Microrna-155 Promotes Tumor Growth of Human Hepatocellular Carcinoma by Targeting Arid2
Microrna-155 Promotes Tumor Growth of Human Hepatocellular Carcinoma by Targeting Arid2
Microrna-155 Promotes Tumor Growth of Human Hepatocellular Carcinoma by Targeting Arid2
1
Department of General Surgery, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an,
Shaanxi 710004; 2Department of General Surgery, The First Affiliated Hospital of Xi'an Jiaotong University;
3
Breast Cancer Program, Shaanxi Provincial Tumor Hospital, Xi'an, Shaanxi 710061; 4Department
of General Surgery, Shanxi Province People's Hospital, Xi'an, Shaanxi 710068, P.R. China
DOI: 10.3892/ijo.2016.3465
Abstract. Aberrant expression of microRNA-155 (miR‑155) tumor growth of HCC by targeting ARID2-mediated Akt
has been reported in several human cancers and is associated phosphorylation pathway, and potentially serves as a novel
with prognosis of patients. However, the clinical significance prognostic biomarker and therapeutic target for HCC.
of miR‑155 and its underlying mechanisms involved in hepa-
tocarcinogenesis remain to be determined. In this study, we Introduction
demonstrated that the expression of miR‑155 was elevated in
both hepatocellular carcinoma (HCC) tissues and cell lines. Hepatocellular carcinoma (HCC) is the dominant pathologic
Clinical association analysis revealed that high expression of type of primary liver cancer, which is the third leading
miR‑155 was correlated with malignant clinicopathological cause of cancer-related death worldwide (1). Despite recent
characteristics including large tumor size, high Edmondson- advances in the diagnosis and treatment of HCC, including
Steiner grading and TNM tumor stage. Furthermore, its high hepatectomy and liver transplantation, the prognosis for HCC
expression conferred a reduced 5-year overall survival and patients remains poor due to the high recurrence rate and early
disease-free survival of HCC patients. Gain- and loss-of metastasis (2). However, the detailed mechanism underlying
function studies revealed that miR‑155 promoted cell cycle the development and progression of HCC is still not clear (3).
progression, cell proliferation and inhibited apoptosis. Therefore, it is urgent to clarify the molecular mechanisms
Mechanistically, we identified AT-rich interactive domain 2 of HCC and identify novel prognostic biomarkers to provide
(ARID2) as a direct downstream target and functional medi- potential therapeutic targets for the patients with HCC.
ator of miR‑155 in HCC cells. Notably, alterations of ARID2 Increasing evidence suggests that microRNAs (miRNAs),
expression abrogated the effects of miR‑155 on HCC cell a class of non-coding RNAs composed of ~22 nucleotides, can
proliferation, cell cycle and apoptosis. Moreover, we demon- act as suitable biomarkers with diagnostic, prognostic, and
strated that Akt phosphorylation is essential for the functional predictive potential (4). miRNAs may function as oncogenes
roles of miR‑155 through altering Cyclin D1 and p27, which or tumor suppressors by regulating protein expression by
were key components of cell cycle machinery. Finally, we interacting with complementary sites within the 3'-untrans-
disclosed that the downregulation of miR‑155 suppressed lated region (UTR) of target mRNA transcripts (5). Emerging
tumor growth of HCC by inhibiting Akt signaling pathway. studies have documented that aberrant miRNAs play important
In conclusion, our results indicate that miR‑155 promotes roles in various biological processes (6), including cell prolif-
eration, differentiation, apoptosis, drug-resistance, migration
and invasion (7-10). Recently, miRNAs have shown high
stability in tissues and body fluids, which reveal their potential
Correspondence to: Dr Xuejun Sun, Department of General as tumor markers. In addition, it has been widely recognized
Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, that dysregulation of miRNAs contribute to the development
