Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20

Citrus peel flavonoids improve lipid metabolism

by inhibiting miR-33 and miR-122 expression in
HepG2 cells

Dongxiao Su, Hesheng Liu, Xiangyang Qi, Lihong Dong, Ruifen Zhang & Jie
Zhang

To cite this article: Dongxiao Su, Hesheng Liu, Xiangyang Qi, Lihong Dong, Ruifen Zhang
& Jie Zhang (2019): Citrus peel flavonoids improve lipid metabolism by inhibiting miR-33
and miR-122 expression in HepG2 cells, Bioscience, Biotechnology, and Biochemistry, DOI:
10.1080/09168451.2019.1608807

To link to this article: https://doi.org/10.1080/09168451.2019.1608807

Published online: 24 Apr 2019.

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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2019.1608807

Citrus peel flavonoids improve lipid metabolism by inhibiting miR-33 and

miR-122 expression in HepG2 cells
Dongxiao Sua,b*, Hesheng Liub,c*, Xiangyang Qib,c, Lihong Dongd, Ruifen Zhangd and Jie Zhangb,c
a
School of Chemistry and Chemical Engineering, Guangzhou University, Guangzhou, China; bZhejiang Provincial Top Discipline of
Biological Engineering (Level A), Zhejiang Wanli University, Ningbo, China; cCollege of Biological and Environmental Sciences, Zhejiang
Wanli University, Ningbo, China; dSericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences/Key
Laboratory of Functional Foods, Ministry of Agriculture, Guangzhou, P.R. China

ABSTRACT ARTICLE HISTORY

Citrus plants are rich in flavonoids and beneficial for lipid metabolism. However, the mechan- Received 13 February 2019
ism has not been fully elucidated. Both citrus peel flavonoid extracts (CPFE) and a mixture of Accepted 10 April 2019
their primary flavonoid compounds, namely, nobiletin, tangeretin and hesperidin, citrus KEYWORDS
flavonoid purity mixture (CFPM), were found to have lipid-lowering effects on oleic acid- Citrus; flavonoid; lipid
induced lipid accumulation in HepG2 cells. The carnitine palmitoyltransferase 1α (CPT1α) metabolism; miRNA; HepG2
gene was markedly increased, while the fatty acid synthase (FAS) gene was significantly
decreased by both CPFE and CFPM in oleic acid-treated HepG2 cells. Flavonoid compounds
from citrus peel suppressed miR-122 and miR-33 expression, which were induced by oleic
acid. Changes in miR-122 and miR-33 expression, which subsequently affect the expression of
their target mRNAs FAS and CPT1α, are most likely the principal mechanisms leading to
decreased lipid accumulation in HepG2 cells. Citrus flavonoids likely regulate lipid metabolism
by modulating the expression levels of miR-122 and miR-33.

Citrus plants are rich in flavonoids. Citrus flavonoids
are polyphenolic compounds encompassing several
subgroups of flavonoids, including flavanones (narin-
genin, naringin, hesperitin, and hesperidin), poly-
methoxylated flavones (nobiletin and tangeretin),
flavones, flavonols, favans, and anthocyanins [1].
Previous studies have confirmed that citrus flavo-
noids have antioxidant, anti-inflammatory, and anti-
tumor activities [2,3]. Dietary citrus flavonoids are
beneficial for individuals with metabolic syndrome
and cardiovascular and neurodegenerative dis-
eases [4,5].
Cholesterol-related diseases, including Alzheimer’s
disease, Huntington’s disease, and Parkinson’s dis-
ease, can be treated by antagonizing the miRNA
expression involved in cholesterol biosynthesis and
cholesterol efflux [6]. Regulation of the cholesterol
metabolism of microRNAs in the brain has been
associated with neurodegeneration [6]. MicroRNAs
would need to be the molecular target of phenolics
to exert their biological effects [7].
MicroRNAs (miRNAs) are endogenous, small,
noncoding, single-stranded 18- to 24-nucleotide
RNAs. They help modulate gene expression by direct
posttranscriptional repression and/or by decreasing
the stability of target mRNAs [8,9]. More than 60%
of mammalian mRNAs are conserved targets of
microRNAs [8].
In recent years, it has become evident both in vitro
and in vivo that phenolics modify cell functionality
through a new mechanism by modulating miRNA
levels [10]. The flavonoid tanshinone IIA reduced
miR-155, miR-147, miR-184, miR-29b, and miR-34c
expression levels to exert anti-inflammatory activity
[11]. Troxerutin, a natural flavonoid rutin, exerts
protective effects against ultraviolet light B-induced
damage in HaCaT cells by regulating miRNA expres-
sion [12].
Lipid metabolism is regulated by miRNAs [13],
including miR-103, miR-107, and miR-122 [14,15].
MiRNAs 33, 122, and 208 could be potential targets
for treating obesity and diabetes [16]. The expres-
sion of miRNAs was regulated by flavonoid com-
pounds [11,12,17]. An ECG-type dimer and an
EGCG dimer of persimmon tannin improved hepa-
tic steatosis in L02 cells by regulating miR-122 and
miR-33b [17]. Multiple targeted miRNAs could be
regulated by flavonoids. Wen et al. reported that
the expression level of 25 miRNAs, including 17
upregulated and 8 downregulated miRNAs in
HepG2 cancer cells, was regulated by ellagitannin,
a natural polyphenol compound isolated from
Balanophora [18].
The total (upregulated and downregulated) num-
ber of miRNAs, including miR-33, in the livers of
apoE-/- mice after hesperidin or naringin

