状分布。提示miR-92a-3p反向调控PECAM1的表达。5.应用生物信息学软件RNA22对miR-92a-3p与PECAM1的靶向结合关系进行预测,结果显示在PECAM1 m RNA 3’UTR有潜在的miR-92a-3p结合位点。6.双荧光素酶报告基因实验结果显示,与阴性对照组相比,转染miR-92a-3p mimic后可显著抑制荧光素酶活性,表明miR-92a-3p可以与PECAM1的3’UTR靶向结合,抑制PECAM1的表达。然而,在结合位点突变后,miR-92a-3p对PECAM1突变型3’UTR的荧光素酶表达仍具有调控作用,但该调控关系有所减弱。由此推测,虽然PECAM1是miR-92a-3p的直接靶点,但除本实验突变的结合位点以外,还可能存在其他非典型的结合位点,有待于进一步验证。7.挽救实验结果显示,与mi-NC+Vector组相比,miR-92a-3p mimics+Vector组中PECAM1的表达量显著降低,HLEC/HUVEC通透性明显增加;与miR-92a-3p mimics+Vector组相比,miR-92a-3p mimics+PECAM1 OE组中PECAM1的表达量显著升高,HLEC/HUVEC通透性明显降低。由此提示,PECAM1的过表达挽救了miR-92a-3p对HLEC/HUVEC中PECAM1表达的抑制作用,从而挽救了miR-92a-3p对内皮细胞屏障功能的破坏作用。结论:1.将正常的内皮细胞(N-HLEC/N-HUVEC)与5-8F间接接触共培养后,可将其诱导成为NPCECs(C-HLEC/C-HUVEC)。2.TEM8和miR-92a-3p在NPCECs(C-HLEC/C-HUVEC)中表达上调。3.NPCECs(C-HLEC/C-HUVEC)的迁移及成管能力均显著增强。4.miR-92a-3p增加内皮细胞(HLEC和HUVEC)的通透性,但对内皮细胞的增殖能力无明显影响。5.NPCECs(C-HLEC/C-HUVEC)中miR-92a-3p与PECAM1的表达量存在负相关性。6.miR-92a-3p反向调控PECAM1的表达。7.PECAM1为miR-92a-3p的靶基因。8.PECAM1在维持内皮细胞屏障功能中发挥重要作用。9.miR-92a-3p通过靶向PECAM1抑制HLEC/HUVEC中PECAM1的表达,从而破坏内皮细胞屏障,增加内皮细胞通透性。更多还原

【Abstract】 Objective: In this study,we started with the induction of Tumor-associated endothelial cells to establish a nasopharyngeal carcinoma(NPC)-associated endothelial cell(NPCEC)model.To determine the effect of miR-92a-3p expression on the barrier function of NPC-associated endothelial cells(NPCEC s).To predict and verify the target gene and regulatory mechanism of miR-92a-3p,and to understand the relationship between miR-92a-3p and NPC transfer through the circulatory system.Methods: 1.Human lymphatic endothelial cell(HLEC),human umbilical vein endothelial cells(HUVEC)and human highly metastatic NPC cells(5-8F)were cultured.2.Establishment of the NPCEC model.Two co-culture systems,including embedded cell co-culture system and conditioned medium(CM)co-culture system,were established.And 5-8F was co-cultured with HLEC/HUVEC respectively.After 48 hours,the morphological changes of the two kinds of endothelial cells were observed by inverted phase contrast microscope,the expression of Tumor Endothelial Marker8(TEM8)and miR-92a-3p in the two kinds of endothelial cells were detected by real-time fluorescence quantitative polymerase chain reaction(q RT-PCR),and the migration and tube formation ability of the two kinds of endothelial cells were detected by scratch test and tubule formation test.3.miR-92a-3p mimic,miR-92a-3p inhibitor and their corresponding negative controls were transfected into HLEC/HUVEC to up-regulate or down-regulate the expression of miR-92a-3p in endothelial cells.After 48 hours,the effect of miR-92a-3p on endothelial cell proliferation was detected by CCK-8 and Ed U assay,and the effect of miR-92a-3p on endothelial cell permeability was detected by Transwell assay.4.QRT-PCR experiment was used to verify the expression correlation between miR-92a-3p and PECAM1 in NPCECs(C-HLEC/C-HUVEC).Western Blot and immunofluorescence experiments were used to verify the reverse regulation of miR-92a-3p on PECAM1.The targeted binding relationship between miR-92a-3p and PECAM1 was analyzed and predicted by bioinformatics software,and verified by double luciferase reporter gene experiment.5.HLEC and HUVEC cell lines stably overexpressing PECAM1 were constructed,and the infection efficiency of lentivirus was verified by green fluorescent protein(GFP)and q RT-PCR experiments.6.Rescue experiment: miR-92a-3p mimic or miRNA mimic negative control were co-transfected with PECAM1 overexpressed recombinant lentiviral particles or negative control lentiviral particles into HLEC/HUVEC,respectively.48 hours later,q RT-PCR and immunofluorescence assay were used to detect the expression of PECAM1 gene after co-transfection,and Transwell assay was used to detect the effect of co-transfection on HLEC/HUVEC permeability.Results: 1.After co-cultured with 5-8F for 48 hours,the morphology of HLEC/HUVEC elongated,the antennae increased,became polygonal,the connection between the cells was loose,and