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摘要：
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目的 探讨NS5ATP3在非酒精性脂肪性肝病（nonalcoholic fatty liver disease，NAFLD）
相关肝细胞癌（hepatic cell carcinoma，HCC）发生发展中的作用机制。方法 转染
pNS5ATP3或si-NS5ATP3后检测HepG2细胞内总胆固醇（total cholesterol，TC）水
平，分别采用CCK8法、划痕实验、Caspase-3活性检测法检测细胞增殖、凋亡及迁
移情况。通过聚合酶链式反应（quantitative real-time polymerase chain reaction，qRTPCR）和Western blot检测胆固醇合成、细胞增殖、凋亡及细胞周期相关基因和蛋白
（SREBP2、HMGCR、FoxM1）的表达。比较共转染pNS5ATP3和si-HMGCR与单纯转
染pNS5ATP3时下游HCC关键基因FoxM1的表达水平及细胞增殖能力。结果 NS5ATP3
过表达增加了细胞内TC水平 [（0.044 ± 0.008）pmol/mg vs（0.034 ± 0.004）pmol/mg；
t = 3.231，P = 0.023）]，而NS5ATP3沉默后降低了TC水平 [（0.025 ± 0.002）pmol/mg vs
（0.033 ± 0.003）pmol/mg；t = 3.846，P = 0.009）]。Western blot表明，与对照组相
比，过表达NS5ATP3可引起SREBP2和HMGCR蛋白水平增加，p-AMPKα水平显著降
低；沉默NS5ATP3后，SREBP2和HMGCR蛋白水平下降，p-AMPKα水平增高。qRTPCR结果也显示，与对照组相比，NS5ATP3过表达上调SREBP2 mRNA（2.45 ± 0.75 vs
1 ± 0.33；t = 5.159，P = 0.037）和HMGCR mRNA（2.30 ± 0.30 vs 1 ± 0.10；t = 4.432，
P = 0.047）的表达，而沉默NS5ATP3后，SREBP2 mRNA（0.24 ± 0.21 vs 1 ± 0.26；t =
4.769，P = 0.041）和HMGCR mRNA（0.47 ± 0.13 vs 1 ± 0.21；t = 4.522，P = 0.046）
表达水平显著下降。在48 h和72 h时，过表达NS5ATP3组细胞相对活力显著高于对照组
（1.85 ± 0.06 vs 1.56 ± 0.12，t = 4.583，P = 0.010；3.08 ± 0.19 vs 2.61 ± 0.21，t = 4.790，
P = 0.009），24 h时差异无统计学意义（1.10 ± 0.06 vs 1 ± 0.08，t = 1.873，P = 0.088）。
在24 h、48 h和72 h时，沉默NS5ATP3组细胞相对活力均显著低于对照组（0.90 ± 0.07
vs 0.98 ± 0.09，t = 3.378，P = 0.020；1.57 ± 0.05 vs 1.63 ± 0.11，t = 2.717，P = 0.035；
1.82 ± 0.23 vs 2.61 ± 0.21，t = 5.010，P = 0.004）。与对照组相比，过表达NS5ATP3后，
细胞中caspase-3/7水平降低（0.69 ± 0.09 vs 1 ± 0.15；t = 3.128，P = 0.026），而沉默
siNS5ATP3后，caspase-3/7活性增高（1.34 ± 0.11 vs 1 ± 0.05；t = 5.141，P = 0.004）。
在24 h、48 h和72 h，过表达NS5ATP3后，细胞迁移率显著升高（21.00 ± 2.08 vs 33.33 ±
2.40，t = 3.879，P = 0.018；67.33 ±1.20 vs 78.00 ±1.53，t = 5.488，P = 0.005；85.33 ±
2.60 vs 99.00 ± 0.58，t = 5.125，P = 0.007）；沉默NS5ATP3后，细胞迁移能力显著下
降（36.00 ± 2.65 vs 24.33 ± 2.91，t = 2.969，P = 0.041；67.33 ± 1.20 vs 48.00 ± 3.22，
t =5.633，P = 0.005；92.33 ± 1.45 vs 83.33 ± 1.67，t = 4.070，P = 0.015）。过表达
NS5ATP可显著上调FoxM1 mRNA（1.43 ± 0.10 vs 1 ± 0.06；t = 3.533，P = 0.024）和
CCNB1 mRNA表达（2.07 ± 0.16 vs 1 ± 0.28；t = 4.305，P = 0.013）；沉默NS5ATP3可显
著下调FoxM1 mRNA（0.47 ± 0.17 vs 1 ± 0.21；t = 3.153，P = 0.034）和CCNB1 mRNA
（0.49 ± 0.20 vs 1 ± 0.11；t = 3.676，P = 0.021）的表达水平。Western blot表明，过表达
NS5ATP3后，FoxM1和Bcl-2的蛋白质表达水平上调；沉默NS5ATP3后，FoxM1和Bcl-2
的蛋白表达水平下调。HMGCR基因沉默后FoxM1 mRNA（0.63 ± 0.18 vs 1 ± 0.17；t =
2.698，P = 0.036）和蛋白质表达水平均显著下降，提示FoxM1受HMGCR调控。与单
纯过表达NS5ATP3相比，同时过表达NS5ATP3和沉默HMGCR，HMGCR mRNA（0.83 ±
0.17 vs 2.13 ± 0.26；t = 7.776，P = 0.016）和FoxM1 mRNA（0.92 ± 0.21 vs 1.48 ± 0.10；
t = 4.323，P = 0.049）表达水平均显著下降，48 h细胞增殖相对活力也显著降低（0.91 ±
