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cbin.10896

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Research Article
Cell Biology International
10.1002/cbin.10896
miR-23b-3p and miR-125b-5p downregulate apo(a) expression by targeting Ets1 in
HepG2 cells†
Running title: miR-23b-3p and miR-125b-5p downregulate apo(a) expression
Jun-fa Zeng1, Zhao-lin Zeng2#, Kai Zhang1, Yue Zhao2, Ya-mi Liu2, Jiao-jiao Chen2, Hai Tong3,
Dang-heng Wei 2, Zhi-sheng Jiang2, Zuo Wang2*
1
The Second Hospital Affiliated to University of South China, Hengyang, Hunan 421001, PR
China
2
Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan
Province, University of South China, Hengyang, Hunan 421001, PR China
3
The First Hospital Affiliated to University of South China, Hengyang, Hunan 421001, PR
China
#These authors contributed equally to this work
*Corresponding author: Zuo Wang, PhD, Professor, Institute of Cardiovascular Research,
Key Laboratory for Atherosclerology of Hunan Province, University of South China,
Hengyang, Hunan 421001, China
E-mail: smt121101@163.com
†
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: [10.1002/cbin.10896]
This article is protected by copyright. All rights reserved
Received: 13 September 2017; Revised: 11 October 2017; Accepted: 22 October 2017
ABSTRACT
High concentrations of plasma lipoprotein(a) [Lp(a)] have been inferred to be an independent
risk factor for cardiovascular and cerebrovascular diseases, such as coronary artery diseases,
restenosis, and stroke. Apolipoprotein(a) [apo(a)] is one of the most important components of
Lp(a) and contributes greatly to the increased concentration of plasma Lp(a). As a critical
positive transacting factor of apo(a) gene, Ets1 has been proven as a target gene of several
miRNAs, such as miR-193b, miR-125b-5p, miR-200b, miR-1, and miR-499. In this study, a
series of experiments on miRNAs and relative miRNAs inhibitor delivered HepG2 cells were
conducted, and two miRNAs that downregulate the apo(a) by targeting the 3′-UTR of Ets1
were identified. Results showed that apo(a) and Ets1 were differentially expressed in
SMMC7721 and HepG2 cell lines. Meanwhile, apo(a) and Ets1 were inversely correlated
with several hepatic endogenous miRNAs, such as miR-125b-5p, miR-23b-3p, miR-26a-5p,
and miR-423-5p, which were predicted to bind to Ets1. Results show that miR-125b-5p and
miR-23b-3p mimics could inhibit the synthesis of apo(a) by directly targeting Ets1 in HepG2,
thereby reducing the plasma Lp (a) concentration.
Key words: atherosclerosis; Lipoprotein (a); miR-23b-3p; miR-125b-5p
Abbreviations
Lp(a): Lipoprotein(a)
FBS: Fetal bovine serum
Apo(a): Apolipoprotein(a)
DMEM: Dulbecco’s modified Eagle’s medium
MiRNAs: MicroRNAs
ELISA: Enzyme-linked immunosorbent assay
ApoB-100: ApolipoproteinB-100
FXR: Farnesoid X receptor
HNF-1α: Hepatocyte nuclear factor 1α
LDL: Low-density lipoprotein
Ets1: Erythroblastosis-twenty six 1
siRNA: Small interfering RNA
UTR: Untranslated Region
PVDF: Polyvinylidene fluoride
1. Introduction
As a highly glycosylated component of lipoprotein(a) [Lp(a)], apolipoprotein(a) [apo(a)] has extremely
minimal affinity with lipids and forms a disulfide linkage with apolipoprotein B100 (apoB100).