277 Yanta West Road, Xi'an, Shaanxi 710061, P.R. China and progression of HCC.
E-mail: sunxj1234@sina.com miR‑155 is located within a region known as B cell inte-
Dr Li Zhang, Department of General Surgery, The Second Affiliated gration cluster on chromosome 21, which plays a critical role
Hospital of Xi'an Jiaotong University, 157 Xiwu Road, Xi'an, in the progression of gastric cancer (11), colorectal carcinoma
Shaanxi 710004, P.R. China (12), lung cancer (13), breast cancer (14), bladder cancer (15)
E-mail: zl6403222@sina.com and B-cell lymphoma (16). Previous studies show that miR‑155
functions as a regulator in the pathogenesis of cell prolif-
Key words: miR‑155, hepatocellular carcinoma, AT-rich interactive eration, apoptosis, drug-resistance, migration, invasion and
domain 2 , proliferation, cell cycle, apoptosis epithelial-mesenchymal transition (EMT). miR‑155 promotes
oral squamous cell carcinoma metastasis and correlates a poor
2426 Zhang et al: THE ROLE OF miR-155 IN HCC
prognosis (17). miR‑155 promotes the B-cell lymphoma cell and Homo sapiens snRNA U6 qPCR Primer (HmiRQP9001)
proliferation and inhibits cell apoptosis by targeting NIAM were purchased from GeneCopoeia (Guangzhou, China).
phenocopies (16). Moreover, direct quantitative detection for
cell-free miR‑155 in urine could be a potential novel biomarker Western blot analysis. Total protein was extracted from whole
in diagnosis and prognosis for non-muscle invasive bladder cells and 40 µg of isolated protein was separated by 10%
cancer (15). miR‑155 mediates anti-Warburg effect of rosma- SDS-PAGE and transferred onto a PVDF membrane (Bio‑Rad
rinic acid in colorectal carcinoma and gastric cancer (18). Laboratories, Hercules, CA, USA). The membranes were
miR‑155 presents tamoxifen-resistance by modulating SOCS6- probed with antibodies: anti-Akt, anti-p-Akt, anti‑ARID2,
STAT3 signaling pathway in breast cancer (19). However, the anti-Cyclin D, and anti-p27 primary antibodies (Cell Signaling,
clinical significance of miR‑155 and the underlying mechanisms Danvers, MA, USA) overnight. Then the membranes were
involved in the development of HCC remain to be investigated. incubated with the HRP-conjugated goat anti-mouse or
In this study, we demonstrated that the expression of anti-rabbit IgG antibody (ZSGB-BIO, China). Protein bands
miR‑155 was upregulated in HCC tissues and its high expres- were visualized using an enhanced chemiluminescence kit
sion was associated with poor clinicopathological features (Amersham, Little Chalfont, UK).
and the reduced survival of HCC patients. miR‑155 promoted
cell proliferation, cell cycle and apoptosis resistance in vitro. Immunohistochemical staining. Immunohistochemistry was
Moreover, the downregulation of miR‑155 inhibited tumor performed on paraformaldehyde-fixed paraffin sections.
growth of HCC in vivo. Notably, AT-rich interactive domain 2 ARID2 (1:100, #13594 Cell Signaling Technology, Inc.) anti-
(ARID2) was identified as a direct target of miR‑155. The body was used in immunohistochemistry by a streptavidin
results showed a new role for miR‑155 in prediction of prog- peroxidase-conjugated (SP-IHC) method. The percentage
nosis and promoting tumor growth of HCC. of positive tumor cells was graded as: 0, <10%; 1, 10-30%;
2, 31-50%; 3, >50%.
Materials and methods
Plasmids and cell transfection. miRNA vectors, including
Clinical samples and cell lines. HCC samples (124) and miR‑155 expression vector (HmiR0358-MR02), the control
matched normal tumor-adjacent samples (>2 cm distance from vector for miR‑155 (CmiR0001-MR04 and miR‑control),
the margin of the resection) were obtained during surgery and miR‑155 inhibitor (HmiR-AN0220-AM03 and anti‑miR‑155)
used after obtaining informed consent. All patients underwent and the negative control for the miR‑155 inhibitor
resection of their primary HCC in the Department of General (CmiR-AN0001-AM04 and anti-miR-NC), and ARID2
Surgery at The Second Affiliated Hospital of Xi'an Jiaotong expression plasmid were purchased from GeneCopoeia.
University, from January 2006 to December 2008. None of The targeted sequences for ARID2 siRNA sense, 5'-AGCT
the patients received preoperative chemo- or radiotherapy. CCAATTCCTTGTGAAGTTTT-3' and antisense, 5'-ACTTCA
The stage of cancer was determined according to the cancer CAAGGAATTGCAGCTTTT-3' or a non-specific duplex
staging system published in 2010 by the Union for International oligonucleotide as a negative control were produced by Sangon
Cancer Control (UICC). The Xi'an Jiaotong University Ethics Biotech Co., Ltd. (Shanghai, China). The cells were transfected
Committee approved all protocols according to the 1975 with the vectors mentioned above using Lipofectamine 2000
Helsinki Declaration. according to the manufacturer's instructions (Invitrogen).
