摘要
目的探讨长链非编码RNA LINC00665在肝癌中的表达和对肝癌血管生成的调控作用及潜在分子机制。方法收集2016年5月至2017年4月南京医科大学第一附属医院100例肝细胞癌患者的癌组织和相应的癌旁组织,并比较生存预后。实时定量聚合酶链反应检测LINC00665在肝癌组织和细胞中的表达。通过小管形成实验和鸡胚尿囊膜实验检测过表达LINC00665的Hep-3B肝癌细胞血管生成情况。生物信息学数据库预测LINC00665下游微RNA(miRNA)及靶基因,聚合酶链反应、Western印迹、双荧光素酶报告基因进一步检测LINC00665、miR-126-5p和血管内皮生长因子A(VEGFA)的关系。结果LINC00665在肝癌组织中的表达(6.5±2.8)明显高于癌旁组织(4.8±3.1),差异有统计学意义(t=4.12,P<0.001)。100例肝癌患者根据LINC00665表达的中位数分组,LINC00665高表达组(n=50)的累积生存率低于LINC00665低表达组(n=50),差异有统计学意义(χ2=3.79,P=0.008)。与LINC00665组(过表达LINC00665的Hep-3B肝癌细胞)共培养后,人脐静脉内皮细胞小管形成的长度(596.0±22.3)μm、数量(36.3±4.5)个,均高于与NC组(仅感染空载体的Hep-3B肝癌细胞)共培养的人脐静脉内皮细胞小管形成长度(127.0±13.5)μm和数量(9.3±1.5)个,差异均有统计学意义(t=31.15、9.82,P<0.001、P=0.001)。鸡胚尿囊膜实验与小管形成实验结果一致。Western印迹检测LINC00665组VEGFA蛋白的相对表达高于NC组,差异有统计学意义(t=7.15,P<0.001)。使用StarBase和DIANA数据库预测和筛选LINC00665下游miR-126-5p。使用StarBase数据库对LINC00665/miR-126-5p/VEGFA轴的结合位点进行预测。双荧光素酶报告基因实验结果中,与miR-126-5p模拟物共转染的LINC00665、VEGFA载体荧光强度均下降。结论LINC00665在肝癌中高表达,并且与肝癌患者的不良预后相关。LINC00665通过调控miR-126-5p/VEGFA轴促进肝癌的血管生成。
Objective To investigate the expression of long non-coding RNA 00665(LINC00665)in hepatocellular carcinoma(HCC)and its regulatory effect on angiogenesis of hepatocellular carcinoma cells and its potential molecular mechanism.Methods HCC tissues and corresponding paracancerous tissues of 100 patients with HCC in the First Affiliated Hospital of Nanjing Medical University from May 2016 to April 2017 were collected,and the survival prognosis was compared.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of LINC00665 in HCC tissues and cells.The effect of LINC00665 overexpressed Hep-3B cells on the angiogenesis of human umbilical vein endothelial cells(HUVEC)was examined by tube formation assay and chick chorioallantoic membrane assay.Bioinformatics database predicted the downstream microRNA(miRNA)and target genes of LINC00665,and the relationship between LINC00665,miR-126-5p and vascular endothelial growth factor A(VEGFA)was verified by RT-qPCR,Western blot and dual-luciferase reporter gene assay.Results The expression level of LINC00665 in HCC(6.5±2.8)was significantly higher than that in paracancer tissues(4.8±3.1),the difference was statistically significant(t=4.12,P<0.001).According to the median LINC00665 expression level of 100 patients with HCC,the cumulative survival rate of LINC00665 high expression group(n=50)was lower than that of LINC00665 low expression group(n=50),and the difference was statistically significant(χ2=3.79,P=0.008).After co-culture with LINC00665 group(Hep-3B cells overexpressing LINC00665),the length of HUVEC cell tubule formation was(596.0±22.3)μm,and the number of HUVEC cell tubules was(36.3±4.5),which were both higher than NC group with the tubule formation length(127.0±13.5)μm and the number(9.3±1.5)of HUVEC cells co-cultured with Hep-3B cells of control group,and the differences were statistically significant(t=31.15,9.82,P<0.001,P=0.001).The chick chorioallantoic membrane assay results were similar to tube formation assay.Western blot detected that the relative expression of VEGFA in LINC00665 group was higher than that in NC group,the difference was statistically significant(t=7.15,P<0.001).StarBase and DIANA database were used to predict and screen LINC00665 downstream miR-126-5p.StarBase database was used to predict the binding sites of LINC00665/miR-126-5p/VEGFA axis.In dual-luciferase reporter gene assay,the fluorescence intensity of LINC00665 and VEGFA vector co-transfected with miR-126-5p mimics decreased.Conclusion LINC00665 is highly expressed in HCC and is associated with poor prognosis in patients with HCC.LINC00665 promotes angiogenesis of HCC by regulating the miR-126-5p/VEGFA axis.
作者
王金宜
周永平
华志元
王浩
闫勇
丁治杰
孔连宝
戴途
Wang Jinyi;Zhou Yongping;Hua Zhiyuan;Wang Hao;Yan Yong;Ding Zhijie;Kong Lianbao;Dai Tu(Hepatobiliary Center,the First Affiliated Hospital of Nanjing Medical University,Key Laboratory of Liver Transplantation,Chinese Academy of Medical Sciences,NHC Key Laboratory of Living Donor Liver Transplantation,Nanjing 210019,China;Wuxi Clinical College of Nantong University,Wuxi 214000,China;Wuxi Second Affiliated Hospital of Nanjing Medical University,Wuxi 214000,China)
出处
《中华肝胆外科杂志》
CAS
CSCD
北大核心
2023年第9期687-693,共7页
Chinese Journal of Hepatobiliary Surgery
基金
无锡市卫健委科研项目(M202106、M202110)
"太湖之光"科技攻关基金(Y20222002)
江苏省研究生实践创新计划基金(SJCX22_0645)。
关键词
癌
肝细胞
RNA
长链非编码
血管生成
Carcinoma,hepatocellular
RNA,long noncoding
Angiogenesis