ApoB100 is a congener to plasminogen with similar serine proteinase activity and is known for
modulating thrombosis and fibrinolysis response(Tsimikas et al. , 2005). The plasma Lp(a) level varies
among individuals by approximately 1000-fold, and the variation is mainly dependent on the regulation
of gene expression. APOA gene polymorphism, especially kringle IV-type 2, largely determines the Lp
(a) plasma concentration. (Bucci et al., 2016; Santos, 2017). Hence, manipulation of apo(a) gene
expression is considered an effective method to control pathological Lp(a) plasma levels. Regarding the
regulation of apo(a) level, approximately half of the variation in the apo(a) plasma concentration
among individuals is due to transcriptional efficiency, while the rest is due to post-transcriptional
processes, such as secretion efficacy and catabolism. Several transcriptional factors bind to different
loci with enhancer activities at the 40k bp apo(a)-plasminogen intergenic region. A transacting factor
HNF-1α abundantly expressed in hepatocytes was shown to bind to the -98 to 130 bp regions of apo(a)
gene and transactivate apo(a) gene (Negi et al., 2004; Wade et al., 1997). An enhancer element within a
LINE retro-transposon has been identified at 18 kb away from the apo(a) promoter 5′-UTR, and an
enhancer associated with transcription factors Sp1 and PPAR1 are located at 26 kb upstream of apo(a)
promoter (Haas et al., 2004; Qu et al., 2017). An apo(a) transcriptional control region at 20 kb 5′ from
the starting site also contained an enhancer element that mediated the effect of the Ets transcription
factor families on apo(a) gene (Yang et al., 1998). Furthermore, a retinoid response element and an
estrogen response element (Boffelli, 1999) have also been identified in the apo(a) promoter upstream.
In a previous study, basal transcription of the apo(a) gene was shown to be dependent on the
transactivating action of liver-enriched transcription factor HNF-1α in the promoter HNF-1α
specifically to express in the liver and a few other tissues (Qu et al., 2016). This phenomenon partly
accounts for the specificity of the apo(a) gene expression in the liver. However, the transactivating
capacity mediated by the apo(a) promoter is extremely weak, such that very low basal apo(a) level can
only be determined by conducting series of experiments on transiently transfected HepG2 cells. Thus,
the more active cis-acting element of apo(a) gene, similar to several genes, might exist in the region far
from the basal promoter. Elevated Lp(a) is an important risk factor for premature atherosclerosis, and
high Lp(a) levels are also associated with autoimmune diseases (Missala et al., 2012). However,
Ets1-related signal pathways are correlated with autoimmune diseases and play an important role in
inflammatory cell recruitment and vascular remodeling in response to systemic administration of the
vasoactive peptide angiotensin II. As a transacting factor of apo(a) gene, Ets1 and apo(a) are correlated
with autoimmune diseases and vessel inflammatory response, implying the Ets1 exert prominent
function in the synthesis of apo(a) and consequent Lp(a) level. Related large-scale epidemiological
investigation showed remarkable variation in the plasma apo(a) among individuals. These findings
suggest that variation in the apo(a) synthesis in hepatocyte determines the majority of Lp(a) variation
among individuals. Therefore, the apo(a) levels in two hepatoma carcinoma cell lines, namely, HepG2
and SMMC7721 cells were analyzed.
MicroRNAs (miRNAs) are a wide range of small non-coding RNA molecules that originate from
longer primary transcripts termed as pri-miRNAs. MiRNAs underwent successive enzymatic
maturation steps (by Drosha in the nucleus and Dicer in the cytoplasm) from hairpin-loop structures.
MiRNAs can regulate gene expression at messenger RNA degradation and translation suppression to
inhibit gene expression (Mulholland et al. , 2017; Kowara et al. , 2017). These molecules are involved
in many intracellular processes, such as cell cycle regulation, differentiation, development, metabolism,
and ageing, through post-transcriptional suppression (Li et al., 2017; Spizzo et al., 2009). To date,
several miRNAs, such as miR-1, miR-499 (Wei et al., 2012), miR-193b (Xu et al., 2010), miR-125b-5p
(Zhang et al., 2011a), and miR-200b(Chan et al., 2011a), have been confirmed to target Ets1 gene.
However, whether these miRNAs can regulate apo(a) expression remains largely unknown.
In this study, miRNA overexpression and underexpression cell lines with transient delivered HepG2
cells were constructed. Western blot analysis was performed to detect the expression of apo(a) and Ets1.
Intracellular miRNA level was measured using qRT-PCR. Dual-luciferase reporter gene was used to
verify the binding site of certain miRNA located at the target gene. A series of experiments were
conducted to demonstrate that miR-125b-5p and miR-23b-3p lower the apo(a) level in HepG2 cells by
targeting the Ets1 gene.
