lncRNA LUCAT1通过miR-199b-5p/AKAP1信号轴促进肝癌进展的机制

Mechanism of lncRNA LUCAT1 on promoting metastasis of liver cancer through miR-199b-5p/AKAP1 signal axis

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(Huh7:0.20±0.03比0.90±0.05,t=20.793,P=0.002;HepG2:0.05±0.02比0.21±0.02,t=9.798,P=0.010)、MMP-9(Huh7:0.25±0.04比0.75±0.05,t=13.525,P=0.005;HepG2:0.15±0.03比0.59±0.04,t=15.242,P=0.004)表达水平显著降低;共转染pHRi-si-LUCAT1和pHRi-anti-miR-199b-5p后,miR-199b-5p水平显著降低(Huh7:1.42±0.11比3.65±0.25,t=14.142,P=0.005;HepG2:1.30±0.05比3.71±0.20,t=20.248,P=0.002),LUCAT1(Huh7:0.85±0.10比0.40±0.06,t=6.683,P=0.022;HepG2:0.90±0.08比0.45±0.04,t=8.714,P=0.013)和AKAP1 mRNA水平显著升高(Huh7:0.80±0.07比0.55±0.04,t=5.371,P=0.033;HepG2:0.85±0.08比0.51±0.04,t=6.584,P=0.022);细胞EdU阳性率、划痕愈合率和细胞侵袭数均显著升高(P均<0.05);Ki67(Huh7:0.91±0.06比0.25±0.04,t=15.853,P=0.004;HepG2:0.92±0.07比0.18±0.03,t=16.830,P=0.004)、MMP-2(Huh7:0.62±0.05比0.22±0.03,t=11.882,P=0.007;HepG2:0.75±0.05比0.39±0.05,t=8.818,P=0.013)、MMP-9(Huh7:0.51±0.05比0.18±0.02,t=10.614,P=0.009;HepG2:0.89±0.06比0.34±0.04,t=13.211,P=0.006)表达水平显著升高;共转染pHRi-si-LUCAT1和pHRi-AKAP1后,miR-199b-5p水平显著降低(Huh7:1.82±0.12比3.55±0.30,t=9.274,P=0.011;HepG2:1.70±0.14比3.62±0.25,t=11.606,P=0.007),LUCAT1(Huh7:0.71±0.03比0.30±0.03,t=16.738,P=0.004;HepG2:0.75±0.05比0.35±0.04,t=10.820,P=0.008)和AKAP1 mRNA水平显著升高(Huh7:0.87±0.05比0.51±0.03,t=10.694,P=0.009;HepG2:0.90±0.09比0.54±0.04,t=6.331,P=0.024);细胞EdU阳性率、划痕愈合率和细胞侵袭数均显著升高(P均<0.05);Ki67(Huh7:0.64±0.06比0.30±0.03,t=8.779,P=0.013;HepG2:0.75±0.06比0.25±0.03,t=12.910,P=0.006)、MMP-2(Huh7:0.80±0.05比0.34±0.04,t=12.443,P=0.002;HepG2:0.84±0.08比0.40±0.03,t=8.920,P=0.012)、MMP-9(Huh7:0.76±0.05比0.23±0.04,t=14.337,P=0.005;HepG2:0.76±0.05比0.31±0.04,t=12.173,P=0.007)表达水平显著升高;③转染pHRi-si-LUCAT1后,肿瘤体积[(523.67±64.33)mm^(3)比(1542.21±201.51)mm^(3),t=8.340,P=0.014)]和质量[(0.67±0.15)g比(1.87±0.22)g,t=7.806,P=0.016)]均显著减小,LUCAT1(0.47±0.10比1.00±0.14,t=5.336,P=0.033)、AKAP1(0.12±0.03比0.51±0.05,t=11.585,P=0.007)、Ki67(2.45±0.28比5.93±0.55,t=9.766,P=0.010)、MMP-2(2.35±0.25比5.74±0.51,t=10.338,P=0.009)、MMP-9(3.55±0.34比6.42±0.84,t=5.486,P=0.032)蛋白水平均显著降低,miR-199b-5p(1.68±0.17比1.00±0.16,t=5.045,P=0.037)水平显著升高。结论LncRNA LUCAT1通过miR-199b-5p/AKAP1信号轴促进HCC细胞增殖、迁移和侵袭。

