Cardiovascular Journal of Africa: Vol 27 No 4 (July/August 2016)

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 4, July/August 2016 AFRICA 229
Methods
All relevant ethical and clinical approvals were obtained for
this study. A total of 88 patients with coronary atherosclerotic
disease (CAD) (63 males; mean age 60.80 ± 9.27 years) and 88
unrelated control individuals (53 males; mean age 58.18 ± 10.43
years) were retrospectively enrolled from among in-patients at
the Anhui Cancer Hospital of Bengbu Medical College, Bengbu
City, Anhui Province, China. All participants were of Han
Chinese descent.
The criterion for inclusion in the CAD group was ≥ 50%
stenosis in at least one major segment of the coronary arteries,
determined by coronary artery angiography. Individuals in
the control group had negative coronary artery angiography
results (used to rule out CAD). A history of conventional risk
factors for CAD or hypercholesterolaemia (total cholesterol ≥
5.7 mmol/l) was obtained from the medical records. Exclusion
criteria for both groups were familial hypercholesterolaemia,
diabetes mellitus, cancer, renal disease and any other chronic
illness.
For lipid analysis, whole blood samples were drawn from all
participants in the morning after a 12-hour fast. Fasting plasma
glucose (FPG), triglyceride (TG), HDL and LDL cholesterol
(HDL-C and LDL-C), and total cholesterol (TC) levels were
determined for each subject using an automated chemistry
analyser (AU2000; Olympus Promarketing, Tokyo, Japan).
For ApoM analysis, 5-ml blood samples were collected in
EDTA (as anticoagulant) after an overnight fast. Samples were
centrifuged at 3 000 rpm for 10 minutes at room temperature.
Separated sera were stored at –40°C. The serum ApoM level
was quantified with ELISA, using horseradish peroxidase and
the anti-ApoM antibodies 1E2 and 8F12 (Hunan Far Tai
Biotechnology Co, Ltd). To minimise errors, all samples were
used in the same reaction system, each test was repeated twice,
and the average value was used in the final analysis.
Genotyping of ApoM T-778C and T-855C
polymorphisms
Genomic DNA was extracted from peripheral blood using a
salting-out protocol. Single-nucleotide polymorphisms (SNPs)
selected for this study are recorded in the public dbSNP database
). Two SNPs, rs9404941
(T-855C) and rs805296 (T-778C), representing at least 5% of
the minor allele frequency in the promoter region of the ApoM
gene in the Han Chinese, were genotyped with the PCR-RFLP
method. PCR primers for the ApoM T-855C and T-778C SNPs
were designed based on GeneBank sequences (Table 1).
PCR products were digested in a total volume of 20 μl
containing 1 μg of PCR product, 5 U of restriction endonuclease
HaeIII (-855) or Rsa I (-778) (Fermentas, USA), 2 μg acetylated
bovine serum albumin (BSA), and 2 μl restriction enzyme buffer.
ApoM polymorphisms were detected with 2.5% agarose gel
(Promega, USA) electrophoresis and visualised with ethidium
bromide staining.
In the T-778C polymorphism of the ApoM gene, the T →C
substitution creates an Rsa I restriction site. The PCR product
with the C allele was digested into two fragments (272 and 164
bp), whereas the T allele was not cut by RsaI. Because T-855C
contains GGC(C/T) and HaeIII recognises the GGCC sequence,
digestion of TT homozygotes with HaeIII produced 496-,
352- and 84-bp fragments, CC homozygotes gave 84-, 157- and
195-bp products, and CT heterozygotes produced 84-, 157-, 195-
and 436-bp fragments (Fig. 1C). To confirm detection of the
T-778C polymorphism of the ApoM gene by PCR-RFLP, the
PCR products were also sequenced (Shengong Bio Company,
Shanghai, China; Fig. 1C).
Construction of luciferase reporter plasmids
Four reporter plasmids, encompassing −1334 to +227 bp of the
human ApoM promoter, were constructed. Plasmids containing
the regions −855T to −778T, −855T to –778C, −855C to −778T
and −855C to −778C were named PGL3-TT, PGL3-TC, PGL3-
CT and PGL3-CC, respectively (Fig. 2).
PCR primers containing a Kpn I- and HindIII-inserted forward
and reverse primer, respectively, used in the construction of these
plasmids, are shown in Table 1. PCR products were first ligated into
the PMD®8-T vector (Takara, Dalian, China) and then subcloned
into the pGL3-basic vector (Promega). All constructs used in this
study were sequenced to confirm their authenticity (Fig. 2).
Cell culture, transfection and reporter gene assays
The HepG2 hepatoma cell line was propagated in Dulbecco’s
modified Eagle’s medium (DMEM; Hyclone) with 10% foetal
calf serum (FCS) and maintained in 5% CO2 at 37°C. HepG2
cells were seeded in 24-well plates at 5 × 104 HepG2 cells/
well, respectively. After 24 hours, the cells were transfected
with pGL3-basic (a promoterless control), pGL3-promoter (a
promoter control), and two pGL3-basic constructed plasmids.
The pRL-SV40 plasmid (Promega) was co-transfected as a
normalising control.
To prevent transcription of ApoM by endogenous liver
cell extracts, recombinant vector transfection groups were set
up with and without liver cell extracts. After six hours, the
AP-2α siRNA and AP-2α siRNA negative control fragments
(Gima) were co-transfected into the two pGL3-basic constructed
plasmid groups. The AP-2α expression vector (GeneChem)
Table 1. Primer, probe and oligonucleotide sequences used in this study
Primer/probe/oligonucleotide Sequence
ApoM T-855C and T-778C SNPs 5′-GGTACCGTCTTTGCTAAGGGCTTTATGTGCATTA-3′
(forward)
5′-AAGCTTTGTTGGTGTCAGGCAGAATGTGTCCAA-3 ′
(reverse)
Luciferase reporter plasmids 5′-AAGCTTCTCCTACTCGGGAATCAT-3′ (forward)
5′ -GGTACCTCCAGAGCCTCCACCATA-3′ (reverse)
Wild-type T1 probe synthesis 5′ -TCGACATCCCAGGCTCAAGCAATCCT-3′
Wild-type T2 probe synthesis 5′-AGGATTGCTTGAGCCTGGGATGTCGA-3′
Mutant-type C1 probe synthesis 5′-TCGACATCCCAGGCCCAAGCAATCCT-3 ′
Mutant-type C2 probe synthesis 5′-AGGATTGCTTGGGCCTGGGATGTCGA-3′
Sense 5′-UUCUCCGAACGUGUCACGUTT-3′
Negative control Anti-sense 5 ′-ACGUGACACGUUCGGAGAATT-3′
Interference fragment 1 Sense 5′-CCAGAUCAAACUGUAAUUATT-3′
Anti-sense 5 ′-UAAUUACAAGUUUGAUCUGGTT-3′
Interference fragment 2 Sense 5′-GGAAGAUCUUUAAGAGAAATT-3′
Anti-sense 5 ′-UUUCUCUUAAAGAUCUUCCTT-3′
Interference fragment 3 Sense 5′-CCUGCUCAUCACUAGUATT-3′
Anti-sense 5 ′-UACUAGUGAUGUGAGCAGGTT-3′
AP-2α forward primer 5′-CTGGGCACTGTAGGTCAATCT-3′
AP-2α reverse primer 5′-CCTCCTCGATGGCGTGAG-3′
GAPDH forward primer 5¢-CAAGGTCATCCATGACAACTTTG-3¢
GAPDH reverse primer 5¢-GTCCACCACCCTGTTGCTGTAG-3¢