European Review for Medical and Pharmacological Sciences

2019; 23: 7863-7873

MiR-302 a/b/c suppresses tumor angiogenesis in
hepatocellular carcinoma by targeting MACC1
Y.-P. CAO1, M. PAN1, Y.-L. SONG2, H.-L. ZHANG1, H.-T. SUI1,
B.-C. SHAN1, H.-X. PIAO3
Department of Internal Medicine, Yanbian University, Yanji, Jilin, China
Department of Clinical Laboratory, Yanbian University Hospital, Yanji, Jilin, China
3
Department of Liver Diseases, Yanbian University Hospital, Yanji, Jilin, China
1
2

Abstract. – OBJECTIVE: Hepatocellular car-cinoma (HCC) is a hypervascularized tumor. Aberrant angiogenesis is the main cause, which results in cancer growth and progression. It has
been showed that microRNA-302 cluster (miR302) may be associated with angiogenesis. Here,
we aimed to identify the role of miR-302a/b/c in
the regulation of cell angiogenesis in HCC.
PATIENTS AND METHODS: MRNA expression of miR-302a/b/c and MACC1 was detected by quantitative Real Time-Polymerase Chain
Reaction (qRT-PCR). The protein of MACC1 wasmeasured using Western blot. Cells proliferation, migration, and invasion abilities were investigated via Cell Counting Kit-8 (CCK-8) assay
or transwell assay, respectively. Tube formatting
assays were used to explore the tube formation
capacity. The interaction among miR-302a/b/c
was analyzed by luciferase assay.
RESULTS: The expression of miR-302a/b/c
was greatly reduced while MACC1 expression,
whether mRNA or protein was conspicuously
elevated in HCC tissues and cells. Then, functional experiment results showed miR-302a/b/c
overexpression and MACC1 down-regulation
inhibited the proliferation, migration, invasion
ability, and tube formation capacity of HUVECs.
In addition, we detected that miR-302a/b/c directly targeted MACC1 and suppressed MACC1
expression, and miR-302a/b/c could suppress
tumor angiogenesis in HCC by targeting MACC1.
CONCLUSIONS: MiR-302a/b/c may function
as a potential suppressor of tumor angiogenesis
in HCC by targeting MACC1, indicating a promising target for HCC therapy.
Key Words:
MiR-302, MACC1, Angiogenesis, Invasion, Metastasis, HCC.

Abbreviations
HCC = Hepatocellular carcinoma; VEGF = vascular endothelial growth factor; PDGF = platelet derived growth

factor; FGF = fibroblast growth factor; EGF = epidermal
growth factor; hESCs = human embryonic stem cells;
ECs = endothelial cells, HUVECs = umbilical vein
endothelial cells; TCM = tumor-conditioned medium;
qRT-PCR = quantitative Real Time-Polymerase Chain
Reaction; FBS=fetal bovine serum.

Introduction
Hepatocellular carcinoma (HCC), a leading
cause of cancer-related death worldwide, is themost common type of primary liver cancer1. HCC
is a hypervascularized tumor, and most tumors
above 20 mm are arterially hypervascularized2.
Angiogenesis has an important effect on thepathophysiology of chronic liver disease3. Aberrant angiogenesis is the main cause, which resultsin cancer growth and progression, and the angiogenic switch is pivotal for HCC dedifferentiationand progression. Secretion of proangiogenic factors, such as vascular endothelial growth factor(VEGF), platelet-derived growth factor (PDGF),
fibroblast growth factor (FGF), epidermal growth
factor (EGF), etc., and suppression of antiangiogenic factors, such as angiostatin, endostatin,etc., stimulate endothelial cells to generate newblood vessels (sprouting) and facilitate tumor
growth and aggressiveness2,4. Therefore, antiangiogenic drugs are a prerequisite.
MicroRNAs (miRNAs) have emerged as critical regulators of gene expression via post-transcriptional inhibition and RNA decay, and havebeen involved in the regulation of cellular differentiation, proliferation, metastasis, apoptosis, andangiogenesis5,6. The miR-302/367 cluster, mainlycomposed of five members, miR-302a, miR-302b,
miR-302c, miR-302d, and miR-367, is ubiquitously distributed in vertebrates and occupies an in-