0.18 vs 1.33 ± 0.04；t = 4.946，P = 0.016）。结论 NS5ATP3通过HMGCR-FoxM1轴参与
HCC的发生发展，部分解释了NAFLD相关HCC的分子机制。
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Abstract: Objective To investigate the mechanism of NS5ATP3 in the occurrence and
development of nonalcoholic fatty liver disease (NAFLD)-related hepatocellular carcinoma
(HCC). Methods The HepG2 cell line was transfected with pNS5ATP3 or si-NS5ATP3, and the
concentration of total cholesterol (TC), cell proliferation, apoptosis and migration were detected by
TC test kit, CCK8 method, scratch test and caspase-3 activity detection, respectively. Meanwhile,
key genes and proteins (SREBP2, HMGCR, FoxM1) involved in cholesterol synthesis, cell
proliferation, apoptosis and cell cycle were dectected by quantitative real-time polymerase chain
reaction (qRT-PCR) and Western blot assay. Expression of FoxM1, which had important roles
in the tumorigenesis of HCC and cell proliferation were compared between HepG2 cell line
transfected with pNS5ATP3 or co-transfected with pNS5ATP3 and si-HMGCR. Results NS5ATP3
overexpression increased the intracellular TC level [(0.044 ± 0.008) pmol/mg vs (0.034 ±
0.004) pmol/mg; t = 3.231, P = 0.023)], while NS5ATP3 silencing decreased the TC level [(0.025 ±
0.002) pmol/mg vs (0.033 ± 0.003) pmol/mg; t = 3.846, P = 0.009)]. Western blot showed that
overexpression of NS5ATP3 could increase the protein levels of SREBP2 and HMGCR and
decrease the protein level of p-AMPKα; after silencing NS5ATP3, the protein levels of SREBP2
and HMGCR decreased and p-AMPKα increased. qRT-PCR results also showed that compared
with the control group, NS5ATP3 overexpression could up-regulate the mRNA expression of
SREBP2 (2.45 ± 0.75 vs 1 ± 0.33; t = 5.159, P = 0.037) and HMGCR (2.30 ± 0.30 vs 1 ± 0.10; t =
4.432, P = 0.047). After NS5ATP3 was silenced, the mRNA level of SREBP2 (0.24 ± 0.21 vs 1 ± 0.26;
t = 4.769, P = 0.041) and HMGCR (0.47± 0.13 vs 1 ± 0.21; t = 4.522, P = 0.046) decreased. At 48 h
and 72 h, the relative viability of cells in NS5ATP3 overexpression group was significantly higher
than that in control group (1.85 ± 0.06 vs 1.56 ± 0.12, t = 4.583, P = 0.010; 3.08 ± 0.19 vs 2.61 ± 0.21,
t = 4.790, P = 0.009) and there was no significant difference at 24 h (1.10 ± 0.06 vs 1 ± 0.08, t = 1.873,
P = 0.088). At 24 h, 48 h and 72 h, the relative viability of cells in NS5ATP3 silenced group was
significantly lower than that in control group (0.90 ± 0.07 vs 0.98 ± 0.09, t = 3.378, P = 0.020;
1.57 ± 0.05 vs 1.63 ± 0.11, t = 2.717, P = 0.035; 1.82 ± 0.23 vs 2.61 ± 0.21, t = 5.010, P = 0.004).