2. Materials and Methods
2.1 Materials
Human hepatoma cell line HepG2 and SMMC7721 were obtained from Peking University. HEK293
cell was obtained from Fudan University. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s
medium (DMEM) containing high glucose, and trypsin were purchased from Hyclone (Thermo Fisher
Scientific, Logan, USA). The miR mimics, miR inhibitors, and siRNA were purchased from Ruibo
Biology (Guang Zhou, China). Total RNA purification kit and Rever AidTM First Strand cDNA
synthesis kit were purchased from Thermo Fisher Scientific (Logan, USA). Apo(a) ELISA kit was
purchased from Cell Biolabs (Santiago, USA). The rabbit monoclonal antibody against β-actin and
human polyclonal antibody against apo(a) were all purchased from Bioworld Technology (USA). The
β-actin secondary antibody (Goat anti rabbit) and apo(a) secondary antibody (Goat anti human) were
purchased from CWBIO (Peking, China). The rabbit polyclonal antibody against Ets-1 and secondary
antibody (Goat anti rabbit) was purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Dual
Fluorescent Enzyme Reporter Detection Kit was purchased from Promega Corporation (Wisconsin,
USA). Real-time PCR Assay Kit was purchased from CWBIO (Beijing, China).
2.2 Cell culture
HepG2, SMMC7721, and HEK293 cell line were grown in DMEM containing 5% FBS at 37 °C with 5%
CO2. The miR mimics, miR inhibitors, siRNA, or Ets1-expressing plasmid were transfected. Cells were
seeded in a six-well plate, and transfection was performed at 70% confluence. The miRNA mimics
labeled with 5′ Cy3 and siRNAs were designed and synthesized by RiboBio (Guangzhou, China).
Transfections for siRNAs, miRNA mimics, miR inhibitors, and Ets1-expressing plasmids were all
performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.
2.3 RT-PCR
Total RNA was prepared 48 h after transfection. Total RNA was extracted using Ultrapure RNA kit
(CWBIO, China). The first cDNA of total RNA was synthesized using Revert Aid First Strand cDNA
synthesis kit (Thermo). PCR primers were as follows: for apo(a),
F 5′-GACGGGAGACAGAGTGAAGC-3′, R 5′-TCAAATCAAAATAAGTGCAGAGTT-3′, product
length 243 bp; for β-actin, F 5′-ACACTGTGCCCATCTACGAGGGG-3′,
R 5′-ATGATGGAGTTGAAGGTAGTTTCGTGGAT-3′, product length 367 bp. PCR conditions were
as follows: pre-denaturation at 94 °C for 5 min; 35 cycles (denaturation at 94 °C for 30 s, annealing at
56 °C for 30 s, extension at 72 °C for 40 s). Finally, the reaction was terminated by heating at 72 °C for
5 min. Electrophoresis was performed on the agarose gel (2%).
2.4 Western blot analysis and immunocytochemistry
The protein was prepared 48 h after transfection and used for Western blot analysis. Western blot
analysis and immunocytochemistry were performed using polyclonal rabbit antibody against Ets-1
(Santa Cruz Biotechnology) and polyclonal human antibody against apo(a) (Abnova). Briefly, the cells
were harvested in lysis buffer that contains the active ingredient, 1% Triton X-100. Subsequently,
40 μg of the protein sample per lane was separated on SDS-PAGE. Afterward, the protein on the
separation gel was transferred to a PVDF membrane (Millipore), blocked, and incubated with primary
antibody overnight at 4 °C and subsequently with secondary antibody at room temperature for 2 h, with
the following antibody concentrations: For Ets1, primary antibody was at 1:5000 concentration,
secondary antibody at 1:5000 concentration; for apo(a), primary antibody at 1:3000 concentration,
secondary antibody at 1:3000 concentration; and β-actin (Sigma, 1:10000) serves as loading control.
Protein bands were visualized using ECL Plus Western blot detection system. Immunocytochemistry
was performed using SP Rabbit HRP kit (CWBIO, China). Cells were fixed with 95% ethanol,
permeabilized with 0.3% Triton X-100, and blocked with endogenous peroxidase confining buffer and
normal goat serum. Then, the cells were incubated with primary antibody against Ets-1 at a 1:500
concentration at 4 °C and then the secondary antibody, which was labeled by biotin and
streptavidin-HRP, respectively. The signal was visualized using DAB. Images were captured by the
microscope using Image-Pro Plus 6.0.