Human HCC cell lines (Hep3B, Bel-7402, MHCC-97L,
HepG2, SMMC-7721) and human immortalized normal Cell cycle, proliferation and detection of apoptosis. Flow
hepatic cell line LO2 were obtained from the Institute of cytometry was performed using the fluorescence-activated
Biochemistry and Cell Biology, Chinese Academy of Sciences cell sorting (FACS) Calibur and CellQuest software (both
(Shanghai, China). The cells were maintained in Dulbecco's from Becton-Dickinson, San Jose, CA, USA). For cell cycle
modified Eagle's medium (DMEM, Gibco, Grand Island, analysis, the cells were seeded in 6-well plates at 2x105/well.
NY, USA) containing 10% fetal bovine serum (FBS, Gibco) Forty-eight hours after transfection, the cells were fixed in
with 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma, 70% ethanol at 4˚C for 24 h and stained with 50 µg/ml prop-
St. Louis, MO, USA) and cultured in a humidified 5% CO2 idium iodide (Keygen, Nanjing, China). An Annexin V-Fluor
incubator at 37˚C. Staining kit (Roche) was used to analyze apoptosis levels.
For the proliferation assay, bromodeoxyuridine labeling and
Real-time quantitative reverse transcription polymerase chain immunofluorescence was used. Cells grown on coverslips
reaction (qRT-PCR). Total RNA was extracted from clinical (Fisher, Pittsburgh, PA, USA) were incubated with bromode-
specimens or HCC cells using TRIzol reagent (Invitrogen, oxyuridine (BrdU) for 1 h and stained with anti-BrdU antibody
Carlsbad, CA, USA) according to the manufacturer's instruc- (Sigma) according to the manufacturer's instructions. Gray
tions. cDNA was synthesized from 1 µg RNA with the level images were acquired under a laser scanning microscope
PrimeScript RT Master Mix (Takara, Osaka, Japan). The PCR (Axioskop 2 plus, Carl Zeiss Co. Ltd., Jena, Germany).
amplification for the quantification of the miR‑155 and U6 was
performed using the TaqMan miRNA Reverse Transcription kit Luciferase reporter assay. The 3'-UTR sequence of ARID2
(Applied Biosystems, Foster City, CA, USA) and TaqMan Human predicted to interact with miR‑155 or the mutated sequence
miRNA Assay kit (Applied Biosystems). The relative expres- within the predicted target sites was synthesized and inserted
sion of miR‑155 was shown as the fold difference relative to U6. into the pGL3 control vector (Promega, Madison, WI,
qPCR primer against mature miRNA miR‑155 (HmiRQP0221) USA). These constructs were named as wt ARID2-3'UTR
INTERNATIONAL JOURNAL OF ONCOLOGY 48: 2425-2434, 2016 2427
Table I. Clinical correlation of miR-155 expression in HCC and luciferase activity was measured using the dual-luciferase
(n=124). reporter assay system (Promega). Firefly luciferase activity
was normalized to the Renilla luciferase activity. Results were
Expression level obtained from three independent experiments performed in
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑
triplicate.
Clinical Cases miR-155high miR-155low P-value
parameters (n) (n=68) (n=56) (p<0.05)a
In vivo experiments. Four-to-six-week-old female BALB/c
Age nude mice (Centre of Laboratory Animals, The Medical
College of Xi'an Jiaotong University, Xi'an, China) were used
<50 years 39 20 19 0.698
to establish the nude mouse xenograft model. SMMC-7721
≥50 years 85 48 37 (5x106) cells that were transfected with anti-miR‑155 or anti-
Gender miR-NC vectors were mixed in 150 µl of Matrigel and were
Male 92 50 42 0.852 inoculated subcutaneously into the flank of nude mice. The
tumor volume for each mouse was determined by measuring
Female 32 18 14
two of its dimensions and then calculated as tumor volume
Tumor size = length x width x width/2. After 3 weeks, the mice were
(cm) 0.001a sacrificed by cervical dislocation under anesthesia with ether
<5 cm 88 40 48 and the xenograft tumor tissue was explanted for examination.