CONTACT Dongxiao Su dongxsu@126.com; Jie Zhang zhangjiezh@foxmail.com

*Dongxiao Su and Hesheng Liu should be considered joint first author.
© 2019 Japan Society for Bioscience, Biotechnology, and Agrochemistry
2 D. SU ET AL.

supplementation for two weeks was 97 and 69, Citrus peel flavonoid extraction, identification,
respectively, and the total number of mRNAs in and quantification
the corresponding livers (4101 and 953, respectively)
Mature Ponkan mandarin (Citrus reticulata Blanco cv.
was much larger than the number of miRNAs [19].
Ponkan) peel was obtained from a local fruit market in
Previous studies confirmed that citrus flavonoids
Jingzhou, China. The fruit peel was washed with tap
regulate gene expression related to lipid metabolism
water, hot air-dried and finely ground to pass through
[20,21]. The mechanisms responsible for citrus fla-
a 0.3-mm screen. The flavonoids were extracted as pre-
vonoid action have not been fully elucidated.
viously described [22]. The evaporated concentrated
Modulating miRNA expression by dietary supple-
ethanol extracts were subjected to chromatography
mentation with citrus flavonoids would be a new
using an octadecylsilyl silica gel column (Cangzhou
mechanism of action. Therefore, the aim of the
Bonchem Co., Ltd., Cangzhou, China), washed with
present study was to investigate how citrus flavo-
water and eluted with 80% methanol. They were then
noids regulate lipid metabolism by regulating the
collected, concentrated and vacuum-freeze dried.
expression of lipid metabolism-related miRNAs.
The primary flavonoid constituents in the refined
Ponkan peel extract were analyzed by high-
performance liquid chromatography with a diode
Materials and methods
array detector (HPLC-DAD) using a model 1260 series
Chemicals and reagents LC system (Agilent Technologies, Palo Alto, CA, USA)
and a previously reported method [23]. The HPLC
Minimum Eagle medium (MEM), fetal bovine serum
separations were performed using an Agilent Zorbox
(FBS), nonessential amino acid nutrient solution, and
SB-C18 column. The standards (hesperidin, nobiletin,
penicillin-streptomycin were purchased from Thermo
and tangeretin, Figure 1) were purchased from Sigma-
Fisher Scientific (Waltham, MA, USA). Bovine serum
Aldrich (Shanghai, China). CPFE were identified and
albumin (BSA), oleic acid (OA), SNC50 were
quantified using external calibration curves for hesper-
obtained from Sigma-Aldrich Corp. (St. Louis, MO,
idin, nobiletin, and tangeretin, respectively, based on
USA). A triacylglycerols (TG) commercial kit was
the retention time and peak area. This CPFE contained
provided by Applygen Technologies Inc. (Beijing,
hesperidin (52.74%), nobiletin (14.32%), and tangeretin
China). A TaqMan® microRNA reverse transcription
(10.14%).
kit, a TaqMan® microRNA assay kit, TaqMan probes,
and TaqMan® universal master mix II were purchased
from Life Technologies (NY, USA). An MTT cyto-
Cytotoxicity assay
toxicity assay kit, a total protein assay kit (BCA
method), and a triglyceride assay kit were purchased Human hepatoma cell line HepG2 was purchased
from Nanjing Jiancheng Bioengineering Institute from the American Type Culture Collection
(Nanjing, China). (Manassas, VA, USA). The cells were cultured in