2.5 ELISA
After 48 h post-transfection, the medium was collected and centrifuged (3000 rpm for 10 min), and the
supernatants were harvested for direct apo(a) analysis using ELISA. The concentration of apo(a) in the
cell culture supernatants was determined using commercially available ELISA kits (Cell Biolabs, USA)
according to the manufacturer's instruction. Absorption was identified with the Elx800 microplate
reader (Bio-Tek, USA) at 450 nm. The detection limit for this kit was 40 µg/L to 1600 µg/L.
2.6 Dual-luciferase reporter assays
HEK293 cells were plated in six-well plates to reach 70%–80% confluence. Cells were transfected with
luciferase reporter vectors (400 ng) containing the Ets1 3′-UTR (pGL3-Ets1-3′-UTR with 900 bp
starting from 1300 to 2200) or deletion of Ets1 3′-UTR (as control plasmid). Normalization was
achieved by co-transfection with Renilla plasmid (10 ng) using Lipofectamine 2000. Cells were lysed
after 48 h, and luciferase activity was determined using the dual-luciferase reporter assay system
(Promega). Data were presented as the ratio of firefly to Renilla luciferase assay.
2.7 MiRNA microassay
Total RNA was extracted using TRIzol (Invitrogen) and miR Neasy mini kit (QIAGEN) according to
the manufacturer’s instructions. The RNA quantity was measured using the NanoDrop 1000. Then, the
samples were labeled using miR CURY™ Hy3™/Hy5™ Power labeling kit and hybridized on
miRCURY™ LNA Array (v.18.0). After being washed, the slides were scanned using the Axon
GenePix 4000B microarray scanner. Scanned images were then imported into GenePix Pro 6.0
software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged, and
miRNAs (with ≥50 intensities) in all samples were chosen to calculate the normalization factor.
Expressed data were normalized using median normalization. After normalization, differentially
expressed miRNAs were identified through fold-change filtering. Finally, hierarchical clustering was
performed to show distinguishable miRNA expression profiling among samples.
2.8 MiRNA real-time RT-PCR
Small RNA was prepared 48 h after transfection and used for qRT-PCR. Small RNA was extracted
using miRNA purification. Reactions were performed in triplicate using a Light Cycler 3.0 real-time
PCR instrument (Roche). The first cDNA for small RNA was synthesized using miRNA cDNA kit
(CWBIO, China). Real-time PCR for miRNA was performed using miRNA Real-time PCR assay kit
(CWBIO, China). The experiment was performed according to the manufacturer’s protocol. The
primers of miRNAs were as follows: miR-16-5p (as internal reference miRNA for data normalization),
5′-TAGCAGCACGTAAATATTGG-3′; miR-26a-5p, 5′-TTCAAGTAATCCAGGATAGG-3′;
miR-423-5p, 5′-TGAGGGGCAGAGAGCGAGAC-3′; miR-125b-5p,
5′-TCCCTGAGACCCTAACTTGT-3′; and miR-23b-3p, 5′-ATCACATTGCCAGGGATTAC-3′.
Expression level was calculated according to the ΔΔCt method, normalizing to the least variable
miR-16-5p.
2.9 Statistical analyses
Data are reported as mean ± S.E. of at least three independent experiments. Difference between two
means was tested using Student’s t test, whereas one-way ANOVA was used to compare three groups
or more. P value < 0.05 was considered statistically significant.
3. Results
3.1 Differential expression of apo(a) and Ets1 in HepG2 and SMMC7721 cells
Western blot analysis was performed to assess the apo(a) level in HepG2 and SMMC7721 cell lines.
Apo(a) expression in HepG2 was dramatically higher than those in SMMC7721cells (Fig. 1A). Further
study displays the differential apo(a) mRNA expression level between the two cell lines (Fig. 1C). The
expression levels of Ets1 in both cell lines were further examined. The results showed that the
differential expression of Ets1 between the two cell lines was consistent with that of apo(a). Thus, Ets1
expression was higher in HepG2 cells than in SMMC7721 cells, and the difference was considered
statistically significant (Fig. 1B). To confirm that Ets1 was an important transcriptional factor of apo(a)
gene, the Ets1 gene in HepG2 cells was knocked down via Ets1 siRNA transfection, and the change in
intracellular apo(a) was observed. The results revealed that Ets1 siRNA could notably inhibit Ets1 and
apo(a) expression in HepG2 cells (Fig. 2). Thus, Ets1 was demonstrated to modulate apo(a) expression
as an important transcriptional factor of ETS1 gene.