≥5 cm 36 28 8 Animal protocols were approved by the Institutional Animal
Care and Use Committee of Xi'an Jiaotong University.
Tumor
number 0.947 Statistical analysis. Data are presented as the mean ± SD
Solitary 106 58 48 from at least three independent replicates. SPSS software, 16.0
Multiple 18 10 8 (SPSS, Inc, Chicago, IL, USA) was used to conduct the
analysis, and a two-tailed Student's t-test was employed to
Edmondson analyze the differences between two groups. Pearson's corre-
Ⅰ+Ⅱ 77 36 41 0.021a lation analysis was used to analyze the correlation between
Ⅲ+Ⅳ 47 32 15 two indices. Survival curves were plotted by the Kaplan-Meier
method and compared by the log-rank test. Differences were
TNM stage 0.020a
considered statistically significant at P<0.05.
Ⅰ+Ⅱ 102 51 51
Ⅲ+Ⅳ 22 17 5 Results
Capsular
infiltration 0.826 miR‑155 is upregulated in HCC tissues and cells. To evaluate
the potential role of miR‑155 in HCC, we first quantified
Present 81 45 36
miR‑155 in 124 pairs of HCC tissues and matched adjacent
Absent 43 23 20 non-tumor tissues using qRT-PCR methods. As shown in
Venous Fig. 1A, miR‑155 was significantly increased in the HCC tissues
infiltration 0.668 compared with adjacent non-tumor tissues. Furthermore, we
determined the expression level of miR‑155 in HCC cell lines
Present 15 9 6
and the normal hepatocyte cell line LO2. Similarly, miR‑155
Absent 109 59 50 was significantly upregulated in all HCC cell lines compared
AFP 0.569 with LO2 cells (P<0.05, Fig. 1B). These results suggest that
<400 ng/ml 41 21 20 miR‑155 expression is upregulated in HCC and may contribute
≥400 ng/ml 83 47 36 to the development of HCC.
Figure 1. miR‑155 is highly expressed in HCC tissues and cell lines. (A) Relative miR‑155 expression levels in HCC tissues and matched adjacent non-tumor
tissues were determined by qRT-PCR (n=124). (B) The expression of miR‑155 in HCC cell lines was significantly increased compared to that in the LO2 cells.
U6 snRNA was used as internal control. *P<0.05, **P<0.01.
Figure 2. The prognostic value of miR‑155 for HCC patients assessed by Kaplan-Meier analysis. HCC patients with high expression of miR‑155 had worse (A)
overall survival (OS) and (B) disease-free survival (DFS). **P<0.01.
Figure 3. miR‑155 promotes HCC cell cycle transition, cell proliferation and apoptosis resistance. (A) Hep3B and SMMC-7721 cells that were transfected with
corresponding miRNA vectors were subjected to qRT-PCR for miR‑155. (B) As assessed by flow cytometry, overexpression of miR‑155 promoted cell cycle
transition from G1 to S-phase in Hep3B cells and knockdown of miR‑155 induced G1 phase arrest in SMMC-7721 cells. (C) Quantification of the apoptotic
cell population by flow cytometry. Overexpression of miR‑155 inhibited apoptosis in Hep3B cells and knockdown of miR‑155 increased the percentage of
apoptotic SMMC-7721 cells compared with the control cells. (D) Cell proliferation as measured by BrdU incorporation assays was increased by overexpression
of miR‑155 in Hep3B cells and inhibited by knockdown of miR‑155 in SMMC-7721 cells. Representative micrographs (left) and quantification (right) of BrdU
incorporating-cells of indicated cells. Experiments were repeated at least 3 times with similar results, and error bars represent mean ± SD. *P<0.05, **P<0.01.
INTERNATIONAL JOURNAL OF ONCOLOGY 48: 2425-2434, 2016 2429
Figure 4. ARID2 is a direct target of miR‑155 in HCC cells. (A) TargetScan demonstrated that 3'-UTR of ARID2 contained the highly conserved putative
miR‑155 binding sites. (B) qRT-PCR analysis of ARID2 mRNA expression in Hep3B cells with miR‑155 or control (miR-control) transfection and SMMC‑7721
cells with anti-miR‑155 or anti-miR-NC (negative control) vector transfection. (C) Overexpression of miR‑155 reduced the expression of ARID2 protein in
Hep3B cells and knockdown of miR‑155 increases the level of ARID2 protein in SMMC-7721 cells. (D) miR‑155 significantly suppresses the luciferase activity
that carried wild-type (wt) but not mutant (mt) 3'-UTR of ARID2. Anti-miR‑155 led to a notable increase in the luciferase activity of wt 3'-UTR of ARID2.