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(Huh7:0.52±0.05 vs.1.00±0.09,t=8.075,P=0.015;HepG2:0.55±0.06 vs.1.00±0.13,t=5.444,P=0.032)decreased significantly.EdU positive rate of cells,the scratch healing rate and the number of invasion cells decreased significantly(all P<0.05).The expression of Ki67(Huh7:0.24±0.03 vs.0.92±0.06,t=17.558,P=0.003;HepG2:0.10±0.03 vs.0.51±0.03,t=16.738,P=0.004),MMP-2(Huh7:0.20±0.03 vs.0.90±0.05,t=20.793,P=0.002;HepG2:0.05±0.02 vs.0.21±0.02,t=9.798,P=0.010)and MMP-9(Huh7:0.25±0.04 vs.0.75±0.05,t=13.525,P=0.005;HepG2:0.15±0.03 vs.0.59±0.04,t=15.242,P=0.004)decreased significantly.After cotransfection of pHRi-si-LUCAT1 and pHRi-anti-miR-199b-5p,the levels of miR-199b-5p decreased significantly(Huh7:1.42±0.11 vs.3.65±0.25,t=14.142,P=0.005;HepG2:1.30±0.05 vs.3.71±0.20,t=20.248,P=0.002),the levels of LUCAT1(Huh7:0.85±0.10 vs.0.40±0.06,t=6.683,P=0.022;HepG2:0.90±0.08 vs.0.45±0.04,t=8.714,P=0.013)and AKAP1 mRNA(Huh7:0.80±0.07 vs.0.55±0.04,t=5.371,P=0.033;HepG2:0.85±0.08 vs.0.51±0.04,t=6.584,P=0.022)increased significantly.EdU positive rate of cells,the scratch healing rate and the number of invasion cells increased significantly(all P<0.05).The expression of Ki67(Huh7:0.91±0.06 vs.0.25±0.04,t=15.853,P=0.004;HepG2:0.92±0.07 vs.0.18±0.03,t=16.830,P=0.004),MMP-2(Huh7:0.62±0.05 vs.0.22±0.03,t=11.882,P=0.007;HepG2:0.75±0.05 vs.0.39±0.05,t=8.818,P=0.013)and MMP-9(Huh7:0.51±0.05 vs.0.18±0.02,t=10.614,P=0.009;HepG2:0.89±0.06 vs.0.34±0.04,t=13.211,P=0.006)increased significantly.After cotransfection of pHRi-si-LUCAT1 and pHRi-AKAP1,the levels of miR-199b-5p decreased significantly(Huh7:1.82±0.12 vs.3.55±0.30,t=9.274,P=0.011;HepG2:1.70±0.14 vs.3.62±0.25,t=11.606,P=0.007),the levels of LUCAT1(Huh7:0.71±0.03 vs.0.30±0.03,t=16.738,P=0.004;HepG2:0.75±0.05 vs.0.35±0.04,t=10.820,P=0.008)and AKAP1 mRNA(Huh7:0.87±0.05 vs.0.51±0.03,t=10.694,P=0.009;HepG2:0.90±0.09 vs.0.54±0.04,t=6.331,P=0.024)increased significantly.EdU positive rate of cells,the scratch healing rate and the number of invasion cells increased significantly(all P<0.05).The expression of Ki67(Huh7:0.64±0.06 vs.0.30±0.03,t=8.779,P=0.013;HepG2:0.75±0.06 vs.0.25±0.03,t=12.910,P=0.006),MMP-2(Huh7:0.80±0.05 vs.0.34±0.04,t=12.443,P=0.002;HepG2:0.84±0.08 vs.0.40±0.03,t=8.920,P=0.012)and MMP-9(Huh7:0.76±0.05 vs.0.23±0.04,t=14.337,P=0.005;HepG2:0.76±0.05 vs.0.31±0.04,t=12.173,P=0.007)increased significantly.③After transfection of pHRi-si-LUCAT1,the tumor volume[(523.67±64.33)mm^(3)vs.(1542.21±201.51)mm^(3),t=8.340,P=0.014]and weight[(0.67±0.15)g vs.(1.87±0.22)g,t=7.806,P=0.016]reduced significantly.LUCAT1(0.47±0.10 vs.1.00±0.14,t=5.336,P=0.033),AKAP1(0.12±0.03 vs.0.51±0.05,t=11.585,P=0.007),Ki67(2.45±0.28 vs.5.93±0.55,t=9.766,P=0.010),MMP-2(2.35±0.25 vs.5.74±0.51,t=10.338,P=0.009)and MMP-9(3.55±0.34 vs.6.42±0.84,t=5.486,P=0.032)protein levels reduced significantly,while miR-199b-5p levels(1.68±0.17 vs.1.00±0.16,t=5.045,P=0.037)increased significantly.Conclusions LncRNA LUCAT1 promoted the proliferation,migration,and invasion ability of HCC cells through miR-199b-5p/AKAP1 signal axis.