3.2 Differential expression of miRNAs predicted to bind to 3′-UTR of target gene Ets1
Due to the different Ets1 level in the two cell lines, and many microRNA can targeting the 3'UTR of
Ets1,so we hypothesized that the differential expression of Ets1 in the Hep G2 and SMMC7721 cells is
partially due to the distinct profiles of the miRNAs. Therefore, we used miRNA microassay to
semiquantitatively analyze miRNA expression profile in the Hep G2 and SMMC7721 cells. The result
consistent with our hypothesis (Fig. 3). High miRNA expression could induce the downregulation of
related target gene. In SMC7721 cells Ets1 gene expression is low, so the level of miRNA inhibiting
Ets1 expression shall be elevated. Unlike in HepG2 cells, where Ets1 gene expression is high, therefore
Ets1-inhibiting miRNA expression level is supposed to be decreased. Among the reported miRNA,
miR-125b-5p was the only miRNA highly expressed in SMMC7721 cells but lowly expressed in
HepG2 compared with other two groups of miRNA profiles (Li et al.,2015). Similar to miR-125b-5p,
the other miRNAs with possible interaction with the 3′-UTR of Ets1 mRNA predicted by Target Scan
program included miR-23b-3p, miR-26a-5p, and miR-423-5p. Moreover, these miRNAs had
expression tendency similar to that of miR-125b-5p between the two cell lines. The difference in the
miRNA concentrations between HepG2 and SMMC7721 cell lines were further confirmed by miRNA
real-time RT-PCR.
MiR-125b-5p, miR-23b-3p, miR-26a-5p, and miR-423-5p mimics or inhibitors were transiently
transfected into HepG2 to build related miRNA overexpression and underexpression HepG2 cell lines.
The miRNA 5′ labeled with Cy3 exhibit fluorescence when stimulated by green ray. Thus, miRNA
transfection efficiency was evaluated by measuring intracellular fluorescence intensity, and statistical
analysis showed no significant difference in the fluorescence intensities among the four transfection
groups (Fig. 4). Moreover, miRNA level at 48 h post-transfection was assessed using miRNA real-time
RT-PCR. The results showed that the transfection groups had higher level of transfected miRNA than
that of the control group, and the difference was statistically significant (Fig. 5B). Subsequently, apo(a)
expression level was analyzed by Western blot and RT-PCR, and Ets1 expression was detected using
Western blot. The result showed that miR-125b-5p and miR-23b-3p significantly decreased the protein
and mRNA levels of apo(a) and the protein level of Ets1 compared with the control group (Figs. 7A,
7B, and 7E). Conversely, the corresponding miRNA inhibitor notably upregulated the expression of
apo(a) and Ets1 (Figs. 7C, 7D, and 7F). In addition, we examined the apo(a) concentration of culture
medium using the apo(a) ELISA kit. The results revealed that the extracellular levels of apo(a) in all
miRNA mimic transfection groups had similar concentration tendencies and intracellular levels of
apo(a), and its miRNA inhibitor groups had the same results (Fig. 6). Results indicated that Ets1 served
as shared target gene of both miR-125b-5p and miR-23b-3p. Thus, Ets1 may be a target gene for
miR-125b- 5p, and this result is consistent with the previous studies (Zhang et al., 2011; Luo et al.,
2013). Moreover, we further confirmed the Ets1 level in HepG2 cells at 48 h post-transfection through
immunocytochemistry. After DAB staining and hematoxylin re-dyeing, miR-125b- 5p and miR-23b-3p
transfection groups had the lowest Ets1 level, and relative inhibitor transfection groups had the highest
Ets1 level among overexpression, underexpression, and control groups.
