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摘要：
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摘要：目的 探讨核纤层蛋白B1（lamin B1，LMNB1）对肝癌进展的影响，发掘肝癌新
的治疗靶点。方法 利用工具网站基因表达水平值的交互式分析平台（Gene Expression
Profiling Interactive Analysis，GEPIA）分析癌症基因组图谱数据库（The Cancer
Genome Atlas，TCGA）中肝癌组织及癌旁组织LMNB1的表达水平，使用Kaplan-Meier
分析LMNB1表达水平差异对肝癌患者预后的影响。采用实时定量聚合酶链式反应
（polymerase chain reaction，PCR）检测正常肝细胞系及肝癌细胞系中LMNB1表达水
平。选取LMNB1表达水平较高的两种人肝癌细胞株Hep3B、HepG2，转染小干扰RNA
敲低LMNB1表达，在LMNB1表达水平较低的细胞SUN475中构建LMNB1稳定过表达细
胞系并使用Western blot验证转染效率，采用CCK8法检测敲低或过表达LMNB1后肝癌细
胞的增殖能力，采用平板克隆形成检测敲低或过表达LMNB1后细胞形成克隆的能力。
采用Transwell检测敲低或过表达LMNB1后细胞侵袭及迁移能力的变化。采用Western
blot实验检测磷酸化蛋白激酶B（phosphorylated protein kinase B，p-AKT）、蛋白激酶
B（protein kinase B，AKT）、磷酸化细胞外调节蛋白激酶（phosphorylated extracellular
regulated protein kinases，p-ERK）及细胞外调节蛋白激酶（extracellular regulated
protein kinases，ERK）的变化。结果 TCGA数据库分析表明肝癌组织中LMNB1表达
水平较癌旁正常组织高（P ＜ 0.05），且LMNB1表达水平与肝癌患者预后呈负相关
（P = 0.003）。实时定量PCR显示相对正常人肝细胞系，肝癌细胞中LMNB1 mRNA表
达量分别为MHCC97H：1.80 ± 0.10（t = 11.08，P ＜ 0.001）；SUN475：1.29 ± 0.03
（t = 5.88，P = 0.004）；HepG2：2.97 ± 0.04（t = 39.68，P ＜ 0.001）；PLC/Prf/5：
1.74 ± 0.04（t = 14.51，P ＜ 0.001）；HuH7：1.70 ± 0.02（t = 15.22，P ＜ 0.001）；
SK-HEP-1：1.59 ± 0.05（t = 11.17，P ＜ 0.001）；Hep3B：2.27 ± 0.09（t = 18.50，P ＜
0.001）；均显著高于正常人肝细胞系。Western blot结果显示在HepG2及Hep3B细胞系
中，与siScramble阴性对照组相比，转染siLMNB1实验组的LMNB1表达水平显著降低
（HepG2：0.60 ± 0.10 vs 1.60 ± 0.10，t = 11.55，P ＜ 0.001；Hep3B：0.40 ± 0.10 vs 1.70 ±
0.10；t = 15.61，P ＜ 0.001）；而SUN475中转染质粒后LMNB1表达显著增加（0.70 ±
0.20 vs 0.07 ± 0.04；t = 5.541，P = 0.005）。敲低LMNB1后培养96 h，与对照组相比，
Hep3B（10.81 ± 0.67 vs 15.48 ± 0.62；t = 8.86，P ＜ 0.001）及HepG2（9.45 ± 0.61 vs
17.08 ± 0.75；t = 13.67，P ＜ 0.001）细胞增殖能力被显著抑制；而在SUN475中过表
达LMNB1后，实验组细胞增殖活性显著增加（16.94 ± 1.52 vs 12.65 ± 1.06；t = 3.99，
P = 0.016）。在HepG2及Hep3B细胞系中敲低LMNB1后，细胞克隆形成数显著减少
[HepG2：（90.30 ± 7.24）个 vs （382.01 ± 25.27）个，t = 19.24，P ＜ 0.001；Hep3B：
（128.03 ± 8.24）个 vs （395.85 ± 28.27）个，t = 15.76，P ＜ 0.001]，在SUN475中过
表达LMNB1组比对照组克隆形成个数显著增加 [（467.82 ± 42.45）个 vs （85.31 ± 15.32）个；
t = 14.87，P = 0.001]。在HepG2及Hep3B细胞系中敲低LMNB1后，肝癌细胞迁移数量
显著减少 [HepG2：（75.25 ± 8.10）个 vs （15.02 ± 3.50）个，t = 11.90，P ＜ 0.001；
Hep3B：（168.20 ± 12.26）个 vs （34.83 ± 7.61）个，t = 15.96，P ＜ 0.001]，侵袭
的细胞数量同样显著减少 [HepG2：（110.21 ± 12.01）个 vs （25.76 ± 4.03）个，t =
11.50，P ＜ 0.001；Hep3B：（150.22 ± 15.16）个 vs （22.03 ± 14.26）个，t = 10.65，
P ＜ 0.001]；在SUN475中，过表达LMNB1组比对照组细胞迁移数量显著增加 [（50.11 ±
5.55）个 vs （349.85 ± 25.26）个；t = 11.33，P ＜ 0.001]，侵袭的细胞数量同样显著增
加 [（40.11 ± 5.26）个 vs （80.13 ± 12.20）个；t = 5.21，P = 0.007]。与对照组相比，
敲低LMNB1后，AKT的磷酸化活性形式p-AKT表达水平显著下降，而过表达LMNB1可
导致p-AKT表达升高（P均＜ 0.05）。而敲低或过表达LMNB1后，p-ERK表达水平无显
著变化，AKT和ERK的本底表达水平均未发生明显变化。结论 抑制LMNB1可通过抑制
PI3K/AKT信号转导通路的激活抑制肝癌的发生发展，可作为诊断肝癌的生物标记物和
潜在的治疗靶点。
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Abstract: Objective To investigate the effect of LMNB1 on the progression of hepatocellular
carcinoma (HCC), and to explore new therapeutic targets for HCC. Methods The expression
level of LMNB1 in HCC tissues and normal tissues in The Cancer Genome Atlas (TCGA)
database were analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) website,
and Kaplan-Meier curve was used to analyze the effect of LMNB1 expression on prognosis.
Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression
level of LMNB1 in normal liver cell lines and HCC cell lines. Two human HCC cell lines
Hep3B and HepG2 with higher expression levels of LMNB1 were selected and transfected
with small interfering RNA to knockdown LMNB1 expression, LMNB1 stable overexpression
cell lines were constructed in SUN475 cells with low level of LMNB1 expression and the
transfection efficiency was verified by Western blot. The proliferation capacity of HCC cells
after knockdown or overexpression LMNB1 was measured by CCK8 assay, and plate clone
formation assay was used to detect the ability of clone formation of the cells. Western blot
was used to detect the expression of phosphorylated protein kinase B (p-AKT), protein kinase
B (AKT), phosphorylated extracellular regulated protein kinases (p-ERK) and extracellular
regulated protein kinases (ERK). Results The results of TCGA database showed that the
expression level of LMNB1 was higher in HCC tissues than that of normal tissues (P ＜ 0.05), and
the expression level of LMNB1 was negatively correlated with the prognosis of patients with
HCC (P = 0.003). Real-time quantitative PCR showed that compared with the normal human
liver cell lines, the mRNA expression level of LMNB1 in MHCC97H (1.80 ± 0.10; t = 11.08,
P ＜ 0.001), SUN475 (1.29 ± 0.03; t = 5.88, P = 0.004), HepG2 (2.97 ± 0.04; t = 39.68, P ＜
0.001), PLC/Prf/5 (1.74 ± 0.04; t = 14.51, P ＜ 0.001), HuH7 (1.70 ± 0.02; t = 15.22, P ＜
0.001), SK-HEP-1 (1.59 ± 0.05; t = 11.17, P ＜ 0.001) and Hep3B (2.27 ± 0.09; t = 18.50,
P ＜ 0.001) were significantly higher than those in normal human liver cell lines. Western blot
showed that in HepG2 and Hep3B cell lines, the expression level of LMNB1 in the siLMNB1
group reduced significantly compared to that of the siScramble control group (HepG2: 0.60 ±
0.10 vs 1.60 ± 0.10; t = 11.55, P ＜ 0.001; Hep3B: 0.40 ± 0.10 vs 1.70 ± 0.10; t = 15.61, P ＜
0.001). The expression of LMNB1 increased significantly after transfection of the plasmid in
SUN475 (0.70 ± 0.20 vs 0.07 ± 0.04; t = 5.541, P = 0.005). After LMNB1 knockdown and
cultured for 96 h, the proliferation ability of Hep3B (10.81 ± 0.67 vs 15.48 ± 0.62; t = 8.86, P ＜
0.001) and HepG2 (9.45 ± 0.61 vs 17.08 ± 0.75; t = 13.67, P ＜ 0.001) cells were significantly
inhibited compared with the control group, however, after the overexpression of LMNB1 in
SUN475, the cell proliferation activity was significantly increased in the experimental group
(16.94 ± 1.52 vs 12.65 ± 1.06; t = 3.99, P = 0.016). After LMNB1 knockdown in HepG2
and Hep3B cell lines, the number of cell clone formation decreased significantly (HepG2:
90.30 ± 7.24 vs 382.01 ± 25.27, t = 19.24, P ＜ 0.001; Hep3B: 128.03 ± 8.24 vs 395.85 ±
28.27, t = 15.76, P ＜ 0.001). In SUN475, LMNB1 overexpression significantly increased
the number of clones compared with control group (467.82 ± 42.45 vs 85.31 ± 15.32, t =
14.87, P = 0.001). After LMNB1 knockdown in HepG2 and Hep3B cells, migration (HepG2:
75.25 ± 8.10 vs 15.02 ± 3.50, t = 11.90, P ＜ 0.001; Hep3B: 168.20 ± 12.26 vs 34.83 ± 7.61,
t = 15.96, P ＜ 0.001) and invasion (HepG2: 110.21 ± 12.01 vs 25.76 ± 4.03, t = 11.50,
P ＜ 0.001; Hep3B: 150.22 ± 15.16 vs 22.03 ± 14.26, t = 10.65, P ＜ 0.001) of HCC cells
decreased significantly. In SUN475, after LMNB1 overexpression, migration (50.11 ± 5.55
vs 349.85 ± 25.26); t = 11.33, P ＜ 0.001) and invasion (40.11 ± 5.26 vs 80.13 ± 12.20; t =
5.21, P = 0.007) of the cells increased significantly. Compared with the control group, LMNB1
knockdown resulted in a significant decrease in the phosphorylation activity form of AKT
(p-AKT expression level), while overexpression of LMNB1 resulted in an increase in p-AKT
expression (all P ＜ 0.05). After knocking down or overexpressing LMNB1, there was no
significant change in the expression level of p-ERK, and there were no significant change in
the background expression levels of AKT and ERK. Conclusions Inhibition of LMNB1 can
inhibit the occurrence and development of HCC by inhibiting the activation of PI3K/Akt
signaling pathway, which can be used as a new biomarker for diagnosis of liver cancer and a
potential therapeutic target.
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