Corresponding Author: Hongxin Piao, MD; e-mail: wmeqol@163.com

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Y.-P. Cao, M. Pan, Y.-L. Song, H.-L. Zhang, H.-T. Sui, B.-C. Shan, H.-X. Piao

tragenic cluster located in intron 8 of Larp7 gene7.
MiR302/367 is highly expressed in embryonicstem cells and plays an important role in diverse
biological processes, such as the pluripotency ofhuman embryonic stem cells (hESCs), self-renewal, and reprogramming8,9. Pi et al10 recently
demonstrated that miR302/367 regulation of an
Erk1/2-Klf2-S1pr1 pathway could restrict tumor
development and angiogenesis, improve vascularstability, and potently retard tumor growth in an
endothelial cells (ECs) autonomous manner. MiR302 cluster can inhibit angiogenesis and growthof K562 leukemia cells by targeting VEGFA11.
However, the role of miR-302 and the underlying
mechanisms in HCC angiogenesis have not yet
been elucidated.
Metastasis-associated in colon cancer-1(MACC1), located on chromosome 7 at position
7p21.1, was initially identified by genome-wide
expression analysis in primary and metastatic
tissues of human colon cancer12. The effects ofderegulation of MACC1 on the progression ofcancers have been revealed, such as MACC1 induces autophagy to regulate proliferation, apoptosis, migration, and invasion of squamous cell
carcinoma through the underlying signaling pathway13,14. MACC1 may regulate the expression of
HGF, MMP-2, and MMP-9 in HSCs, and may
thus promote migration and invasion of gastriccancer15. MACC1 downregulation significantly
induced G1 phase arrest, suppressed endometrial carcinoma cell proliferation, migration, andinvasion16. MACC1 suppressed cell apoptosis in
hepatocellular carcinoma by targeting the HGF/c-MET/AKT pathway17. Thus, MACC1 may be
a useful target for developing cancer treatment
strategies.
Here, we took miR-302a/b/c as the researchtarget, and we found miR-302a/b/c might function as a potential suppressor of tumor angiogenesis in HCC by targeting MACC1, indicating anovel strategy to improve HCC therapy.

Patients and Methods
Patients and Specimens
In this study, a total of 33 patients undersetting
hepatectomy for HCC at the Yanbian University
Hospital were collected. All patients were diagnosed with primary HCC, and none of themhad received any preoperative cancer treatment.
Tumor specimens were immediately stored at–80°C until further study. Informed consent had
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been obtained from patients and hospital, and thework was approved by the Ethics Committee of
Yanbian University Hospital.
Cell Culture and Transfection
The human hepatoma cell lines (Huh7 and
PLC/PRF/5) and normal human hepatoma cellline THLE-2 were purchased from American
Type Culture Collection (ATCC; Manassas, VA,
USA). The umbilical vein endothelial cells (HUVECs) were purchased from AllCells (Shanghai,
China). Huh7 and PLC/PRF/5 cells were incubated with Dulbecco’s Modified Eagle’s Medium(DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS;
Gibco, Rockville, MD, USA). HUVECs and
THLE-2 cells were maintained in Roswell Park
Memorial Institute-1640 (RPMI-1640) medium(Gibco, Rockville, MD, USA) containing 10%
phosphate-buffered saline (PBS). All cells were
cultured in a humidified atmosphere with 5% CO2
at 37°C.
The miRNA mimic (miR-302a/b/c) and corresponding negative control (miR-NC) were purchased from RiboBio (Guangzhou, China). Smallinterfering RNA (siRNA) targeting MACC1 (siMACC1), siRNA negative control (si-NC), pcDNA3.1, and pcDNA3.1-MACC1 overexpression
vector (MACC1) were synthesized by Genepharma (Shanghai, China). These oligonucleotides
or vectors were transfected into Huh7 and PLC/
PRF/5 cells using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA)according to the manufacturer’s instruction. Cells
were harvested at 24 h for the following experiments.
Collection of the Tumor-Conditioned
Medium (TCM)
Huh7 and PLC/PRF/5 cells were transfectedwith or without corresponding miRNA mimic,
siRNAs, or plasmids or their matched control,
alone or in combination for 24 h, and then maintained in DMEM supplemented with serum-free
medium. After culture for further 14 h, the supernatant was collected and sequentially centrifuged
at 500 g and 12,000 g to remove cell debris at 4°C
for 15 min each. Then, aliquots of the TCM werestored at –80°C until needed.
Quantitative Real-Time Polymerase
Chain Reaction (qRT-PCR)
Once transfected under various conditions, total RNA was extracted from the Huh7 and PLC/MiR-302 a/b/c suppresses tumor angiogenesis in hepatocellular carcinoma by targeting MACC1