the cell structure was disordered.The m RNA expression levels of TEM8 and miR-92a-3p were significantly increased in the co-cultured HLEC/HUVEC cells,and their migration ability and tube formation ability were both significantly enhanced.2.Compared with the corresponding negative control group,overexpression of miR-92a-3p significantly increased endothelial cell permeability;on the contrary,down-regulating the expression of miR-92a-3p significantly reduced endothelial cell permeability.However,up-regulation or down-regulation of miR-92a-3p expression in endothelial cells had no significant effect on the proliferation of HLEC/HUVEC.3.After indirect contact co-culture,compared with the control group,the expression of PECAM1 in NPCECs induced by the two co-culture methods was significantly decreased,while the expression of miR-92a-3p was significantly increased,indicating there was a negative correlation between PECAM1 and miR-92a-3p.4.The results of Western Blot and immunofluorescence experiments showed that after down-regulation of miR-92a-3p,the expression of PECAM1 protein increased significantly,which showed that the fluorescence signal on the cell membrane was significantly enhanced.After overexpression of miR-92a-3p,the expression of PECAM1 protein decreased significantly,which showed that the junction between endothelial cells was destroyed,the fluorescence signal was weakened,broken,and showed zigzag distribution.It is suggested that miR-92a-3p inversely regulates the expression of PECAM1.5.The bioinformatics software RNA22 was used to predict the targeted binding relationship between miR-92a-3p and PECAM1.The results showed that there were potential miR-92a-3p binding sites in PECAM1 m RNA 3’UTR.6.The results of double luciferase reporter gene assay showed that compared with the negative control group,the transfection of miR-92a-3p mimics could significantly inhibit the luciferase activity,indicating that miR-92a-3p could bind to the 3’UTR of PECAM1 and inhibit the expression of PECAM1.However,after a mutation at the binding site,miR-92a-3p still had a regulatory effect on the expression of luciferase in PECAM1 mutant 3’UTR,but the regulation relationship was significantly weakened.It is speculated that although PECAM1 is the direct target of miR-92a-3p,there may be other atypical binding sites besides the mutant binding sites in this experiment,which need to be further verified.7.The results of Rescue experiment showed that compared with mi-NC +Vector group,the expression of PECAM1 was significantly decreased and HLEC/HUVEC permeability was significantly increased in miR-92a-3p mimics+ Vector group;compared with miR-92a-3p mimics + Vector group,the expression of PECAM1 in miR-92a-3p mimics + PECAM1 OE group was significantly increased and HLEC/HUVEC permeability was significantly decreased.It is suggested that the overexpression of PECAM1 rescues the inhibitory effect of miR-92a-3p on the expression of PECAM1 in HLEC/HUVEC,and thus rescues the destructive effect of miR-92a-3p on the barrier function of endothelial cells.Conclusion: 1.After indirect contact co-cultured with 5-8F,normal endothelial cells(N-HLEC/N-HUVEC)can be induced into NPCECs(C-HLEC/C-HUVEC).2.The expressions of TEM8 and miR-92a-3p were up-regulated in NPCECs.3.The migration and tube-forming ability of NPCECs were significantly enhanced.4.miR-92a-3p increased the permeability of endothelial cells(HLEC and HUVEC),but had no significant effect on the proliferation of endothelial cells.5.There was a negative correlation between the expression of miR-92a-3p and PECAM1 in NPCECs.6.miR-92a-3p inversely regulates the expression of PECAM1.7.PECAM1 is the target gene of miR-92a-3p.8.PECAM1 plays an important role in maintaining endothelial barrier function.9.miR-92a-3p inhibits the expression of PECAM1 in HLEC/HUVEC by targeting PECAM1,thus breaking the endothelial cell barrier and increasing endothelial cell permeability.更多还原