Figure 1. Types and chemical structures of citrus peel flavonoids.


MEM containing 10% FBS, 1% nonessential amino
acid nutrient solution, and 1% penicillin-
streptomycin at 37°C in a humidified 5% CO2 atmo-
sphere in a HeraCell 240i CO2 incubator (Thermo
Fisher Scientific, Waltham, MA USA).
The HepG2 cell cytotoxicity was assessed in cells
stimulated with various sample concentrations using
the MTT assay [24]. HepG2 cells were seeded onto
96-well plates at a density of 2.5 × 103 per well in
MEM plus 10% FBS. The cells were incubated with
various concentrations of samples for 24 or 48
h. Then, a specific dye solution (10 μL) for the
MTT assay was added and incubated for an addi-
tional 4 h at 37°C. After DMSO addition (100 μL/
well), the absorbance was measured at 570 nm using
an Infinite M200 Pro multifunctional microplate
reader (Tecan, Männedorf Switzerland).
Cell treatment
The HepG2 cells were seeded in 12-well plates in
1-mL volumes at a concentration of 5 × 105/mL for
24 h. The cells were then allowed to grow to 70–80%
confluence. The culture medium was replaced with
1 mL of 1% BSA serum-free medium and maintained
for 16 h. Then, the cells were treated with 1 mL of 1%
BSA serum-free medium containing different concen-
trations of CPFE or CFPM (0, 0.5, 1, 10, 20 40, 60, 80,
and 100 μg/mL) and 0.4 mmol/L oleic acid for 24
h. The cells incubated with oleic acid in the absence
of citrus flavonoids were used as the control group.
Or HepG2 cells were exposed to CPFE and CFPM at
the indicated 10.0 μg/mL concentrations for 0.5, 1, 3
and 6 h. The CFPM, hesperidin, nobiletin, and tan-
geretin, were mixed at the same proportion as that of
the compounds found in citrus peel. The CPFE and
CFPM were dissolved in DMSO, and the final con-
centration of the DMSO was 0.1%.
Triacylglycerol measurement
For triacylglycerol content detection, the cells were
washed extensively with phosphate-buffered saline
(PBS). Then, the cells were lysed and centrifuged. The
intracellular TG content in the supernatant was mea-
sured using a commercial kit. Total protein measure-
ments were determined using a BCA reagent
commercial kit.
mRNA and miRNA preparation
The cells (5 × 105) were incubated with or without
the CPFE or CFPM and, after 48 h, were harvested
and centrifuged at 10,000 rpm for 10 min. Total RNA
containing miRNA was extracted and isolated using
the mirPremier mi/mRNA isolation reagent kit
SNC50 (Sigma-Aldrich, USA) according to the
CITRUS FLAVONOID CHANGE LIPID METABOLISM MIRNA 3
manufacturer’s instructions. The RNA concentration
and purity were further assessed at 260 and 280 nm
using a NanoDrop 1000 spectrophotometer (Thermo
Scientific, USA).
RT-qPCR for mRNA (FAS and CPT1α) and miRNA
(miR-33, miR-122) expression
The relative mRNA levels of FAS and CPT1α were
quantified using Real-Time PCR with a CFX
Connect™ Real-Time PCR Detection System (Bio-Rad,
USA). The total RNA (1 μg) was reverse-transcribed
into cDNA using random hexamer primers. The
reverse-transcribed cDNA (10 ng) for RT-qPCR ampli-
fication was mixed with specific TaqMan probes. The
total RNA reverse transcription and quantitative RT-
PCR amplification were conducted according to the
manufacturer’s protocols. The endogenous control
cyclophilin (PPIA) was similarly measured, and it
served as the reference gene.
For miR-33 and miR-122 RT-qPCR analysis, RNA
was reverse-transcribed into cDNA using a TaqMan®
MicroRNA Reverse Transcription Kit. Then, the
cDNA was amplified using a TaqMan® MicroRNA
Assay Kit with specific TaqMan probes and
TaqMan® Universal Master Mix II according to the
manufacturer’s instructions. The expression levels of
the target genes were normalized to that of small
nuclear RNA endogenous control U6. The relative
mRNA and miRNA transcript levels were calculated
according to the 2−ΔΔCT method.
Statistical analysis
All the results are expressed as the means ± SD.
Significant differences between groups were assessed
by one-way analysis of variance (one-way ANOVA)
followed by the Tukey (Figures 2–4) or LSD (Figure 5)
test. Significant differences were defined as p values of
less than 0.05. The statistical analysis was performed
using SPSS 24.0 (SPSS Inc., Chicago, IL, USA).
Results
Cytotoxicity of citrus peel flavonoids in human
hepatoma cells
Before determining whether the CPFE and the mix-
ture of primary citrus peel flavonoid compounds
composed of nobiletin, tangeretin, and hesperidin
had any lipid-lowering activities, the cytotoxicity of
CPFE and CFPM was measured in the HepG2 cells
by the MTT assay, and the results are shown in
Figure 2. After incubation for 24 h, both citrus peel