(E) The expression of ARID2 mRNA in miR‑155 high-expressing tumors was significantly lower than that in miR‑155 low-expressing tumors. (F) A significant
inverse correlation between miR‑155 and ARID2 protein expression was observed in HCC tissues. Representative immunohistochemical staining showed
a weak staining of ARID2 in miR‑155 high-expressing HCC tissue and strong staining of ARID2 in the miR‑155 low-expressing tumor. (G) A statistically
significant inverse correlation was shown between miR‑155 and ARID2 mRNA levels in HCC specimens by Spearman's correlation analysis. Experiments
were repeated at least 3 times with similar results. *P<0.05, **P<0.01.
overall survival (P=0.0004) and disease-free survival cells, while the anti-miR‑155 vector significantly reduced the
(P=0.0006). These data indicate that miR‑155 could serve as a expression of miR‑155 in SMMC-7721 cells (P<0.01, Fig. 3A).
valuable indicator for predicting the prognosis of HCC. As determined by flow cytometric analysis, the upregulation
of miR‑155 promoted cell cycle transition from G1 to S phase
miR‑155 promotes cell cycle progression, cell proliferation and (P<0.05, Fig. 3B) and apoptosis resistance (P<0.05, Fig. 3C)
inhibits apoptosis in HCC cells. To investigate the biological in Hep3B cells. Furthermore, the overexpression of miR‑155
function of miR‑155 in the development and progression of significantly increased cell proliferation examined with incor-
HCC, we transduced a miR‑155 expression vector or a miR‑155 poration assay in Hep3B cells (P<0.05, Fig. 3D). By contrast,
inhibitor (anti-miR‑155) into Hep3B and SMMC-7721 cells, the downregulation of miR‑155 resulted in G1 arrest, apoptosis
respectively. As measured by qRT-PCR, miR‑155 expression promotion and proliferation reduction in SMMC-7721 cells
vector significantly increased the level of miR‑155 in Hep3B (P<0.05, respectively, Fig. 3B-D). These results demonstrated
2430 Zhang et al: THE ROLE OF miR-155 IN HCC
Figure 5. Alterations of ARID2 partially abolish miR‑155-mediated HCC cell cycle progression, proliferation and apoptosis. (A) miR‑155-overexpressing
Hep3B that were transfected with EV or ARID2 expression plasmid and miR‑155-suppressive SMMC-7721 cells that were transfected with control siRNA or
ARID2 siRNA were subjected to western blot analysis for ARID2. (B) G1 phase arrest was induced after ARID2 overexpression in miR‑155-overexpressing
Hep3B cells. The cell cycle transition from G1 to S-phase was increased by ARID2 knockdown in miR‑155-suppressive SMMC-7721 cells. The correlations
between miR‑155 effects and ARID2 overexpression or knockdown are shown in (C) apoptosis and (D) cell proliferation. ARID2 overexpression induced
effects that were opposite to those stimulated by miR‑155 overexpression in Hep3B cells. ARID2 knockdown abrogated the effects of miR‑155 knockdown on
SMMC-7721 cells. n, three independent experiments. *P<0.05, **P<0.01.
that miR‑155 regulates the cell cycle progression, apoptosis expression tumors were significantly lower than those in the
and proliferation of HCC cells. low miR‑155 expression tumors (P<0.05, respectively, Fig. 4E
and F). Notably, the expression level of miR‑155 was inversely
ARID2 is a direct target of miR‑155 in HCC. To explore the correlated with the level of ARID2 mRNA in HCC tissues
mechanism of miR‑155 regulation in HCC, we used publicly (R2=0.8393, P<0.0001, Fig. 4G). On the basis of these data,
available databases TargetScan 6.2 and miRanda to search we conclude that ARID2 is a direct target gene for miR‑155
predicted genes. Among them, the binding sites for miR‑155 and that miR‑155 downregulates ARID2 expression.
