【作者】 汪洋；

【导师】 欧敬民；

【作者基本信息】 上海交通大学 ， 外科学（普外科）， 2018， 硕士

【摘要】 目的研究miR-199a-5p(miR-199a)在血管瘤发生发展中的作用机制。方法收集血管瘤组织及瘤旁组织样本,利用qRT-PCR检测组织miR-199a和PCNA(增殖细胞核抗原)的表达水平,采用Spearman相关分析不同阶段血管瘤miR-199a和PCNA的相关性。然后,检测转染了miR-199a mimic或miR-199a inhibitor的血管瘤来源的内皮细胞(HDEC)和小鼠血管内皮瘤细胞(CRL-2586EOMA)的增殖和凋亡水平。最后,通过生物信息学分析确定miR-199a的目标基因。用qRT-PCR和western blotting分析miR-199a mimic或miR-199a inhibitor转染HDEC和CRL-2586EOMA细胞后的HIF1α表达水平。使用双荧光素酶检测探索miR-199a与靶基因HIF1α的关系。结果(1)增生期血管瘤组织的miR-199a的表达量降低,PCNA表达量升高,Spearman相关分析表明增生期血管瘤组织miR-199a与PCNA表达量具有负相关性。(2)miR-199a mimic抑制HDEC和CRL-2586EOMA细胞增殖活性,促进细胞凋亡,western blotting检测显示PCNA表达水平减少,cleaved caspase-3表达水平增高。而miR-199a inhibitor的结果相反。(3)使用生物信息学分析,HIF1α被认为是最有可能与miR-199a结合的靶基因。用qRT-PCR和western blotting分析显示转染了miR-199a mimic的HDEC和CRL-2586EOMA细胞HIF1α的表达减少,而miR-199a inhibitor增加表达。双荧光素酶报告显示miR-199a mimic降低了WT 3’-UTR HIF1α的荧光素酶活性,而miR-199a inhibitor增加了这种效果。结论(1)miR-199a可能具有抑制血管瘤组织增殖能力。(2)miR-199a mimic抑制细胞增殖,诱导细胞凋亡,而miR-199a inhibitor促进细胞增殖并抑制细胞凋亡。(3)HIF1α被认为是miR-199a作用于血管瘤细胞的目标基因。更多还原

【Abstract】 Objective To investigate the function of miR-199a-5p(miR-199a)in HAs progression.Methods We collected normal skin tissues(n=15),proliferating phase HAs(n=15)and involunting phase HAs(n=15),qRT-PCR analysis of expression level of miR-199 a and PCNA(Proliferating Cell nuclear A antigen).Spearman correlation analysis of association of miR-199 a and PCNA expression in proliferating phase HAs and involunting phase HAs.After HDEC and CRL-2586 EOMA cells were transfected with miR-199 a mimic or miR-199 a inhibitor,evaluation of the cell proliferation activity and analysis of the cell apoptosis index.We screened the target genes of miR-199 a by using the starBase v2.o for bioinformatic analysis.After HDEC and CRL-2586 EOMA cells were transfected with miR-199 a mimic or miR-199 a inhibitor,qRT-PCR and western blotting analysis the expression of HIF1α.PmirGLO report vector carrying wild type 3′-UTR or mutated 3′-UTR of HIF1α was co-transfected with miR-199 a mimic or inhibitor into HDEC and CRL-2586 EOMA cells.Luciferase activities were examined with a Dual-luciferase Reporter System.Results(1)The expression level of miR-199 a was decreased,while that of PCNA was increased in proliferating phase HAs compare with the normal skin tissues and the involuting phase HAs.In addition,Spearman correlation analysis indicated that miR-199 a had a negative correlation with PCNA expresstion in proliferating phase HAs,but had no correlation in involuting phase HAs.(2)miR-199 a mimic suppressed cell proliferation activity and induced cell apoptosis in HDEC and CRL-2586 EOMA cells.The expression levels of PCNA and cleaved caspase-3 were detected by western blotting,indicating a decreased expression of PCNA and increased expression of cleaved caspased-3 caused by miR-199 a mimic.The results of miR-199 a inhibitor is the opposite.(3)After bioinformatic analysis,HIF1α ranked the first place and was considered to have the greatest potential to bind with miR-199 a.Besides,qRT-PCR and western blotting analysis showed that miR-199 a mimic decreased the expression of HIF1α,whereas miR-199 a inhibitor increased its expression in HDEC and CRL-2586 EOMA cells.The results of Dual-luciferaseReporter System illustrated that miR-199 a mimic reduced the luciferase activity of WT 3`UTR of HIF1α,while miR-199 a inhibitor increased this effect,but both of them had no effect on the luciferase activity of mutation 3` UTR of HIF1α.Conclusion(1)miR-199 a may inhibited HAs cell proliferation.(2)miR-199 a mimic inhibited cell proliferation and induced apoptosis,but miR-199 a inhibitor promoted proliferation and prevented apoptosis.(3)HIF1α was identified as a direct target of miR-199a.更多还原