flavonoid extracts (Figure 2(a)) and the mixture of
primary citrus peel flavonoid compounds (Figure 2
(b)) showed obvious cytotoxicity in HepG2 cells at
4 D. SU ET AL.
Figure 2. Effects of citrus peel flavonoid extracts (CPFE) and
primary citrus peel flavonoid compounds, nobiletin, tanger-
etin and hesperidin, citrus flavonoid purity mixture (CFPM),
on HepG2 cell cytotoxicity.
(a) The cells were treated with CPFE for 24 and 48 h, and the cell
cytotoxicity was assessed by the MTT assay. (b) The cells were treated
with CFPM for 24 h and 48 h and their cell cytotoxicity was assessed by
MTT assay. Bars with different letters in common within the same group
are significantly different (p < 0.05) according to the Tukey test (N = 6).
a concentration up to 80 μg/mL. However, at con-
centrations lower than 40 μg/mL, neither the citrus
peel flavonoid extracts nor the mixture of primary
citrus peel flavonoid compounds was cytotoxic. As
shown in Figure 2, the mixture of primary citrus peel
flavonoid compounds was cytotoxic above 60 μg/mL
over a 48 h treatment period. Therefore, the preli-
minary concentrations used for citrus peel flavonoid
extracts and the mixture of primary citrus peel flavo-
noid compounds were less than 40 μg/mL in subse-
quent lipid-lowering assays.
Lipid-lowering effects of citrus peel flavonoids on
lipid accumulation in oleic acid-induced HepG2
cells
To evaluate whether the CPFE and CFPM had a lipid-
lowering effect on oleic acid-treated HepG2 cells, the
cells were incubated in nontoxic doses of CPFE (0.1–20
μg/mL) or CFPM (0.1–20 μg/mL). The suppressive
effects of citrus peel flavonoids on OA-induced lipid
accumulation in HepG2 cells are shown in Figure 3.
The intracellular TG content was decreased by both
CPFE and CFPM at concentrations ranging from 1.0 to
20.0 μg/mL. Within 6 h, the concentration of 1.0 μg/
mL showed an inhibitory effect on TG accumulation
(Figure 3(a,b)). However, from 12 to 24 h, the
suppression of the TG dose had to be increased to
10.0 μg/mL (Figure 3(c,d)). The inhibitory effect of
CPF on intracellular TG accumulation would be partly
time dependent at a low dose. After incubation for 24
h, CPFE and CFPM at the 10.0 μg/mL concentration
decreased TG accumulation levels by 18% and 14%,
respectively. There was no statistically significant dif-
ference in the inhibitory effects between CPFE and
CFPM. These results clearly illustrate that both CPFE
and CFPM had significant lipid-lowering effects on
oleic acid-induced lipid accumulation in HepG2 cells.
Downregulation effects of citrus peel flavonoids
on miR-122 and miR-33 expression in oleic
acid-treated HepG2 cell
s
To understand whether the suppressive effects of
CPFE and CFPM were due to the regulation of the
miRNA expression, the effects of these compounds on
the expression levels of miR-122 and miR-33 were deter-
mined in HepG2 cells at the indicated concentrations.
As shown in Figure 4, pretreating HepG2 cells with
CPFE and CFPM at concentrations ranging from 1.0
to 20.0 μg/mL significantly inhibited miR-122 expres-
sion. Although the inhibitory effect of a higher dose
(10.0 and 20.0) is better than that of a lower dose (0.5
and 1.0), it was difficult to draw the conclusion that
CPFE and CFPM regulate the expression of miRNA in
a concentration-dependent manner since there was no
significant difference between 10.0 and 20.0 μg/mL.
Regarding the expression level of miR-33, only the
higher dose (10.0 and 20.0 μg/mL) exhibited
a markedly inhibitory effect. A lower dose also inhibited
the gene expression, but the effect was not significant
except for CPFE at 1.0 μg/mL compared to control (0.0
μg/mL). After 24 h of treatment, the expression levels of
miR-122 and miR-33 decreased 32% and 28% by CPFE
and 25% and 23% by CFPM, respectively. There was no
significant difference between CPFE and CFPM in the
suppressive effects of miRNA expression at higher dose
(10.0 and 20.0 μg/mL). These results demonstrate that
flavonoid compounds from citrus peel repressed
miRNA expression induced by oleic acid in HepG2 cells.
Citrus peel flavonoids inhibit lipid accumulation
by regulating lipogenesis gene expression
To examine whether the observed lipid-lowering effects
of citrus peel flavonoid compounds were due to the
regulation of lipogenesis gene expression, HepG2 cells
were exposed to CPFE and CFPM at the indicated
concentrations and at 0.5, 1, 3 and 6 h. As shown in
Figure 5, the HepG2 cells treated with oleic acid results
in increased expression levels of both miR-122 and
miR-33 after 6 h of treatment. Neither CPFE nor
CFPM could markedly decrease the expression levels
 CITRUS FLAVONOID CHANGE LIPID METABOLISM MIRNA 5