PRF/5 cells, which were lysed with TRI reagent(Sigma-Aldrich, St. Louis, MO, USA). Then, thecDNA was synthesized using a High Capacity
cDNA Archive kit (Applied Biosystems, Foster
City, CA, USA) according to the manufacturer’s
instruction. SYBR Green qPCR Master Mix kit(Qiagen, Hilden, Germany) was used to estimate
the relative expression of mRNA following the
manufacturer’s instruction. The expression levels were normalized using U6 for miR-302/b/c
and β-actin for MACC1. Fold change was analyzed using the 2-ΔΔCt equation. The specificprimers were listed as follows: MACC1 Forward 5’-CTTGCGGAGGTCACCATAGC-3’ andReserve 5’-GATTTCCAACAACGGGCTCA-3’;
β-actin Forward 5’-GGGAAATCGTGC- GTGACAT-3’ and Reserve 5’-CTGGAAGGTGGACAGCGAG-3’.
Cells Viability
Cell proliferation analyses were conducted using Cell Counting Kit-8 (Dojindo Laboratories,
Kumamoto, Japan). HUVECs cells were seededin 96-well plates at a density of 5x103 cells perwell at 6 h. Next, cells were incubated with
the TCM gathered from different backgroundof miR-302a/b/c, or MACC1 expression for theindicated times (0, 24, 48, and 72 h). Subsequently, 10 µl of CCK-8 reagents were added to eachwell for further 2 h incubation at 37°C. Finally,
the absorbance at 450 nm value of each test well
was measured using Microplate reader (Bio-Rad,
Hercules, CA, USA).
Tube Formation
HUVECs at the density of 2×104 were maintained in the absence or presence of 100% TCM
for 24 h at 37°C in a 96-well plate pre-coated with
Matrigel (BD Biosciences, Franklin Lakes, NJ,
USA). The formation of capillary-like structures
was analyzed under an inverted microscope. The
branch points of the formed tubes were counted
to evaluate tube formation.
Transwell Assay
Cell migration and invasion were evaluated bytranswell assays. Brieﬂy, HUVECs in serum-free
medium were seeded on the upper chamber, andthen the TCM collected from different background of miR-302a/b/c or MACC1 expression,
was added into the lower insert. After 12 h ofincubation, cells on the upper chamber were removed gently, and the migrated and invaded cells

were fixed with methanol for 20 min and stainedby 0.1% crystal violet 30 min. Five fields randomly selected were photographed and counted using
a microscope.
Western Blot
Cells from transfection HCC cells lines weresolubilized using Radio Immunoprecipitation
Assay (RIPA) Lysis (Beyotime, Shanghai, China), and the extracted protein concentration wasquantified using the bicinchoninic acid (BCA)
assay (Beyotime, Shanghai, China). Then, about
50 µg of proteins was subjected to 12% sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and followed by the transfer
to polyvinylidene difluoride (PVDF) membrane(Millipore, Billerica, MA, USA). After blocking
with 5% non-fat milk, the membrane was incubated with primary antibodies against MACC1and β-actin (CST, Danvers, MA, USA) at 4°C
overnight. Subsequently, horseradish peroxidase(HRP)-conjugated secondary antibodies wereadded for further incubation at room temperature for 2 h. Band signals were visualized via anenhanced chemiluminescence (ECL) kit (Pierce,
Minneapolis, MN, USA).
Luciferase Reporter Assay
The MACC1 UTR wild-type or mutant with
miR-302a/b/c binding sequences were cloned into
the pGL3 luciferase reporter vector (Promega,
Madison, WI, USA) to synthesize MACC1-Wt or-Mut luciferase reporter vector (pGL3-MACC1Wt or -Mut). Then, Huh7 and PLC/PRF/5 cells
were co-transfected with corresponding Wt or
Mut luciferase reporter vectors and miR-302a/b/c
mimics or mimic control using Lipofectamine
3000. After transfection for 48 h, luciferase activities were monitored using Dual-Luciferase assay
kit (Biotek Synergy, Winooski, VT, USA).
Statistical Analysis
All the data are expressed as the mean ±SD. All statistical analysis was conducted using
GraphPad Prism 7.0 (GraphPad Inc., San Diego,
CA, USA). All experiments were repeated threetimes. The correlation analysis was analyzed
by Spearman’s correlation. Statistical analysis
was evaluated using Student’s t-test or one-way
ANOVA as appropriate. Tukey’s test was used for
pairwise comparisons. p< 0.05 was considered
statistically significant.
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Y.-P. Cao, M. Pan, Y.-L. Song, H.-L. Zhang, H.-T. Sui, B.-C. Shan, H.-X. Piao