3.3 Ets1 as a shared target of both miR-125b-5p and miR-23b-3p
MiRNA target reporter dual luciferase assay was performed to verify whether miR-125b and
miR-23b-3p directly bind to Ets1 3′-UTR. The sequence of Ets-1 3′-UTR containing a putative site for
miR-125b-5p and miR-23b-3p was cloned into the XbaI site located downstream of the luciferase open
reading frame. Then, pGL3-Ets1 3′-UTR plasmid was cotransfected in HEK-293 cells with miR mimic
or matching control mimic. Apparently, reduced luciferase activity was observed in both miR-125b and
miR-23b-3p mimic groups compared with the control group. HEK-293 transfected with plasmid
without the selected Ets1 3′-UTR sequence was used to confirm the specificity of luciferase activity
repression. Expectedly, the Ets-1 3′-UTR sequence deletion from the construct abolished the inhibitory
effects of miR-125b-5p and miR-23b-3p mimics on luciferase activity (Fig. 9). These results suggest
that miR-125b-5p and miR-23b-3p bind to the Ets-1 3′-UTR to downregulate Ets-1 expression at the
post-transcriptional level.
4. Discussion
Lp(a) is a lipoprotein that is very similar to the structure of low-density lipoprotein (LDL). Lp(a) is
composed of LDL lipid Core, apoB100, and specific components of apo(a) (Hoover-Plow and Huang,
2013). Elevated Lp(a) is an important independent risk factor for atherosclerosis and related diseases,
such as coronary heart disease, calcified aortic stenosis, and ischemic stroke (Tsimikas, 2017). Thus,
reducing the patient's plasma Lp (a) levels will improve the prognosis of related diseases (Norata,
2013). As a secondary priority of LDL-cholesterol reduction, plasma Lp (a) levels is recommended to
be maintained at less than 50 mg/dL (Nordestgaard et al., 2010). However, Lp(a) is difficult to study
because of the limited research model, significantly limiting the development of drugs for reducing
plasma Lp (a) (Ference et al. , 2012). In recent years, significant development has been achieved in the
study of LP(a) in terms of its metabolism and modification, but the specific mechanism remains
unclear.
The direct relation between apo(a) and some miRNAs has not been reported yet. However, as a
transcription factor of apo(a) gene, Ets1 has been proven to serve as a target gene for several miRNAs,
such as miRNA-222/221, miRNA-155, miRNA-193b, miRNA-200b, miRNA-208b, miRNA-370,
miRNA-145, and miRNA-125b (Yang, 1998; Wei, 2012; Xu 2010; Zhang et al., 2011b; Chan et al.,
2011b). The differentially expressed apo(a) and Ets1 have the same differential expression between
HepG2 and SMMC7721 cells, suggesting that one or more miRNAs targeting Ets1 are expressed
differentially to account for the different levels of apo(a) between HepG2 and SMMC7721 cells. After
the miRNA array, 50 kinds of miRNAs were found to be highly expressed in SMMC7721 cells but
lowly expressed in HepG2 cells by comparing the miRNA profiles in these two cells lines. Furthermore,
miRNA target gene prediction software (Targetscan) was used to limit the possible miRNA in a smaller
range, that is, only four kinds of miRNAs, namely, miR-125b-5p, miR-23b-3p, miR-26a-5p, and
miR-423-5p.
Among the four miRNAs predicted to have an effect on Ets1, miR-125b-5p and miR-23b-3p were
demonstrated to downregulate apo(a) expression through Ets1 post-transcription suppression, contrary
to miR-26a-5p and miR-423-5p. The functioning mechanism of miRNAs with Ets1 was verified via
cotransfection of pGL3-Ets1–3′-UTR reporter constructs and miRNA mimic. Thus, miR-125b-5p and
miR-23b-3p downregulated the apo(a) level by Ets1 post-transcriptional suppression in HepG2. Several
cases may be considered. First, miR-26a-5p and miR-423-5p are independent of Ets1, and this
phenomenon could be elucidated by the false positive rate of the target gene prediction. Second,
possible unknown signal pathway mediated by these two miRNAs counteract the downregulation of
Ets1 caused by miRNAs. Third, the effect of miRNAs on Ets1 is very weak such that this effect was
compensated by cell modulation mechanism. Further experiments are needed to verify these cases.