Figure 3. Lipid-lowering effects of citrus peel flavonoid extracts (CPFE) and primary citrus peel flavonoid compounds, namely,
nobiletin, tangeretin and hesperidin, citrus flavonoid purity mixture (CFPM), on intracellular lipid accumulation in HepG2 cells.
The cells (a–d) were treated with CPFE or CFPM for 3, 6, 12 and 24 h, respectively. The intracellular TG content was measured by a commercial kit. Bars
with different letters in common within the same group are significantly different (p < 0.05) according to the Tukey test (N = 3).

of miR-122 and miR-33 within 1 h. However, after the

Figure 4. Effects of citrus peel flavonoid extracts (CPFE) and
primary citrus peel flavonoid compounds, namely, nobiletin,
tangeretin and hesperidin, citrus flavonoid purity mixture
(CFPM), on the expression level of miR-122 and miR-33
after the cells were treated with CPFE or CFPM for 24 h.
Gene expression was determined by real-time RT-PCR. Bars with different
letters in common within the same group are significantly different (p <
0.05) according to the Tukey test (N = 3).
HepG2 cells were incubated with CPFE or CFPM at the
10.0 μg/mL concentration for 3 h, the expression level of
miR-122 decreased significantly, by 33% and 20%, and
the miR-33 expression was decreased notably, by 31%
and 28%, respectively. With further treatment with
CPFE or CFPM for 6 h, the expression of miR-122
was reduced by 40% and 37%, and that of miR-33 was
reduced by 33% and 38%, respectively. FAS and CPT1α
were the target genes of miR-122 and miR-33, and the
expression levels of these genes were also evaluated at
the indicated time, as shown in Figure 5(c,d). The FAS
gene in the cells treated with CPFE or CFPM for 3 and 6
h was markedly suppressed (p < 0.05) compared with
that of the untreated cells, which was coincident with
miR-122. However, the CPT1α gene expression was
markedly increased (p < 0.05) by both CPFE and
CFPM in oleic acid-treated HepG2 cells after 3 or 6
h of incubation, which was inversely proportional to
miR-33 expression. Therefore, changes in miR-122 and
miR-33 expression, which subsequently affect the
expression of their target mRNAs FAS and CPT1α,
are most likely the principal mechanisms leading to
decreased lipid accumulation in HepG2 cells.
Discussion
Citrus flavonoids have complex structural compositions.
Several studies have shown that citrus flavonoids have
a variety of biological activities, including anticancer,
anti-inflammatory and neuroprotective effects [22].
6 D. SU ET AL.