Results
The Expression of MiR-302 a/b/c Is
Reduced in HCC Tissues and Cells
The expression of miR-302 a/b/c was measured in HCC tissues and cell lines. The results
showed that the expression level of miR-302
a/b/c was significantly decreased in HCC tissues
compared with normal group (Figure 1A-C).
Moreover, we found a great reduction of miR302 a/b/c in HCC cell lines (Huh7 and PLC/
PRF/5) compared to the human normal hepatocytes THLE-2 (Figure 1D-F).
MiR-302 a/b/c Evidently Inhibits Tube
Formation, Proliferation, Migration, andInvasion of HUVECs
To explore the biological role of miR-302a/b/c
in HCC angiogenesis, its effects on cell tube
formation, proliferation, migration, and invasionof HUVECs were investigated. After transfectedwith miR-NC or miR-302a/b/c mimic, a significant increase of miR-302a/b/c expression in
Huh7 or PLC/PRF/5 cells was detected (Figure 2A). Subsequently, the tube formation assay
substantiated incubation with TCM from miR302a/b/c-overexpressed Huh7 or PLC/PRF/5 cells
remarkably inhibit the capillary tube formation of
HUVECs (Figure 2B). Additionally, a significant
suppression of HUVECs cells proliferation wasverified when they were grown in TCM obtained
from miR-302a/b/c-overexpressed Huh7 or PLC/
PRF/5 cells (Figure 2C, D). Furthermore, TCM
from miR-302a/b/c-overexpressed Huh7 or PLC/
PRF/5 cell notably attenuated cell migration andinvasion of HUVECs (Figure 2E, F). All the data
showed that miR-302 a/b/c could antagonize tube
formation, proliferation, migration, and invasionof HUVECs.
MiR-302a/b/c Directly Target MACC1and Suppress MACC1 Expression
To investigate the underlying mechanism of
miR-302a/b/c involved in the angiogenesis inhibition, bioinformatics analysis was performed
to predict the potential target of miR-302a/b/c,
and MACC1 was identified as a potential target
with a putative binding site of miR-302a/b/c(Figure 3A). Then, luciferase reporter assay
was conducted, and the result indicated luciferase activity was noticeably abolished following
co-transfection of miR-302a/b/c with MACC1Wt vector, but not in MACC1-Mut groups(Figure 3B, C) in Huh7 and PLC/PRF/5 cells.
Meanwhile, we found that miR-302a/b/c mimictransfection markedly inhibited the expressionof MACC1, whether protein or mRNA, bothin Huh7 and PLC/PRF/5 cells (Figure 3D, E).
Moreover, the expression of MACC1 in HCC
tissues and cells was examined, and the cor-Figure 1. Expression patterns of miR-302a/b/c in HCC tissues and cells. The expression of miR-302a/b/c was detected using
qRT-PCR in HCC, normal tissues (A-C) and HCC cell line (Huh7 and PLC/PRF/5) and human normal hepatocytes THLE-2,
respectively (D-F). *p < 0.05.

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MiR-302 a/b/c suppresses tumor angiogenesis in hepatocellular carcinoma by targeting MACC1

Figure 2. Effect of miR-302a/b/c on angiogenesis of HCC cells. A, Transfection efficiency was measured by qRT-PCR after
transfected with miR-NC or miR-302a/b/c mimic. Then, after incubation with TCM from Huh7 or PLC/PRF/5 cells transfectedwith miR-302a/b/c or miR-NC, (B) the functions in cell capillary tube formation, (C-D) proliferation, (E) migration andinvasion (F) were examined. *p < 0.05.

relation among miR-302a/b/c and MACC1 wasanalyzed. Then, the data exhibited a significant
increase of MACC1 expression in HCC tissues

and cells compared to the normal controls(Figure 4A-C). A perfect negative correlation
among miR-302a/b/c and MACC1 was dis7867