Apo(a) variance among individuals reached 1000-fold. Apo(a) intracellular anabolism is ambiguous. A
technique to efficiently downregulate apo(a) in vivo and in vitro has not been established. To date, the
reported methods to reduce apo(a) contained niacin, estrogen, antisense nucleotide, FXR agonist, and
dialysis (Chapman et al., 2010; Chennamsetty et al., 2011; Jaeger et al., 2009; Merki et al., 2011).
In this study, miR-125b-5p and miR-23b-3p could lower apo(a) and Ets1 levels in HepG2 cells. As a
transcriptional factor for apo(a) gene, depletion of Ets1 notably downregulated apo(a) level in HepG2
cells. Meanwhile, miR-125b-5p and miR-23b-3p were shown to negatively modulate Ets1 expression
by directly binding to the Ets1 mRNA 3′-UTR. This study seems to provide a new way to reduce
apo(a). The endogenous miRNAs in the hepatocyte notably regulated the apo(a) level. Given the
differentially expressed miRNA profiles in the variously derived hepatocytes, apo(a) expression in the
hepatocyte was inferred to be at least partially dependent on several miRNAs. This phenomenon
apparently elucidates a part of the apo(a) variance in people but should be demonstrated by large-scale
epidemiological observation. Therefore, we speculated that the differentially expressed miRNAs in the
two cell lines partially resulted in at least different levels of apo(a), and two or more miRNAs might
have the shared target gene. MiRNA is believed to control 30% protein expression and be involved in
almost all cellular activities (Yue, 2011). Nevertheless, apo(a) metabolism includes transcription,
translation, and transportation processes. Association study of apo(a) and apo(a) metabolism may have
a broad prospect. MiR-125b-5p and miR-23b-3p can lower apo(a) in cells, but this lowering effect and
lowering capacity need to be confirmed by animal experiment. Moreover, large-scale epidemiological
clinic observation might reveal more effective miRNAs that can regulate apo(a) expression.
Limitations of this study: The two cell lines used in this study are hepatocarcinoma cells, we did not
use normal hepatocyte cells for related research. Further, use normal hepatocyte cells, and animal
experiments should be performed to explore the functional mechanism, which will be our topic in
future studies.
5. Conclusion
MiR-125b-5p and miR-23b-3p could lower apo(a) expression level and Ets1 protein level. As a
transcriptional factor for apo(a) gene, depletion of Ets1 notably decreased apo(a) level in HepG2 cells.
Furthermore, miR-125b-5p and miR-23b-3p were identified to negatively modulate Ets1 by directly
binding to Ets1 mRNA 3′-UTR. Therefore, these findings suggest that the downregulation effect of
miR-125b-5p and miR-23b-3p on apo(a) was mediated by their Ets1 gene at the post-transcriptional
level.
Funding
This study was supported by the Natural Science Foundation of China (No. 81070221) and the
Innovative Research Team for Science and Technology in Higher Educational Institutions of Hunan
Province and the Construct Program of the Key Discipline in Hunan Province and Scientific Research
Fund of Hunan Provincial Education Department (15C1201).
Conflict of Interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the
authors.
Informed consent
Informed consent was obtained from all individual participants included in the study.
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LEGENDS
Figure 1: Expression of apo(a) and Ets1 were higher in HepG2 cells, compared with SMMC7721 cells.
A. Western blot analysis of apo(a) protein level in HepG2 and SMMC7721 cell lines
B. Western blot analysis of Ets1 protein level in HepG2 and SMMC7721 cell lines
C. RT-PCR test of apo(a) mRNA level in HepG2 and SMMC-7721 cell lines. β-actin served as
loading control.
Triplicated assay was performed for these experiments. Results were presented as mean ± S.E. ***
indicates p<0.001; ** represents p<0.01.
Figure 2: Ets1 serves as transcriptional factor up-regulating apo(a) in HepG2 cells. Western blot
analysis of apo(a) (A) and Ets1 (B) in Ets1 siRNA delivered HepG2 cells compared with matched
control. Triplicated assay for each experiments. Results were presented as mean ± S.E. *** indicates
p<0.001; ** represents p<0.01.
Figure 3: Partial differential expressed miRNAs between HepG2 cells and SMMC7721 cells. Colour
bar above were the segmental heat maps for miRNAs microassay. The table described the miRNAs
expression difference between two cell lines. 1 represented SMMC7721 cells; 2 represented HepG2
cells. Column "Fold change" contained the ratio of normalized intensities between two cells. Column
"ForeGround-BackGround" meaned the signal of the miRNA after background correction. Column
"Normalized" meaned the normalized signal of the miRNA. We used Median Normalization Method to
perform array normalization.
