摘要
目的探究长链非编码RNA(long non-coding RNA,lncRNA)肺癌相关转录物1(lung cancer associated transcript 1,LUCAT1)通过miR-199b-5p/A激酶锚定蛋白1(A-kinase anchoring protein 1,AKAP1)信号轴促进肝癌转移的机制。方法收集2020年1月至2021年10月在成都市新都区人民医院进行手术治疗的80例肝细胞癌(hepatocellular carcinoma,HCC)患者的肝癌及癌旁组织标本,采用RT-qPCR检测LUCAT1、miR-199b-5p、AKAP1 mRNA水平。将Huh7、HepG2细胞进行不同转染,pHRi-si-NC、pHRi-si-LUCAT1分别转染至Huh7、HepG2细胞,pHRi-si-LUCAT1和pHRi-anti-miR-NC、pHRi-si-LUCAT1和pHRi-anti-miR-199b-5p、pHRi-si-LUCAT1和pHRi-NC、pHRi-si-LUCAT1和pHRi-AKAP1分别共转染至Huh7、HepG2细胞。双荧光素酶报告验证LUCAT1对miR-199b-5p、miR-199b-5p对AKAP1的调控关系;EdU染色、划痕实验和Transwell实验检测细胞增殖、迁移和侵袭能力;RT-qPCR检测细胞LUCAT1、miR-199b-5p和AKAP1 mRNA水平;Western blot检测细胞Ki67、基质金属蛋白酶(matrix metalloproteinase,MMP)-2、MMP-9水平。向20只裸鼠皮下注射已转染pHRi-si-LUCAT1的Huh7细胞悬液,30 d后测定移植瘤体积、质量、LUCAT1、miR-199b-5p、AKAP1 mRNA、Ki67、MMP-2、MMP-9水平。结果①与癌旁组织相比,HCC组织LUCAT1(1.51±0.53比1.13±0.72;t=3.802,P<0.001)、AKAP1 mRNA(3.73±0.97比1.28±0.76;t=17.783,P<0.001)水平显著升高,miR-199b-5p(1.21±0.53比3.56±1.02;t=18.286,P<0.001)水平显著降低。②转染pHRi-si-LUCAT1后,miR-199b-5p水平显著升高(Huh7:3.71±0.28比1.00±0.10,t=15.787,P=0.004;HepG2:3.49±0.25比1.00±0.11,t=15.790,P=0.004),LUCAT1(Huh7:0.34±0.05比1.00±0.06,t=14.637,P=0.005;HepG2:0.41±0.06比1.00±0.07,t=11.084,P=0.008)和AKAP1 mRNA水平显著降低(Huh7:0.52±0.05比1.00±0.09,t=8.075,P=0.015;HepG2:0.55±0.06比1.00±0.13,t=5.444,P=0.032);细胞EdU阳性率、划痕愈合率和细胞侵袭数均显著降低(P均<0.05);Ki67(Huh7:0.24±0.03比0.92±0.06,t=17.558,P=0.003;HepG2:0.10±0.03比0.51±0.03,t=16.738,P=0.004)、MMP-2(H
Objective To investigate the mechanism of long non-coding RNA(lncRNA)lung cancer associated transcript 1(LUCAT1)promoted liver cancer metastasis through miR-199b-5p/A-kinase anchoring protein 1(AKAP1)signal axis.Methods HCC tissues and paracancerous tissues of 80 patients with HCC in Xindu District People’s Hospital of Chengdu from January 2020 to October 2021 were selected.RT-qPCR was used to detect the expression of LUCAT1,miR-199b-5p and AKAP1 mRNA.Huh7 and HepG2 cells were transfected differently,pHRi-si-NC and pHRi-si-LUCAT1 were transfected into Huh7 and HepG2 cells,respectively,pHRi-si-LUCAT1 and pHRi-anti-miR-NC,pHRi-si-LUCAT1 and pHRi-anti-miR-199b-5p,pHRi-si-LUCAT1 and pHRi-NC,pHRi-si-LUCAT1 and pHRi-AKAP1 were co-transfected into Huh7,HepG2 cells,respectively.Double luciferase report was used to verify the regulatory relationship of LUCAT1 on miR-199b-5p and miR-199b-5p on AKAP1.EdU staining,scratch test and Transwell test were used to detect the proliferation,migration and invasion ability.RT-qPCR was used to detect the LUCAT1,miR-199b-5p and AKAP1 mRNA levels.Western blot was used to detect Ki67,matrix metalloproteinase(MMP)-2 and MMP-9 levels.Total of 20 nude mice were subcutaneously injected with Huh7 cell suspension transfected with pHRi-si-LUCAT1,and the tumor volume,weight,LUCAT1,miR-199b-5p,AKAP1,Ki67,MMP-2 and MMP-9 protein levels of transplanted tumor were detected 30 days later.Results①Compared with paracancerous tissues,the expression of LUCAT1(1.51±0.53 vs.1.13±0.72;t=3.802,P<0.001)and AKAP1 mRNA(3.73±0.97 vs.1.28±0.76;t=17.783,P<0.001)in HCC tissues increased significantly,the expression of miR-199b-5p decreased significantly(1.21±0.53 vs.3.56±1.02;t=18.286,P<0.001).②After transfection of pHRi-si-LUCAT1,the levels of miR-199b-5p increased significantly(Huh7:3.71±0.28 vs.1.00±0.10,t=15.787,P=0.004;HepG2:3.49±0.2 vs.1.00±0.11,t=15.790,P=0.004),the levels of LUCAT1(Huh7:0.34±0.05 vs.1.00±0.06,t=14.637,P=0.005;HepG2:0.41±0.06 vs.1.00±0.07,t=11.084,P=0.008)and AKAP1 mRNA(Hu
作者
金璞
谷从阳
陈涛
Jin Pu;Gu Congyang;Chen Tao(Department of Pathology,Xindu District People’s Hospital of Chengdu,Sichuan Chengdu 610500,China;Department of Pathology,The First People’s Hospital of Neijiang,Sichuan Neijiang 641000,China;Department of Clinical Laboratory,Xindu District People’s Hospital of Chengdu,Sichuan Chengdu 610500,China)
出处
《中国肝脏病杂志（电子版）》
CAS
2024年第2期61-72,共12页
Chinese Journal of Liver Diseases：Electronic Version