Figure 5. Effects of citrus peel flavonoid extracts (CPFE) and primary citrus peel flavonoid compounds, namely, nobiletin,
tangeretin and hesperidin, citrus flavonoid purity mixture (CFPM), the concentrations of CPFE and CFPM were 10 μg/mL, on the
expression of miR-122, miR-33 and their target mRNAs FAS and CPT1α after the cells were treated with CPFE or CFPM within 6 h.
The gene expression was determined by real-time RT-PCR. The asterisks and sharps represent significant differences within the same group (Time) which
compared to NC at the same time (p < 0.05) according to the LSD test (N = 3).

Flavonoids reportedly have lipid profile-improving activ-
ity in HepG2 cells [25,26]. Citrus flavonoids (nobiletin,
naringin, and hesperidin) isolated from Korean Citrus
aurantium L. have anti-inflammatory effects that act by
suppressing the expression of cyclooxygenase-2, induci-
ble nitric oxide synthase and cytokines by blocking
nuclear factor-kappa B and mitogen-activated protein
kinase (MAPK) signaling in mouse macrophage RAW
264.7 cells [27]. Huang et al. confirmed that hesperetin
stimulated the activation of MAPKs [28]. Data from
Zygmunt et al. showed that naringenin increases muscle
cell glucose uptake by significantly increasing AMPK
phosphorylation/activation [29]. The underlying
mechanisms of action of all the above results were related
to the regulation of cellular signaling pathways. The
lipid-lowering effect of citrus flavonoids could be related
to the expression levels of proteases and mRNAs.
The lipid-lowering capacities of citrus flavonoids
were determined by their molecular structures. Lin
et al. reported that citrus polymethoxylated flavo-
noids (PMFs), tangeretin and nobiletin inhibited
cholesterol (CH) and triglyceride (TG) synthesis
(IC50 = 13 and 29 μM, respectively). However,
other PMFs (sinensetin) and non-PMFs (hesperetin
and naringenin) had only weak effects on CH and
TG synthesis and apoB secretion in HepG2 cells
(IC50 > 100 μM) [30]. However, Morin et al. found
that hesperetin and nobiletin induced the transcrip-
tion of low-density lipoprotein receptor in HepG2
cells, but with different peak stimulations of 5.3- to
7.5-fold and 3- to 3.8-fold at 150–160 μmol/L
hesperetin and 10–20 μmol/L nobiletin [31],
respectively. The CPFE in the present study con-
tained hesperidin, nobiletin, and tangeretin and
decreased TG at a concentration that was consis-
tent with the results of the above findings, showing
that a combination of hesperidin, nobiletin and
tangeretin still had lipid-lowering activity. Further
studies should be conducted to investigate whether
a synergistic effect exists.
Those investigators speculated that the inhibition
of hepatic apoB secretion would be the primary
underlying mechanism of the nobiletin lipid-
lowering action [30]. However, the present study
showed that the expression levels of other mRNAs
were changed when the FAS gene was markedly
suppressed, but the CPT1α gene level was markedly
increased. Wei et al. found that the expression of
carnitine palmitoyltransferase-1 (CPT-1) and phos-
phorylated acetyl coenzyme A carboxylase alpha
(ACCα) was significantly decreased, but that of
miR-122, sterol response elementary binding pro-
tein-1 (SREBP-1), FAS-1 and ACCα was markedly
elevated in palmitate-treated HepG2 cells [32]. In
the present study, the CPT1α was not significantly
changed, but FAS was markedly increased in OA-
treated HepG2 cells, similar to the findings by Wei
et al. However, after the cells were treated with CPFE
or CFPM, the expression levels of the FAS gene were
markedly reduced, but the CPT1α gene expression
was significantly increased. Different citrus flavonoids
have different lipid-lowering mechanisms. This find-
ing could be related to the molecular structure of
citrus flavonoids. Lin et al. reported that the ability