Y.-P. Cao, M. Pan, Y.-L. Song, H.-L. Zhang, H.-T. Sui, B.-C. Shan, H.-X. Piao

Figure 3. MACC1 was a direct target of miR-302a/b/c. A, Putative complementary sites between MACC1 and miR-302a/b/c.
B-C, Luciferase activity was detected in Huh7 and PLC/PRF/5 cells co-transfected with MACC1-WT or MACC1-Mut andmiR-302a/b/c or miR-NC. D-E, Level of MACC1 mRNA or protein was measured in Huh7 and PLC/PRF/5 cells transfectedwith miR-NC or miR-302a/b/c by qRT-PCR or Western blot, respectively. *p < 0.05.

covered (Figure 4D-F). Thus, based on theseresults, we know that miR-302a/b/c targetedly
suppressed MACC1 expression.
Knockdown of MACC1 Antagonizes Tube
Formation, Proliferation, Migration, andInvasion of HUVECs
To explore the effects of MACC1 on HCC
angiogenesis, the tube formation, proliferation,
migration, and invasion abilities of HUVECs
were determined. After knockdown of MACC1,
a significant reduction of MACC1 was detected(Figure 5A). Subsequently, a notable inhibition of
HUVECs cells tube formation, proliferation, migration, and invasion was verified in the presence
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of TCM obtained from MACC1-depleted Huh7
or PLC/PRF/5 cells (Figure 5B-F). These data revealed that MACC1 induced tube formation, proliferation, migration, and invasion of HUVECs,
while knockdown of MACC1 could hinder theangiogenesis in HCC cells.
Overexpression of MiR-302a/b/c
Attenuates the Promotion Effect of
MACC1 on HUVEC Tube Formation,
Proliferation, Migration, and Invasion
Based on the previous results, cells were transfected with pcDNA or MACC1, or MACC1 andmiR-NC, or MACC1 and miR-302a/b/c to investigate the interaction among miR-302a/b/c
and MACC1 on angiogenesis in HCC cells. TheMiR-302 a/b/c suppresses tumor angiogenesis in hepatocellular carcinoma by targeting MACC1

Figure 4. The correlation between miR-302a/b/c and MACC1. A, Expression of MACC1 mRNA was detected using qRTPCR in HCC and normal tissues (A) and HCC cell line (Huh7 and PLC/PRF/5) and human normal hepatocytes THLE-2,
respectively (B). The protein of MACC1 was examined using Western blot (C). The correlation among miR-302a/b/c and
MACC1 was analyzed via Spearman’s correlation (D-F). *p < 0.05.

transfection efficiency was measured (Figure
6A). Subsequently, we found overexpression of
miR-302a/b/c attenuates the promotion effect of
MACC1 on HUVECs tube formation (Figure

6B), proliferation (Figure 6C, D), migration, andinvasion (Figure 6E, F). These indicated thatoverexpression of miR-302a/b/c could attenuate
the angiogenesis effect of MACC1 on HUVEC.

Figure 5. Effect of MACC1 on angiogenesis of HCC cells. A, Transfection efficiency was determined by qRT-PCR after
transfected with si-MACC1 or si-NC. After incubation with TCM from Huh7 or PLC/PRF/5 cells transfected with si-MACC1
or si-NC, (B) the functions in cell capillary tube formation, (C, D) proliferation, (E) migration and invasion (F) were
investigated. *p < 0.05.

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Y.-P. Cao, M. Pan, Y.-L. Song, H.-L. Zhang, H.-T. Sui, B.-C. Shan, H.-X. Piao

Figure 6. The interaction among miR-302a/b/c and MACC1 on the angiogenesis of HCC cells. A, Transfection efficiency was determined by qRT-PCR after transfected with
pcDNA or MACC1 or MACC1 and miR-NC or MACC1 and miR-302a/b/c. Then after incubation with TCM from Huh7 or PLC/PRF/5 cells transfected with pcDNA or MACC1or MACC1 and miR-NC or MACC1 and miR-302a/b/c, (B) the functions in cell capillary tube formation, (C-D) proliferation, (E) migration and invasion (F) were demonstrated.
*p < 0.05.