on the 3'-UTR of ARID2 were conserved among species
(Fig. 4A). To verify the regulation role of miR‑155 on ARID2, Altering expression of ARID2 influences the effect of miR‑155
qRT-PCR and western blotting were performed to detect on HCC cells. To confirm that ARID2 is a functional target of
the effect of miR‑155 on ARID2 mRNA and protein levels. miR‑155, we restored ARID2 expression in Hep3B-miR‑155
Ectopic expression of miR‑155 markedly decreased, while cells by transfecting ARID2 expression plasmid (P<0.05,
inhibition of miR‑155 increased the ARID2 mRNA (P<0.05, Fig. 5A). Functionally, restoration of ARID2 expression in
Fig. 4B) and protein (P<0.05, Fig. 4C). In addition, the over- Hep3B-miR‑155 cells partially abrogated the effect of exog-
expression of miR‑155 prominently inhibited the luciferase enous miR‑155, resulting in significant increase of apoptosis
activity of ARID2 containing a wild-type (wt) 3'-UTR but did (P<0.01, Fig. 5C) and obvious decrease of cell cycle progres-
not suppress the activity of ARID2 with a mutant (mt) 3'-UTR sion and cell proliferation (P<0.05, respectively, Fig. 5B and D).
(P<0.01, Fig. 4D). Suppression of miR‑155 by anti-miR‑155 Similarly, silencing of ARID2 in SMMC-7721-anti-miR‑155
increased the luciferase activity of wt ARID2 3'-UTR (P<0.01, cells partially abolished the effect of anti-miR‑155 on cell
Fig. 4D). However, with the mt ARID2 3'-UTR constructs, cycle, apoptosis and proliferation (P<0.05, respectively, Fig. 5).
there was no relative increase in activity. Moreover, the expres- These results demonstrate that ARID2 is a downstream
sion levels of ARID2 mRNA and protein in the high miR‑155 mediator for the function of miR‑155 in HCC.
INTERNATIONAL JOURNAL OF ONCOLOGY 48: 2425-2434, 2016 2431
Figure 6. Activated Akt phosphorylation signaling is essential for miR‑155-promoted HCC cell cycle progression, proliferation and miR‑155-inhibited apop-
tosis. (A) Western blot analysis of p-Akt, total Akt, Cyclin D1 and p27 protein levels in indicated cells. Quantification of cell cycle (B), apoptosis (C) and cell
proliferation (D) in indicated HCC cells treated with Akt inhibitor (0.5 µM) or transfected with dominant-active Akt plasmid. (E) The protein expression level
of Cyclin D1 and p27 by western blot analysis in indicated HCC cells treated with Akt inhibitor (0.5 µM) or transfected with dominant-active Akt plasmid.
Experiments were repeated at least 3 times with similar results, and error bars represent mean ± SD. *P<0.05.
Akt phosphorylation is essential for the biological function the expression of Cyclin D1 and p27, which are the down-
of miR‑155 in HCC. Previous studies demonstrated that stream effectors of Akt signaling and the key regulators of
activation of Akt signaling played an important role in HCC cell cycle progression and proliferation in HCC, were also
cell cycle progression, apoptosis and proliferation (20-22), altered in the up- or down-expression of miR‑155 HCC cells
so we further investigated the underlying molecular mecha- (P<0.05). Furthermore, to confirm that Akt phosphorylation
nisms of the miR‑155-mediated promotion of HCC biological contributed to the biological function of miR‑155-mediated
effects. As shown in Fig. 6A, ectopic expression of miR‑155 in HCC cells, we used Akt inhibitor or pmyr-Akt (dominant-
significantly increased, while miR‑155 inhibition decreased, active Akt) plasmid to affect Akt activation. Inactivation of
the Akt phosphorylation in HCC cells (P<0.05). Consistently, Akt phosphorylation by Akt inhibitor significantly decreased
2432 Zhang et al: THE ROLE OF miR-155 IN HCC
Figure 7. miR‑155 promotes HCC growth by activating Akt signaling in vivo. (A) Representative images of HCC xenografts from both SMMC-7721 anti-
miR‑NC (upper panel) and SMMC-7721-anti-miR‑155 cells (lower panel). Tumor growth curve (B) and weight (C) revealed that miR‑155 knockdown
significantly inhibited tumor growth in vivo. (D) Expression of miR‑155 in miR‑155 inhibitor-treated tumors from the subcutaneous model. (E) Expression of
p-Akt, Akt, Cyclin D1, p27 were assessed in the subcutaneous model by western blotting. *P<0.05, **P<0.01.
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