Compared with control group, the level of caspase-3/7 decreased after NS5ATP3 overexpression
(0.69 ± 0.09 vs 1 ± 0.15; t = 3.128, P = 0.026), while the activity of caspase-3/7 increased after
silencing NS5ATP3 (1.34 ± 0.11 vs 1 ± 0.05; t = 5.141, P = 0.004). After overexpression of
NS5ATP3 at 24 h, 48 h and 72 h, the cell migration ability increased significantly (21.00 ± 2.08 vs
33.33 ± 2.40, t = 3.879, P = 0.018; 67.33 ± 1.20 vs 78.00 ± 1.53, t = 5.488, P = 0.005; 85.33 ± 2.60 vs
99.00 ± 0.58, t = 5.125, P = 0.007) and after silencing NS5ATP3, the cell migration ability
decreased significantly (36.00 ± 2.65 vs 24.33 ± 2.91, t = 2.969, P = 0.041; 67.33 ± 1.20
vs 48.00 ± 3.22, t =5.633, P = 0.005; 92.33 ± 1.45 vs 83.33 ± 1.67, t = 4.070, P = 0.015).
Overexpression NS5ATP3 significantly up-regulated the mRNA levels of FoxM1 (1.43 ± 0.10 vs
1 ± 0.06; t = 3.533, P = 0.024) and CCNB1 (2.07 ± 0.16 vs 1 ± 0.28; t = 4.305, P = 0.013),
silencing NS5ATP3 significantly decreased the mRNA levels of FoxM1 (0.47 ± 0.17 vs 1 ±
0.21; t = 3.153, P = 0.034) and CCNB1 (0.49 ± 0.20 vs 1 ± 0.11; t = 3.676, P = 0.021). Western
blot showed that the protein expression levels of FoxM1 and Bcl-2 were up-regulated after
overexpression of NS5ATP3. After silencing NS5ATP3, the protein levels of FoxM1 and Bcl-2 were
down-regulated. After silencing HMGCR, the mRNA (0.63 ± 0.18 vs 1 ± 0.17; t = 2.698, P =
0.036) and protein expression levels of FoxM1 decreased significantly, suggesting that FoxM1
was regulated by HMGCR. Compared with overexpression of NS5ATP3 group, the mRNA levels
of HMGCR (0.83 ± 0.17 vs 2.13 ± 0.26; t = 7.776, P = 0.016) and FoxM1 (0.92 ± 0.21 vs 1.48 ±
0.10; t = 4.323, P = 0.049) in the group of both overexpression NS5ATP3 and silencing HMGCR
decreased significantly, and the ability of cell proliferation decreased significantly at 48 h (0.91 ±
0.18 vs 1.33 ± 0.04; t = 4.946, P = 0.016). Conclusions NS5ATP3 participated in the occurrence
and development of HCC through HMGCR-FoxM1 axis, and ultimately participated in the
transformation of NAFLD to HCC.
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