MiR-302 a/b/c suppresses tumor angiogenesis in hepatocellular carcinoma by targeting MACC1

Discussion
Hepatocarcinogenesis is a multistep process
involving genetic and environmental changes,
permitting the hepatocyte to escape normal
control mechanisms in cell differentiation, proliferation, migration, growth, and death, leading to the development of malignant diseases18. Angiogenesis is a vital biological process
in HCC development and progression due to
HCC, which is one of the most known vascular solid tumors19. At present, antiangiogenic treatments, which can inhibit blood vessel
formation, have been reported to be a promising effective treatment for HCC20. Several
antiangiogenic drugs have been indicated, suchas sorafenib, regorafenib, lenvatinib, cabozantinib, etc. However, the safety and efficacy ofantiangiogenic therapies are still controversial.
Up to date, sorafenib monotherapy is the only global standard of antiangiogenic care foradvanced HCC patients. None of the otherantiangiogenic drugs or combinations tested
to date have improved survival compared withsorafenib monotherapy4,21. Therefore, we shouldtake significant efforts to advance knowledgeof the molecular mechanisms of HCC angiogenic initiation and progression.
MiRNAs, with 21-23 nucleotides in length,
are noncoding, single-stranded RNA molecules
generated endogenously. Recently, miRNAs
have been discovered to modulate various stages of angiogenesis22. MiRNAs can regulate thefunction of ECs via non-autonomous and autonomous manner during tumor angiogenesis, andmiRNAs modulate the expression of pro- or anti-angiogenic factors in tumor cells, thus regulating the proliferation and metastasis of ECs in
a paracrine manner23. Malformation of various
miRNAs mediated processes of angiogenesis
has been found in diverse diseases like cardiovascular diseases24, non-small cell lung cancer25,
multiple sclerosis, type 1, and type 2 diabetes,
diabetic retinopathy22, including HCC26,27. SomemiRNAs, such as miR-17-92 cluster, miR-128,
miR-497, miR-296, miR-125b, etc., targeted thegenes to inhibit or promote ECs tube formation 23,28,29. Therefore, the regulation of angiogenesis by microRNAs in HCC may be a novel therapeutic approach. In this study, the expressionof miR-302 a/b/c was measured in HCC tissues
and cell lines. We found a significant decreaseof miR-302a/b/c expression in HCC tissues andcell lines. Then, HUVECs cells were incubat-

ed with TCM from miR-302a/b/c-overexpressed
Huh7 or PLC/PRF/5 cells. The proliferation,
migration, invasion abilities, and tube formation
capacity of HUVECs were detected, and theresults suggested that overexpression of miR302a/b/c could attenuate the angiogenesis effectof HUVEC in HCC cell lines.
To investigate the underlying molecularmechanism of miR-302a/b/c on the angiogenesis of HUVECs, bioinformatics analysis andluciferase assay were conducted, and MACC1
was identified as a potential target with a putative binding site of miR-302a/b/c. MACC1
was reported to be an independent prognostic biomarker of overall survival for several
digestive system neoplasms (e.g., pancreaticcancer, colorectal cancer, esophageal cancer,
gastric cancer, and hepatocellular carcinoma).
High MACC1 expression markedly associatedwith poorer overall survival, as well as poorer
relapse-free survival30,31. In the angiogenesis,
MACC1 was discovered to promote vascular formation in cholangiocarcinoma, gastriccancer, hepatocellular carcinoma, and cervicalcancer27,32-34. In the present study, a significantly elevated MACC1 mRNA and protein
expression in HCC tissues and cells were determined. Furthermore, after incubated with
TCM obtained from MACC1-depleted Huh7 or
PLC/PRF/5 cells, the inhibition of HUVECs
proliferation, migration, invasion abilities, andtube formation was identified consistently withprevious researches. Additionally, we detectedthat miR-302a/b/c directly targets MACC1 andsuppresses MACC1 expression, and that a perfect negative correlation among miR-302a/b/c
and MACC1 was demonstrated. Meanwhile,
we also illustrated that miR-302a/b/c could
antagonize the angiogenesis effect of MACC1
on HUVEC.

Conclusions
MiR-302a/b/c regulates MACC1 suppression
to counterwork angiogenesis in HUVECs. We
first verified the miR-302/MACC1 signaling
pathway in HCC, indicating a promising target
for the evolvement of anti-angiogenesis drugs.

Conflict of Interest
The Authors declare that they have no conflict of interests.

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Y.-P. Cao, M. Pan, Y.-L. Song, H.-L. Zhang, H.-T. Sui, B.-C. Shan, H.-X. Piao

Ethics Approval and Consent to Participate

This study was approved by the Ethics Committee of Yanbian University Hospital. The methods used in this study
were performed in accordance with the relevant guidelines
and regulations.

Consent for Publication

All authors about this work consent this manuscript to be
published.

Availability of Data and Materials

All original data and materials are available from the corresponding author upon request.

Funding

This work was supported by National Natural Science
Foundation of China (Grant No. 81366075) and the Chinese
Foundation for hepatitis prevention and control (Grant No.
TQGB2015028).

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