of citrus flavonoids to suppress hepatic apoB secre-
tion is related to the molecular structure [30].
Citrus flavonoids attenuated intracellular lipid
accumulation and lipotoxicity in human hepatocellu-
lar carcinoma HepG2 cells [33]. Mosqueda-Solis et al.
analyzed the anti-adipogenic effects of naringenin
and hesperidin on 3T3-L1 preadipocytes and found
that both naringenin and hesperidin at 1 μM were
effective at inducing significant reductions in lipid
accumulation by significantly reducing the expression
of SREBP1c [34], which activates the transcription of
genes involved in fatty acid synthesis [35]. Recent
studies have investigated the idea that fatty acid
synthesis genes (SREBP1c) and FAS are modulated
by miR-122 [36], and mRNA transcripts of CPT1α
involved in the β-oxidation of fatty acids were shown
to be regulated by miR-33 [35].
Flavonoids can regulate miRNA expression.
Differentially expressed miRNAs were detected in
the presence of 1 to 50 μM EGCG ((-)-
epigallocatechin-3-gallate) in a subarachnoid hemor-
rhage model in vitro [37]. Another study reported
that citrus polymethoxylated flavones decrease the
gene expression of liver SREBP1c, FAS, and peroxi-
some proliferator-activated receptor alpha in ham-
sters [38]. The findings in this study showed that
the expression of miR-33 and miR-122 was decreased
in HepG2 cells after treatment with CPFE or CFPM.
The direct targets of miR-122 which modulate lipid
metabolism are essentially unknown and most of the
defined target genes of this miRNA, such as FAS, are
indirectly modulated [39]. Baselga-Escudero et al.
reported that grape seed proanthocyanidins extracts
repressed the expression level of miR-122 and its
target mRNA FAS in FAO cells [40] which was in
consistent with the present study. The overexpression
of miR-33 in HepG2 cells represses CPT1α expres-
sion [35], and CPT1α is negatively correlated with
miR-33. Similar results were found in the A type ECG
dimer and the EGCG dimer of persimmon tannin,
which improved lipid metabolism in L02 cells by
regulating miR-122 and miR-33b [17]. The regulation
of lipid metabolism by citrus flavonoids in vitro may
be due to their modulation of miR-122 and miR-33
expression.
Conclusions
Citrus flavonoids, hesperidin, nobiletin, and tangeretin
attenuated intracellular lipid accumulation. The effec-
tive lipid-lowering dose of citrus peel flavonoid extracts
or its primary flavonoid compounds, including hesper-
idin, nobiletin, and tangeretin, was 10 μg/mL in HepG2
cells. The expression levels of both miR-122 and miR-
33 was downregulated after treatment with CPFE and
CFPM. In addition, the changes in miR-122 and miR-
33 expression after CPFE or CFPM treatment affected
CITRUS FLAVONOID CHANGE LIPID METABOLISM MIRNA 7
their target mRNAs FAS and CPT1α, which are related
to fatty acid synthesis and fatty acid β-oxidation,
respectively. Thus, the in vitro lipid-lowering effect of
citrus flavonoids may be due to the modulation of miR-
122 and miR-33 expression.
Author contributions
Dongxiao Su and Jie Zhang conceived and designed the
experiments. Dongxiao Su and Hesheng Liu performed the
experiments, Dongxiao Su Xiangyang Qi, Lihong Dong,
Ruifen Zhang made contributions to data analysis.
Dongxiao Su and Hesheng Liu wrote the paper.
Disclosure statement
No potential conflict of interest was reported by the
authors.
Funding
This study was supported by the National Natural Science
Foundation of China [31601469]; the Zhejiang Provincial
Top Discipline of Biological Engineering (Level A)
[KF2018002, KF2015004]; the Hubei Provincial Natural
Science Foundation of China [2016CFB179] and the
Guangdong Provincial Science and Technology Project
[2017A070702007].
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