Figure 4: The transfection efficiency among the four miRNAs has no significant difference. The
pictures (A, B, C and D) respectively represented fluorescence images of miRNA-26a-5p,
miRNA-423-5p, miRNA-125b, miRNA-23b-3p at 24h post-transfection (A, B, C and D, magnification
200×). The histogram on the right side was statistically analysis for mean IOD (mean IOD symbolizes
the concentration of miRNAs in cells) of each picture on the left side. Triplicated assay for each
experiments.
Figure 5: The differential expressed miRNAs (miR-125b, miRNA-23b-3p, miRNA-26a-5p,
miRNA-423-5p) between HepG2 cells and SMMC-7721 cells by miRNA microassay were confirmed
by miRNA real time RT-PCR.
A. Real time RT-PCR analysis of miRNAs level between above two cell lines was normalized to
stably expressed miRNA. Triplicated assay for each experiments. Results were presented as
mean ± S.E. *** indicates p<0.001; ** represents p<0.01.
B. Real time RT-PCR analysis of miRNA concentration in miRNA mimic delivered HepG2 cells
and corresponding control. The relative quantity of miRNAs was normalized to the stably
expressed miRNA.
Triplicated assay for each experiments. Results were presented as mean ± S.E. *** indicates p<0.001;
** represents p<0.01 compared with control.
Figure 6: Apo(a) level of medium in miRNA-125b and miRNA-23b-3p groups had the similar trend as
intracellular apo(a) level compared with control. The same results were in inhibitor experiment.
A. ELISA analysis of apo(a) concentration of culture medium at 48h miRNAs mimics
post-transfection.
B. ELISA analysis of apo(a) for inhibitor experiment.
Triplicated assay for each experiments. Results were presented as mean ± S.E. *** indicates p<0.001;
** represents p<0.01 compared with control.
Figure 7: miRNA-125b, miRNA-23b-3p dramatically reduced the expression level of apo(a) and Ets1
in HepG2 cells compared with control. On the other hand, inhibitors for miRNA-125b, and
miRNA-23b-3p significantly enhanced the level of apo(a) and Ets1 in HepG2 cells compared with
control.
A and B. Western blot analysis of apo(a) and Ets1 in miRNAs mimics delivered HepG2 cells.
C and D. Western blot analysis of apo(a) and Ets1 in miRNAs inhibitors delivered HepG2 cells.
E and F. RT-PCR analysis of apo(a) mRNA level in miRNAs mimics and inhibitors delivered
HepG2 cells.
Triplicated assay for each experiments. Results were presented as mean ± S.E. *** indicates p<0.001;
** represents p<0.01 compared with control.
Figure 8: The change of Ets1 expression by miRNA-125b, miRNA-23b-3p and corresponding
inhibitor were verified by ICC assay.
A. a negative control (PBS instead of primary antibody incubation)
B. experimental control
C. miRNA-125b transfection group
D. miRNA-23b-3p transfection group
E. (and F) miRNA-125b inhibitor transfection group and miRNA-23b-3p inhibitor, transfection
group (A, B, C and D, magnification 100×).
The histogram on the right side is statistically analysis for mean IOD (mean IOD symbolizes the
concentration of Ets1 in cells) for each picture on the left side. Triplicated assay for each experiments.
Results were presented as mean ± S.E. *** indicates p<0.001; ** represents p<0.01 compared with
control.
Figure 9: Ets1 served as shared target of miRNA-125b (A) and miRNA-23b-3p (B). miRNAs target
reporter luciferase assay after 48h post-cotransfection. (C) putative binding sites in Ets1 3’-UTR for
miRNA-23b-3p and miRNA-125b. Results were normalized with Renilla luciferase and expressed as
mean ± S.E. *** indicates p<0.001 compared with control mimic-transfected cells or control
plasmid-transfected cells.
Figure. 1
Figure. 2
Figure. 3
Figure. 4
Figure. 5
Figure. 6
Figure. 7
Figure. 7E and F
Figure. 8
Figure. 9
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