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摘要：
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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（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）mm3
比（1542.21 ± 201.51）mm3
，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。
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Abstract: 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 pHRiAKAP1 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. Results ①Compared 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) mm3
vs. (1542.21 ± 201.51) mm3
, 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.
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